Agilent 1290 Infinity LC System
Application Compendium
Introduction
The Agilent 1290 Infinity LC Application Compendium
With the introduction of the Agilent 1290 Infinity LC System, Agilent Technologies
has innovated HPLC as well as UHPLC with new technology, which delivers more
resolution per time, more sensitivity and more flexibility.
This compendium is a collection of applications that Agilent has developed for a
number of key UHPLC applications for various industry and application segments
using the new Agilent 1290 Infinity LC System. Analytical equipment, columns,
separation media and chromatographic conditions are described for each application. All proposed solutions are accompanied by examples, configuration overviews
and method descriptions.
The compendium also includes detailed performance characteristics for the Agilent
1290 Infinity Binary Pump, the Agilent 1290 Infinity Thermostatted Column
Compartment and the Agilent 1290 Infinity Diode Array Detector.
The Agilent 1290 Infinity LC Application Compendium was written to find starting
conditions for your own application development and to make ordering of the entire
HPLC systems easy, correct and complete, so it meets your requirements
and fulfils your needs.
2
Table of Contents
Introduction - Agilent 1290 Infinity LC Application Compendium . . . . . . . . . . . . . . . . .2
The Agilent 1290 Infinity LC System: Powerful – Sensitive – Flexible . . . . . . . . . . . . .5
Ultrafast analysis of synthetic antioxidants in vegetable oils using the
Agilent 1290 Infinity LC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Tryptic digest analysis using the Agilent 1290 Infinity LC System . . . . . . . . . . . . . . . . .13
Developing a green LC method for the determination of the furocoumarins
5-MOP and 8-MOP in citrus oils using the Agilent 1290 Infinity LC System . . . . . . . .17
Increasing productivity in the analysis of impurities in metoclopramide
hydrochloride formulations using the Agilent 1290 Infinity LC System . . . . . . . . . . . .23
Software-assisted, high-throughput identification of main metabolites
of pharmaceutical drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Environmental applications of the Agilent 1290 Infinity UHPLC System:
The evolution of chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Screening impurities in fine chemicals using the Agilent 1290 Infinity
LC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
High-resolution analysis of intact triglycerides by reversed phase HPLC
using the Agilent 1290 Infinity LC UHPLC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Increasing resolution and speed by operating UHPLC columns up to 1200 bar . . . . .67
Metabolic stability study using cassette analysis and polarity switching
in an ultra high performance liquid chromatography (UHPLC)triple quadrupole LC/MS System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Extended ionization capability of thermal gradient focusing ESI in
high-throughput in-vitro ADME assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
High-throughput bioanalytical method development using UHPLC/triple
quadrupole mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
3
Fast analysis of polyaromatic hydrocarbons using the Agilent 1290 Infinity
LC and Eclipse PAH columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Fast analysis of fat soluble vitamins using the Agilent 1290 Infinity LC System and
ZORBAX RRHT and RRHD 1.8 µm columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87
High resolution of complex liquids (triglycerides) using the Agilent 1290
Infinity LC System and ZORBAX RRHT and RRHD 1.8 µm columns . . . . . . . . . . . . . . .89
Analysis of impurities in fine chemicals octyl-dimethyl-4-aminobenzoate
using the Agilent 1290 Infinity LC System and ZORBAX RRHT and
RRHD 1.8 µm columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
New dynamic MRM mode improves data quality and triple quad
quantification in complex analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93
Performance characteristics of the Agilent 1290 Infinity Binary Pump
More resolution and speed for conventional, superficially porous and sub2-micron column packing material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103
Performance characteristics of the Agilent 1290 Infinity Thermostatted
Column Compartment
New QuickChange valves, two heated zones up to 100 °C, improved usability . . . . .111
Performance characteristics of the Agilent 1290 Infinity Diode Array Detector
Low noise, low refractive index, high speed and data security . . . . . . . . . . . . . . . . . . .115
4
The Agilent 1290 Infinity LC System: Powerful – Sensitive – Flexible
With more than enough power to handle any UHPLC or HPLC challenge, the
all-new, advanced technology fluid
delivery system of the Agilent 1290
Infinity LC opens the door to virtually
limitless separation and throughput
possibilities.
To achieve unmatched detector sensitivity for both UHPLC and conventional
HPLC analyses, the Agilent Max-Light
cartridge cells with optofluidic waveguides in the Agilent 1290 Infinity Diode
Array Detector improve light transmission to near 100% efficiency without
sacrificing resolution caused by cell
dispersion effects. Compromising
refractive index and thermal effects are
almost completely eliminated, resulting
in significantly less baseline drift.
Based on an advanced, flow-through
design, the Agilent 1290 Infinity
Autosampler offers high precision
injection of both large and small volumes without the need to change sample loops.
With a temperature range from 10
degrees below ambient up to 100°C,
the Agilent 1290 Infinity Thermostatted
Column Compartment provides infinitely better flexibility to optimize a separation on the speed and selectivity. New
Agilent Quick-Change valves pave the
way for ultra high-throughput, multimethod and automated method development solutions.
Infinitely more powerful
The Agilent 1290 Infinity Binary Pump
gives you maximum chromatographic
performance, compatibility and flexibility. Active damping combines with lowest delay volumes—facilitated by propriety multi-layer technology in the
new Agilent Jet Weaver mixer—to provide ultrafast gradients and superior
LC/UV and LC/MS performance.
Infinitely more sensitive
Featuring an innovative optical
design—including Agilent Max-Light
cartridge flow cells with optofluidic
waveguides— the Agilent 1290 Infinity
Diode Array Detector delivers a new
level of UV sensitivity and baseline
robustness. For LC/MS, Agilent Jet
Stream Thermal Focusing technology
significantly increases MS and MS/MS
sensitivity by improving the spatial
focusing of electrospray droplets.
Infinitely more flexible
New Agilent Quick-Change valves in
the Agilent 1290 Infinity Thermostatted
Column Compartment enable resourcesaving ultra high-throughput, multimethod and automated method development solutions. A totally new module—the Agilent 1290 Infinity Flexible
Cube allows fixed-loop injection mode
for ultrafast cycle times, or automatic
backflushing of the needle seat for a
new benchmark in lowest carryover.
5
6
Ultrafast analysis of synthetic
antioxidants in vegetable oils using
the Agilent 1290 Infinity LC System
Application Note
Food
Authors
THBP
DG
AP
2
BHT
Belgium
4
Ionox-100
OG
B-8500 Kortrijk
6
BHA
Kennedypark 26
8
NDGA
Research Institute for Chromatography
TBHQ
Pat Sandra
mAU
10
PG
Gerd Vanhoenacker, Frank David,
0
0.2
0.4
0.6
0.8
1
1.2
1.4
min
Abstract
The addition of synthetic antioxidants in edible vegetable oils is regulated in Europe
and the US. The official method was translated into an ultrafast LC method using the
Agilent 1290 Infinity LC equipped with an Agilent ZORBAX Rapid Resolution High
Definition (RRHD) column. High throughput is obtained in 2 min with a backpressure
of 1120 bar, which is below the 1200 bar upper limit of the column. Optimization of the
mobile phase composition and the temperature are discussed. The figures of merit are
illustrated using standard solutions and spiked vegetable oil (sunflower, rapeseed, and
olive) extracts. Limits of detection are 1 mg/kg or less in the oil samples. Using a
simple methanol extraction, good recovery was obtained for all antioxidants in the oil
samples.
5990-4378EN
7
Introduction
O
Lipid oxidation causes rancidity and
odor problems and decreases the nutritional value of food products. Synthetic
ascorbyl palmitate and phenolic antioxidants are often added to foods to prevent oxidation of unsaturated fatty
acids in oils and fats. Combinations of
antioxidants are commonly used to
enhance the antioxidative effect. The
structures and abbreviations of the
investigated antioxidants are shown in
Figure 1.
O
O
C 3H 7
O
O
C 8H17
O
C 3H 7
O
C12H25
OH
HO
HO
OH
OH
HO
OH
OH
Propyl gallate
(PG)
HO
OH
OH
OH
Octyl gallate
(OG)
Dodecyl gallate
(DG)
2,4,5-trihydroxybutyrophenone
(THBP)
HO
OH
O
C(CH3) 3
O
C(CH3) 3
OH
HO
C(CH3) 3
OH
OH
tert-butyl-hydroquinone
(TBHQ)
Regulatory agencies in Europe1 and the
US2 have imposed maximum levels for
some antioxidants while the use of others has been forbidden. The determination of antioxidants in foods and food
components is therefore an important
analysis. The limits are given in Table 1.
Nordihydroguaiaretic acid
(NDGA)
2- and 3-tert-butyl-4-hydroxyanisole
(BHA)
OH
OH
(H3C) 3C
OH
OH
C(CH3) 3
(H3C) 3C
OH
O
C(CH3) 3
O
O
HOH
O
OH
C15H31
O
HO
3,5-di-tert-butyl-4hydroxytoluene (BHT)
2,6-di-tert-butyl-4-hydroxymethylphenol
(Ionox-100)
Ascorbyl palmitate
(AP)
Figure 1
Structures and codes of the investigated antioxidants.
Antioxidant
Europe1
US2
AP
Quantum satis
No restriction
PG
OG
DG
BHA
~ 200 mg/kg, individual or combined
~ 200 mg/kg, individual or combined
BHT
~ 100 mg/kg
TBHQ
Not allowed
THBP
Not allowed
Not allowed
NDGA
Not allowed
Not allowed
Ionox-100
Not allowed
Not allowed
Table 1
Limits for antioxidants in edible oils in Europe and US.
5990-4378EN
8
In the official method for the determination of the antioxidants in edible oils,
columns of 15 to 25 cm in length with
an internal diameter of 4.6 mm, and
packed with 5-µm octadecyl silica particles are used.3 The mobile phase is
composed of diluted acetic or phosphoric acid (eluent A) and methanol/ acetonitrile 50/50 volume to volume (eluent B). Analysis times are between 15 to
25 min.
There are two reasons for increasing
the speed of analysis for this application. First, instability of some of the targets (for example, AP) have been
reported and long residence times of
samples in an autosampler can already
lead to significant degradation of the
compounds. Perrin and Meyer could
enhance the stability of sample and
standard solutions by using citric and
isoascorbic acid.4 They were able to
stabilize AP at room temperature for
about 7 h. However, QC laboratories in
edible oil and fat processing industries
have a need for increased analysis
speed. The presence or absence, and
assay of antioxidants have to be carried
out prior to loading or unloading oils
and fats. A fast, accurate, and precise
result is desirable for economical and
practical reasons.
This Application Note describes the
analysis of 10 antioxidants in vegetable
oils using the Agilent 1290 Infinity LC.
The original method was translated into
a high throughput method by optimizing
the mobile phase composition and the
temperature. The figures of merit are
presented for vegetable oil and spiked
oil extracts.
Experimental
Instrumentation and method
An Agilent 1290 Infinity LC System with
the configuration in Table 2 was used:
Solutions and samples
Sample and standard solutions were
prepared according to Perrin and
Meyer.4 The solvent for the standards
and extraction is a solution of citric acid
(1 mg/mL) and isoascorbic acid
(1 mg/mL) in methanol. For the spiked
samples, a stock solution of the antioxidants in the solvent was added prior to
extraction. The extraction was carried
out by weighing 1 g of oil and adding
10 mL of the solvent. This mixture was
vortexed for 30 s, allowed to stand for
2 min, and vortexed once more for 30 s.
The sample was then centrifuged at
5000 x g for 5 min and the supernatant
was transferred into an autosampler
vial for injection.
Part number
Description
G4220A
Agilent 1290 Infinity Binary Pump with integrated vacuum degasser
G4226A
Agilent 1290 Infinity Autosampler
G1316C
Agilent 1290 Infinity Thermostatted Column Compartment
G4212A
Agilent 1290 Infinity Diode Array Detector
Method parameters:
Column
ZORBAX RRHD Eclipse Plus C18, 50 mm L × 2.1 mm id, 1.8 µm dp
Mobile phase
A = 0.02% phosphoric acid in water
B = Acetonitrile/methanol 50/50 or 75/25 v/v
Flow rate
Variable
Gradient
Variable
Temperature
Variable
Injection
2 µL
Detection
DAD, 40 or 80 Hz
Phenolic antioxidants Signal 280/10 nm, Reference 400/50 nm
Ascorbyl palmitate
Signal 255/10 nm, Reference 400/50 nm
Table 2
Conditions
5990-4378EN
9
Results and Discussion
C
75/25
75/25
Flow rate
0.4 mL/min
0.4 mL/min
1.9 mL/min
Gradient
0–7.5 min: 35–100% B
0–7.5 min: 30–100% B
0–1.6 min: 30–100% B
Temperature
30 °C
45 °C
45 °C
Detector speed
40 Hz
40 Hz
80 Hz
Maximum pressure
375 bar
270 bar
1120 bar
A.
mAU
THBP
120
100
80
DG
OG
AP
BHT
20
Ionox-100
TBHQ
40
BHA
PG
NDGA
60
0
1
2
3
4
5
6
7
min
7
min
B.
mAU
THBP
120
100
80
AP
DG
BHT
Ionox-100
NDGA
BHA
20
OG
60
40
TBHQ
An additional advantage of the
increased temperature is the decrease
of the backpressure. When the flow
rate was increased to 1.9 mL/min the
last peak eluted under 1.5 min and the
pressure on the column was 1120 bar
(Figure 2C).
B
50/50
PG
The analysis was first carried out with
the mobile phase used in the official
method. The flow rate was set at a
moderate 0.4 mL/min. The analysis
time was 8 min (see Figure 2A). The
synthetic phenolic antioxidants are all
detected at 280 nm while for ascorbyl
palmitate (AP) 255 nm was used. The
eluent B composition was then modified from methanol/acetonitrile 50/50
to 75/25 volume to volume to lower the
viscosity and enable a faster separation. The selectivity changed considerably with this mobile phase adaptation
and the temperature was optimized to
obtain sufficient separation between all
target antioxidants. Note that at 45 °C,
the elution order of dodecyl gallate (DG)
and BHT is reversed compared to Figure
2A at 30 °C. All compounds were stable
at 45 °C column temperature.
A
Methanol/Acetonitrile
ratio (v/v)
0
1
2
3
4
5
6
C.
THBP
mAU
80
AP
BHT
DG
Ionox-100
OG
20
BHA
NDGA
TBHQ
40
PG
60
0
0.2
0.4
0.6
0.8
1
Figure 2
Analysis of 10 µg/mL standard solution under the various conditions.
5990-4378EN
10
1.2
1.4
1.6 min
The performance of the ultrafast
method was evaluated and the results
are summarized in Table 3. The repeatability and linearity of the method were
investigated using standard solutions of
the antioxidants. The detection limit
was equal to or below 0.1 µg/mL for all
antioxidants. This corresponds to
approximately 1 mg/kg or lower in an oil
or fat sample. Extracts of vegetable oils
and spiked oils were analyzed to determine the recovery and accuracy. The oil
samples were spiked with 10 or
50 mg/kg of each antioxidant and the
detected amounts in the extracts were
compared to standard solutions at the
same concentration.
The chromatograms for the fast analysis of a standard solution and the
spiked oil samples are shown in
Figure 3. Additional peaks originating
from the oil matrix are visible in the
chromatograms but only a few interfere
with the analysis. Most interfering
peaks are present in the olive oil sample, however, the 10 mg/kg spiked oil
can still be differentiated from an
unspiked sample and the recovery is
satisfactory (Table 3).
Repeatability Linearity
(% RSD)(1)
(R²)(2)
Recovery 10 mg/kg
(%)
Recovery 50 mg/kg
(%)
Sunflower Rapeseed
Olive
Sunflower Rapeseed Olive
PG
0.27
0.99988
100.1
105.3
95.1
100.0
100.9
98.3
THBP
0.27
0.99983
97.3
99.1
105.9
98.6
99.1
99.4
TBHQ
0.99
0.99933
90.7
89.7
81.2
97.4
95.8
95.6
NDGA
0.16
0.99983
109.3
89.7
93.6
102.2
98.0
98.9
BHA
0.33
0.99983
104.8
107.0
102.0
98.5
96.4
94.4
Ionox-100
0.40
0.99974
90.7
93.8
89.7
97.5
97.5
93.3
OG
0.41
0.99985
99.3
101.0
95.9
99.7
100.3
98.8
DG
0.56
0.99985
97.8
100.1
101.9
98.4
98.9
98.7
BHT
0.54
0.99960
81.0
89.5
74.4
81.6
83.8
79.0
AP
0.67
0.99934
92.6
85.7
75.4
89.5
91.2
83.7
(1) 6 consecutive injections of 10 µg/mL standard solution
(2) 0.1, 0.2, 0.5, 1, 10 µg/mL standard solution, 1 injection/level
Table 3
Method performance data.
5990-4378EN
11
Conclusion
Standard solution
8
0.2
0.6
0.4
0.6
0.4
0.6
0.4
0.6
AP
BHT
DG
Ionox-100
OG
0.8
1
1.2
1
1.2
1
1.2
1
1.2
1.4
min
THBP
mAU
10
8
AP
BHT
DG
Ionox-100
OG
2
BHA
TBHQ
NDGA
PG
6
0
0.2
0.8
1.4
min
Rapeseed oil
THBP
mAU
10
8
AP
DG
BHT
BHA
NDGA
TBHQ
2
Ionox-100
OG
6
0
0.2
0.8
1.4
min
Olive oil
mAU
10
DG
AP
2
BHT
4
BHA
NDGA
6
Ionox-100
OG
8
TBHQ
4.
Perrin C., Meyer L., J. Am. Oil Chem.
Soc., 80 (2003) 115-118.
0.4
Sunflower oil
4
3.
Official Methods of Analysis of AOAC
International, 17th edition, AOAC
Official Method 983-15, W. Horwitz ed.,
AOAC International, Gaithersburg
(2000).
BHA
0
THBP
2.
Encyclopedia of Food Color and
Additives, Vols. I, II, and III, Burdock G.,
CRC Press, Boca Raton (1997).
NDGA
2
PG
1.
European Parliament and Council
Directive No. 95/2/EC (1995)
TBHQ
4
PG
6
4
References
THBP
mAU
10
PG
Using the Agilent 1290 Infinity LC, an
ultrafast analytical method could be
developed for the determination of
antioxidants in vegetable oils. The analysis time could be reduced to less than
2 min with a backpressure of 1120 bar.
The performance of the high throughput
method (repeatability, linearity, detection limits) was investigated using standard solutions. Oil samples and spiked
oil samples were extracted and the
recovery of the antioxidants was calculated. Satisfactory recovery was
obtained for all antioxidants. The developed method is useful in laboratories
where a fast result is mandatory.
0
0.2
0.8
1.4
min
Figure 3
Analysis of standard solution (1 µg/mL) and spiked oil (10 mg/kg) extracts with the fast method.
5990-4378EN
12
Tryptic digest analysis using the
Agilent 1290 Infinity LC System
Application Note
Drug Development, Production QA/QC
Authors
Gerd Vanhoenacker, Frank David,
mAU
Pat Sandra
Research Institute for Chromatography
mAU
Kennedypark 26
B-8500 Kortrijk
Belgium
500
400
300
200
100
0
3
3.5
4
4.5
5
5.5
6
6.5
7
min
3
3.5
4
4.5
5
5.5
6
6.5
7
min
3
3.5
4
4.5
5
5.5
6
6.5
7
min
200
150
100
50
0
mAU
120
Koen Sandra
80
40
0
Metablys
Kennedypark 26
B-8500 Kortrijk
Belgium
Bernd Glatz and Edgar Naegele
Agilent Technologies R&D and
Marketing GmbH and Co. KG
Hewlett-Packard-Str. 8
76337 Waldbronn
Germany
Abstract
This Application Note demonstrates:
• The applicability of the Agilent 1290 Infinity LC System to resolve peptide mix
tures of higher complexity.
• A bovine serum albumin (BSA) tryptic digest was separated on a 250 mm ×
2.1 mm id × 1.7 µm dp RP-LC column using different gradient slopes and flow
rates.
• The maximum pressure applied was 900 bar. Peak capacities from 188 to 851
within total analysis times of 8 and 260 min, respectively, were obtained.
5990-4031EN
13
Introduction
Note that an in silico digest of BSA
generates approximately 150 peptides
with one miscleavage allowed, and
aspecific cleavages not taken into
account.
Peptide separations are of great importance in a variety of fields ranging from
the characterization of recombinant,
therapeutic proteins to proteomicsbased biomarker discovery and verification. Sample complexity is enormous
with typically hundreds of species
encountered in biopharmaceutical
preparations and many thousands of
peptides in proteomics samples.
Evidently, the chromatographer is confronted with an enormous separation
challenge.
Experimental
Instrumentation and method
An Agilent 1290 Infinity LC system with
the configuration described below was
used.
In this application note, the resolving
power of ultra-high pressure LC (UHPLC)
using the 1290 Infinity LC system is
demonstrated. BSA tryptic digest was
separated on a 250 mm × 2.1 mm ×
1.7 µm dp column. Peak capacity and
peak capacity productivity, two powerful metrics to evaluate the separation,
were determined at different gradient
slopes and flow rates.
Tryptic digestion of BSA was carried
out in an ammonium bicarbonate buffer
at pH 8. Trypsin was added in an
enzyme/substrate ratio of 1/50 and the
mixture was incubated overnight at
37 °C. Another BSA sample (called BSA
RA) was reduced and alkylated prior to
digestion. Both samples were acidified
with mobile phase A to a concentration
of 3 nmol/mL prior to injection. A peptide standard mixture, used to aid in the
calculation of the peak capacity, was
dissolved in mobile phase A and contained bradykinin 1–5 (5 nmol/mL),
angiotensin II (3 nmol/mL), neurotensin
(2 nmol/mL), ACTH clip [18-39]
(2.5 nmol/mL), and bovine insulin chain
B (12.5 nmol/mL).
Results and Discussion
Part number
Description
G4220A
Agilent 1290 Infinity Binary Pump with integrated vacuum degasser
G4226A
Agilent 1290 Infinity Autosampler
G1316C
Agilent 1290 Infinity Thermostatted Column Compartment
G4212A
Agilent 1290 Infinity Diode Array Detector
Method parameters:
Column
C18 150 mm × 2.1 mm 1.7 µm
C18 100 mm × 2.1 mm 1.7 µm
Mobile phase
A = 0.10% TFA in water/acetonitrile 98/2 v/v
B = 0.08% TFA in acetonitrile
Flow rate
0.4 mL/min or 0.2 mL/min
Gradient
0 to 50% B
65% B
0% B
Temperature
60 °C
Injection
10 µL
Detection
DAD, Signal 214/4 nm, Reference 400/60 nm, 40 Hz
variable time
for 10 min
for 5 min
Samples
(gradient elution)
(column rinsing)
(column reconditioning)
5990-4031EN
14
A column length of 250 mm was
obtained by coupling two columns
(150 and 100 mm) using a stainless
steel capillary of 70 mm with an internal
diameter of 0.12 mm. Performing a relatively fast gradient analysis of 8%
B/min resulted in a fast analysis of the
digest (Figure 1). A peak capacity of
approximately 190 was generated with
this short gradient time (6.25 min). This
corresponded to a peak capacity production rate of over 30 peaks/min. Peak
capacity was calculated by dividing the
gradient time with the average peak
width at the base (4s ) determined for
five standard peptides (Figure 1). The
gradient applied in this note was longer
than actually required to elute the last
BSA fragments from the column. The
reason for this is that this gradient is
also applied for the analysis of other
digests with more retentive peptides.
When only the elution window (3 to
7.5 min) is taken into account for the
calculation a peak capacity of 136 is
obtained. The peak capacity productivity is not affected however; the number
of peaks generated per minute remains
the same.
When the flow rate and gradient slope
are reduced to 0.2 mL/min and 0.5%
B/min, respectively, the peak capacity
mAU
mAU
500
400
300
200
100
0
3
3.5
4
4.5
5
5.5
6
6.5
7
min
3
3.5
4
4.5
5
5.5
6
6.5
7
min
3
3.5
4
4.5
5
5.5
6
6.5
7
min
200
150
100
50
0
120
mAU
Applying longer, more shallow gradients increases peak capacity and
therefore the amount of detail visualized in the chromatogram. Evidently,
the price to pay is analysis time. Figure
2 shows the result for the BSA digest
analyzed with four different gradient
slopes. The peak capacity tripled from
188 to 567 when the gradient time was
increased from 6.25 min (8% B/min) to
50 min (1% B/min), respectively. If only
the elution window of the BSA digest
is taken into account for the 50-min
gradient, the peak capacity is 375 in
39 min. It is clear that the chromatogram at the more shallow gradient
reveals much more detail, while analysis time remains acceptable. Further
increasing the gradient time leads to a
higher peak capacity, but the effect of
the flatter gradient becomes less significant from a defined point and the
peak capacity productivity becomes
nearly fixed. This is summarized in
Figure 3. Doubling the gradient time
from 50 to 100 min increases the peak
capacity by approximately 25% (567 to
711). However, an additional increase
in the gradient time to 200 min, produces a gain in peak capacity of only
approximately 15% (711 to 820). In the
last situation, the analysis time is over
3 h and becomes less practical in routine operation. From Figure 3 it can be
deduced that the best compromise
between peak capacity and analysis
time is obtained with a gradient time of
100 to 150 min.
80
40
0
Figure 1
High speed analysis of the peptide standard mixture (upper trace), BSA digest (middle trace) and BSA RA digest
(lower trace). Flow rate: 0.4 mL/min, gradient: 0–50% B in 6.25 min.
mAU
200
175
150
125
100
75
50
25
DAD1 B, DAD1A, DAD: Signal A, 214 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI...90-20090403\BSA-0000003.D)
3.5
4
4.5
5
5.5
6
min
6.5
DAD1 B, DAD1A, DAD: Signal A, 214 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI... 3LIFESCI\BSA25CM000002.D)
mAU
120
100
80
60
40
20
0
5
mAU
6
7
8
9
10
12 min
11
DAD1 B, DAD1A, DAD: Signal A, 214 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI... 3LIFESCI\BSA25CM000004.D)
80
60
40
20
0
6
mAU
70
8
10
12
14
16
18
20
min
DAD1 B, DAD1A, DAD: Signal A, 214 nm/Bw:4 nm Ref 400 nm/Bw:60 nm (B:\RIC\AGI... 3LIFESCI\BSA25CM000005.D)
60
50
40
30
20
10
0
10
15
20
25
30
35
min
Figure 2
Analyses of the BSA digest with different gradients. Flow rate: 0.4 mL/min, gradient: 0-50%B in 6.25 min
(8%/min), in 12.5 min (4%/min), in 25 min (2%/min), and in 50 min (1%/min).
5990-4031EN
15
increases from 567 to 645 compared to
the analysis carried out at 0.4 mL/min
and 1% B/min. However, the peak
capacity production rate is reduced
from 11.3 to 6.4 with this approach.
When samples become more complex
on the other hand, a moderate increase
in resolution can become useful for
detecting minor differences between
related samples, especially when highend qualitative detectors such as a
mass spectrometer are applied.
Conclusion
This Application Note demonstrates the
versatility of Agilent 1290 Infinity LC
system for separating peptide mixtures
of high complexity. Protein digests were
analyzed on a 250 mm long column
packed with 1.7-µm particles and operated at a pressure up to 900 bar.
Depending on the need, high productivity (peak capacity of 188 in less than
10 min) or high resolution (peak capacity exceeding 800 in 3h) can be
obtained.
Production rate
(peaks/min)
Peak Capacity
Peak Capacity Productivity
Peak Capacity
900
700
500
300
100
0
50
100
150
200
Gradient time (min)
250
35
30
25
20
15
10
5
0
0
Peak capacity
(peaks/min)
50
100
150
200
Gradient time (min)
Gradient time
(min)
Gradient slope
(% B/min)
6.25
8
188
30.1
12.5
4
296
23.7
25
2
426
17.0
50
1
567
11.3
100
0.5
711
7.1
150
0.375
788
5.3
200
0.25
820
4.1
250
0.2
851
3.4
Peak capacity productivity
Figure 3
Peak capacity and peak capacity production rate in function of gradient time.
5990-4031EN
16
250
Developing a green LC method for
the determination of the
furocoumarins 5-MOP and 8-MOP in
citrus oils using the Agilent 1290
Infinity LC System
Application Note
Natural Products, Fragrances
Authors
mAU
175
Gerd Vanhoenacker, Frank David, and
150
Pat Sandra
100
125
25
5-MOP
8-MOP
50
Kennedypark 26
O
O
75
Research Institute for Chromatography
O
O
5-MOP (5-methoxypsoralen,
bergapten)
0
B-8500 Kortrijk
0
1
2
3
4
5
6
7 min
Belgium
Bernd Glatz and Edgar Naegele
Agilent Technologies R & D and
Marketing GmbH and Co. KG
Hewlett-Packard-Str. 8
76337 Waldbronn
Germany
Abstract
Lemon and orange oils were analyzed for the presence of the furocoumarins
5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) with the Agilent 1290
Infinity LC system. At 1000 bar, the total analysis time in reversed-phase (RP) liquid
chromatography (LC) with acetonitrile and water as mobile phase components could
be reduced to 4 min. In the frame work of green chromatography, and the present acetonitrile shortage, mobile phases constituted of water/methanol and water/ethanol
were compared to water/acetonitrile. The influence of the nature of organic modifiers
on selectivity and peak capacity was investigated. The figures of merit of the different
methods were compared for the determination of 5-MOP in a lemon oil sample containing approximately 50 mg/kg. 8-MOP was not detected at the ppm level. The
detection limit for both furocoumarins was approximately 1 mg/kg citrus oil.
5990-4033EN
17
Introduction
Furocoumarins are natural products
that may be present in plant extracts
and essential oils used in fragranced
cosmetic products. The furocoumarins
have been identified as photomutagenic
and photocarcinogenic products. The
International Agency for Research on
Cancer (IARC) has classified 5-MOP
(5-methoxypsoralen, bergapten) and
8-MOP (8-methoxypsoralen,
xanthotoxin) when combined with UV
radiation as group 2A (probably carcinogenic to humans) and as group 1 (carcinogenic to humans) risk carcinogens,
respectively.
On that basis, limits have been defined
for the presence of psoralens in cosmetics. The Commission Directive
95/34/DC of 1995 states that furocoumarins should be below
1 mg/kg (1 ppm) in sun protection and
in bronzing products.1 There is an ongoing debate to extend this 1 ppm limit to
all finished cosmetic products.2
Therefore, fast analysis of furocoumarins in cosmetics and in the
essential oils used in cosmetics is of
utmost importance.
The Agilent 1290 Infinity LC system in
combination with UV detection was
evaluated for the determination of
5-MOP and 8-MOP (Figure 1) in a lemon
and orange oil sample. Typical analysis
times for such samples using standard
LC instrumentation are 30 min.3 The
possibility of increasing the analysis
speed by employing the high pressure
capabilities of the Agilent 1290 Infinity
LC was investigated. Mobile phases for
these analyses are commonly composed of water and acetonitrile.
Different organic modifiers (acetonitrile,
methanol, and ethanol) were compared
and a green method with biodegradable
ethanol as the mobile phase constituent was developed.
O
O
O
O
O
O
O
O
5-MOP (5-methoxypsoralen,
bergapten)
8-MOP (8-methoxypsoralen,
xanthotoxin)
Figure 1
Structures of 5-MOP and 8-MOP.
The features of diode array detection
(DAD) and mass spectrometer (MS)
detection for trace analysis of furocoumarins using the Agilent 1290
Infinity LC will be described elsewhere.4
Experimental
Instrumentation and method
An Agilent 1290 Infinity LC system with
the following configuration was used:
Solutions and samples
Stock solutions of 500 µg/mL of 5-MOP
and 8-MOP standards were prepared in
ethanol. The solutions were further
diluted in ethanol prior to injection.
Samples of lemon and orange oil were
used. The oils were analyzed separately, as well as mixed, and they were
diluted 1/10, volume to volume, in
ethanol prior to injection.
Part number
Description
G4220A
Agilent 1290 Infinity Binary Pump with integrated vacuum degasser
G4226A
Agilent 1290 Infinity Autosampler
G1316C
Agilent 1290 Infinity Thermostatted Column Compartment
G4212A
Agilent 1290 Infinity Diode Array Detector
Method parameters:
Column
C18 150 mm L × 2.1 mm id, 1.7 µm dp
C18 100 mm L × 2.1 mm id, 1.7 µm dp
Mobile phase
A = Water
B = Acetonitrile, methanol or ethanol
Flow rate
Variable
Gradient
Variable
Temperature
80 °C
Injection
1 µL
Detection
DAD, Signal 315/4 nm, Reference 500/60 nm, 40 Hz
5990-4033EN
18
5-MOP
8-MOP
2
4
6
8
10
12
14
16
18 min
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090319\CITR-BEH150-102.D)
mAU
B
175
150
125
75
50
25
5-MOP
100
0
0
mAU
1
2
3
4
5
6
7
8
9 min
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090319\CITR-BEH150-301.D)
C
175
150
125
75
50
25
5-MOP
100
0
0
1
2
3
4
5
6
7 min
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090410\CITR-10-045-002.D)
mAU
400
350
300
250
200
150
100
50
0
D
0
5-MOP
A further increase in speed could be
obtained by reducing the column length
to 100 mm and increasing the flow rate
to 1.45 mL/min (Figure 2D). The result
is that the analysis time is approximately 4 min with a pressure of 1000 bar.
The drawback of using a shorter column for high speed analysis is that the
resolution decreases as well. However,
the separation is still sufficient to
detect 5-MOP in the sample.
0
8-MOP
The high pressure capabilities of the
Agilent 1290 Infinity LC system were
utilized to increase speed of analysis.
Starting from a RP-LC method with a
water/acetonitrile gradient, the flow
rate and gradient slope were increased
proportionally to each other while the
isocratic hold time at the beginning of
the analysis was reduced to maintain
the elution profile. The flow rate was
increased from 0.45 to 0.6, 0.9, and
1.2 mL/min resulting in pressure drops
up to 1090 bar on the 150 mm long column. The analysis time was more than
2.5 times faster and could be obtained
without sacrificing resolution.
Figures 2A, B, and C show some typical
profiles for the mixed oil sample. Only
5-MOP was detected in the sample.
Only use the analysis conditions in
Figure 2C if your column and hardware
is rated above 1000 bar.
A
8-MOP
Increase speed
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090319\CITR-BEH150-002.D)
mAU
160
140
120
100
80
60
40
20
0
8-MOP
Results and Discussion
0.5
1
1.5
2
2.5
3
3.5
min
Figure 2
Chromatograms for the analysis of the mixed oil sample with different flow rates and columns. Flow
rate/column length: 0.45 mL/min/150 mm (A), 0.9 mL/min/150 mm (B), 1.2 mL/min/150 mm (C), and
1.45 mL/min/100 mm (D). Mobile phase: water-acetonitrile.
Conditions Figure 2
A
B
C
D
Column length
150 mm
150 mm
150 mm
100 mm
Flow rate
0.45 mL/min
0.9 mL/min
1.2 mL/min
1.45 mL/min
Gradient
0–5 min:
30% B isocratic
5–25 min:
30–100% B
0–2.5 min:
30% B isocratic
2.5–12.5 min:
30–100% B
0–1.9 min:
30% B isocratic
1.9–9.4 min:
30–100% B
0–1 min:
30% B isocratic
1–5.2 min:
30–100% B
Maximum pressure
440 bar
840 bar
1090 bar
1000 bar
5990-4033EN
19
Green chromatography
Analyzing samples with only water and
ethanol as mobile phase components is
green chromatography. This approach is
interesting ecologically as well as economically due to the present acetonitrile shortage. The drawback of using
water/ethanol mobile phases is the
high backpressure that is generated.
For this particular analysis the pressure
reached 770 bar with ethanol while only
440 and 590 bar for acetonitrile and
methanol, respectively. The Agilent
1290 Infinity LC is rated to 1200 bar and
has no problem with these high backpressures.
The presence of 5-MOP could be elucidated in the 3 chromatograms. This
indicates that in the mixed sample,
5-MOP was originating from the lemon
oil sample and not from the orange oil
sample. Note that, identification and
quantification of all furocoumarins at
trace levels is best performed by using
mass spectrometry.4
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090324\CITR-ACN-000003.D)
mAU
A
40
5-MOP
30
20
10
0
2
4
6
8
10
12
14
16
min
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090325\CITR-MEOH-00003.D)
mAU
B
5-MOP
40
30
20
10
0
2
4
6
8
10
12
14
16
min
DAD1 B, DAD1A, DAD: Signal A, 315 nm/Bw:4 nm Ref 500 nm/Bw:60 nm (B:\RIC\AGI...0090325\CITR-ETOH-00003.D)
mAU
C
40
30
5-MOP
In a second application, the Agilent
1290 Infinity LC system was used to
develop a green LC method. The citrus
oil was analyzed with three different
organic modifiers. Solvent composition
and gradients had to be adapted for
each combination to obtain a similar
elution window. The results are shown
in Figure 3. The elution profile showed
significant differences with the sample
analyzed in section 1 (mixed oil sample). The reason is that the orange oil
contains mainly polymethoxylated flavanoids while the lemon oil is mostly
composed of psoralene derivatives.
20
10
0
2
4
6
8
10
12
14
16
min
Figure 3
Chromatograms for the analysis of a lemon oil sample with 3 different modifiers. Acetonitrile (A),
methanol (B), and ethanol (C). Flow rate: 0.45 mL/min.
Conditions Figure 3
A
B
C
Modifier
Acetonitrile
Methanol
Ethanol
Gradient
0-5 min:
30% B isocratic
5–25 min:
30-100% B
0–3.5 min:
40% B isocratic
3.5–23.5 min:
40-100% B
0–5 min:
23% B isocratic
5–25 min:
23–100% B
Maximum pressure
440 bar
590 bar
770 bar
5990-4033EN
20
Quantitative and performance
data
The different methods were applied to
perform the quantitative analysis of
5-MOP in the lemon oil sample and
their performances were compared
(Table 1). Calibration lines were created
by single consecutive injections of
standard solutions containing 0.1, 0.5,
1, 5, and 10 µg/mL of 5-MOP. The data
are summarized in Table 1.
In order to compare the resolving power
of the different methods the peak
capacity was calculated for each. This
was done by dividing the gradient time
with the average peak width at the
base (4s) determined for the 5 µg/mL
5-MOP solution. As expected, the
resolving power on the shorter column
decreased compared to the 150-mm
column. It is noteworthy that the peak
capacity with the water/acetonitrile
was not affected by the increased flow
rate while peak capacities for acetonitrile and ethanol were similar.
Mobile
phase
Column
length (mm)
Flow rate
(mL/min)
Maximum
pressure
(bar)
Peak
capacity
Linearity
(R²)
Assay 5-MOP
(µg/mL)*
Acetonitrile
Methanol
Ethanol
Acetonitrile
Acetonitrile
Acetonitrile
150
150
150
150
150
100
0.45
0.45
0.45
0.90
1.20
1.45
440
590
770
850
1090
1000
219
192
215
218
213
152
>0.9999
>0.9999
>0.9999
>0.9999
>0.9999
>0.9999
Average
5.39
5.60
5.30
5.53
5.44
5.52
5.46
RSD (%)
1.98
*Concentration in sample solution. The oil is diluted 1/10 v/v in ethanol prior to analysis.
Table 1
Comparison of the different methods. Assay was performed on the lemon oil sample.
The assay of 5-MOP was very similar
with all methods and the relative standard deviation (RSD) was 1.98%. In the
sample solution an average concentration of 5.46 µg/mL was detected. This
corresponds to 51 mg/kg 5-MOP in the
original lemon oil.
5990-4033EN
21
Conclusion
The Agilent 1290 Infinity LC system
was used to analyze furocoumarins in
citrus oil samples. The performance of
the original method with a water/acetonitrile mobile phase was compared to
green chromatography methods where
the acetonitrile was replaced with
methanol or with ethanol. Additionally,
the performance of a high speed
method was evaluated. This Application
Note demonstrates that the high pressure capabilities and the high detector
acquisition rate of the Agilent 1290
Infinity LC system are very useful tools
for increasing analysis speed. In addition, it shows that the Agilent 1290
Infinity LC system can be used for
methods that are less toxic and more
environmentally friendly than existing
methods.
References
1.
Eightieth Commission Directive
95/34/EC of 10 July 1995 adapting to
technical progress Annexes II, III, VI
and VII to Council Directive 76/768/EEC
on the approximation of the laws of the
Member States relating to cosmetic
products (1995)
2.
Sixth plenary of 13 December 2005,
Scientific Committee on Consumer
Products SCCP/0942/05, Opinion on
furocoumarins in cosmetic products
(2005)
3
Frérot E., Decorzant E., J. Agric. Food
Chem. 52 (2004) 6879-6886.
4.
G. Vanhoenacker, F. David, P. Sandra, in
preparation.
5990-4033EN
22
Increasing productivity in the
analysis of impurities in
metoclopramide hydrochloride
formulations using the Agilent 1290
Infinity LC System
Application Note
Pharmaceuticals
Authors
Abstract
Gerd Vanhoenacker, Frank David,
This Application Note evaluates the performance of the Agilent 1290 Infinity LC
Pat Sandra
System for the determination of impurities and related substances of metoclopramide
Research Institute for Chromatography
hydrochloride in a pharmaceutical formulation. The translation of a conventional liquid
Kennedypark 26
chromatography (LC) method on a 1200 Series HPLC system to an ultra high pressure
B-8500 Kortrijk
method on an Agilent 1290 Infinity LC System is discussed. Method translation is rela-
Belgium
tively easy and temperature fine-tuning is the most important parameter to obtain the
same selectivity for the different impurities. Pressures as high as 1070 bar were
Bernd Glatz and Edgar Naegele
Agilent Technologies
R&D and Marketing GmbH & Co. KG
Hewlett-Packard-Str. 8
76337 Waldbronn
Germany
applied during method development.
The final high productivity method is carried out at 880 bar in an analysis time of
3.5 min which is approximately 4 times faster than the original HPLC method but with
the same accuracy. A validation study was carried out to demonstrate the performance of the Agilent 1290 Infinity LC System. Limits of detection for the impurities
were as low as 0.001 % w/w relative to the main compound using the new diode
array detector (DAD). This is more than one order of magnitude lower than required.
5990-3981EN
23
Introduction
In pharmaceutical analysis present key
words are high throughput, high productivity and high resolution. In high
productivity, the goal is to develop analytical methods that are approximately
4–5 times faster than those presently
used with the prerequisite that accuracy, precision, and repeatability of developed and validated methods are kept
intact.
In liquid chromatography (LC), ways to
speed up analysis include the use of
particles less than 2 µm and/or operation at elevated temperature. Columns
packed with particles less than 2 µm
can be operated at much higher velocities compared to conventional columns
but dedicated LC instrumentation is
required.
The Agilent 1290 Infinity LC system
was applied for the determination of
impurities in a metoclopramide
hydrochloride formulation. The system
was equipped with a high pressure
pump capable of delivering up to 1200
bar and a new fast and sensitive diode
array detector (DAD). Method
development consisted of the translation of conditions from a conventional
instrument (Agilent 1200 Series LC system) equipped with a column packed
with 3.5-µm particles to an UHPLC system (Agilent 1290 Infinity LC system)
equipped with columns packed with
1.7-µm particles. The data from the two
systems were compared and evaluated
in terms of accuracy, precision, and
repeatability.
Experimental
Solutions
Stock solutions of the impurities and
related substances were prepared in
methanol. The structures of the compounds together with their EP-code are
listed in Table 1. The peak numbering is
used throughout the text. The stock
solutions were mixed and diluted at the
appropriate concentrations with water.
The formulation was a solution in water
for injection of metoclopramide
hydrochloride (5 mg/mL) together with
some other substances (confidential
composition).
Instrumentation
A standard Agilent 1200 Series HPLC
system and an Agilent 1290 Infinity LC
system with the following configurations were used:
Agilent 1200 Series LC System
Agilent 1290 Infinity LC system
G1322A
Vacuum degasser
G4220A
1290 Infinity Binary Pump with
integrated vacuum degasser
G1311A
Quaternary pump
G1313A
Automated liquid sampler
G4226A
1290 Infinity Autosampler
G1316A
Thermostatted column compartment
G1316C
1290 Infinity Thermostatted Column
Compartment
G1315B
Diode Array Detector
G4212A
1290 Infinity Diode Array Detector
5990-3981EN
24
Peak
Name (European Pharmacopoeia code, EP)
Main
Metoclopramide
Structure
CH 3
O
Cl
N
N
H
OCH 3
H 2N
X
Bromated metoclopramide
CH 3
O
Br
N
N
H
4-Amino-5-chloro-2-methoxybenzoic acid (EP C)
2
Cl
COOH
H 2N
OCH 3
4-(Acetylamino)-2-hydroxybenzoic acid (EP H)
COOH
O
OH
N
H
H 3C
3
4-Amino-5-chloro-N-2-(diethylaminoethyl)-2-methoxybenzamide N-oxide (EP G)
CH 3
O
O
Cl
N
H
4-Amino-5-chloro-N-2-(diethylaminoethyl)-2-hydroxybenzamide (EP F)
N
N
H
4-(Acetylamino)-5-chloro-N-2-(diethylaminoethyl)-2-methoxybenzamide (EP A)
CH 3
O
Cl
O
N
H
OCH 3
N
H
H 3C
Methyl 4-(acetylamino)- 2-methoxybenzoate (EP D)
O
OCH 3
O
OCH 3
N
H
H 3C
7
O
Methyl 4-(acetylamino)-2-hydroxybenzoate
OCH 3
O
OH
N
H
H 3C
8
Methyl 4-(acetylamino)-5-chloro-2-methoxybenzoate (EP B)
O
Cl
OCH 3
O
H 3C
9
OCH 3
N
H
O
Methyl 4-amino-2-methoxybenzoate
OCH 3
H 2N
Table 1.
Compounds under investigation.
5990-3981EN
25
CH 3
OH
H2N
6
CH 3
CH 3
O
Cl
5
N
OCH 3
H 2N
4
CH 3
OCH 3
H 2N
1
CH 3
OH
N
CH 3
The original method was developed on
a 1200 Series HPLC with a linear gradient using a quaternary pump. A chromatogram for a spiked formulation at
0.5% w/w level is shown in Figure 1A.
The column used was an XBridge C-18
column packed with 3.5-µm particles.
The initial pressure was 140 bar. The
method was easily transferred to the
1290 Infinity LC as long as some instrumental differences were taken into
account. When the method parameters
were copied to the 1290 Infinity LC system retention times were significantly
shorter. Selectivity changes were noted
and attributed to the difference in delay
volume for the two systems. A quaternary pump has a delay volume of 950
µL, while the 1290 Infinity Binary Pump
has a reduced volume of 10 µL. An initial isocratic hold time was introduced
into the 1290 Infinity LC method to
compensate for this difference. After
this straightforward correction, the
retention times and selectivity were
very similar for both systems, while the
efficiency for the 1290 Infinity LC was
higher than that of the 1200 Series
(Chromatogram not shown).
lution should increase by a factor of 1.4
on the same system since resolution is
related to the square root of the efficiency. In this particular case, the resolution enhancement is much higher
than theoretically predicted (for example, from 2.6 to 5.9 for peaks 1 and 2).
This is due to the lower dead volume
and the superior pump drives of the
Agilent 1290 Infinity LC system.
A) 1200 Series LC
mAU
Main
Method translation from a 1200
Series LC to a 1290 LC Infinity
System
Efficiency and resolution were improved
significantly with the 1290 Infinity
setup. The theoretical efficiency can
roughly be calculated as the ratio
between column length and two times
the particle diameter. Therefore, the
efficiency of the 1.7-µm particle column
should be about double the efficiency of
the 3.5-µm column. Consequently, reso-
80
3
70
7
60
8
6
50
40
4 5
2
30
9
1
20
10
X
0
0
2
4
6
8
10
12
min
12
min
B) 1290 Infinity LC
mAU
3
Main
Results and Discussion
200
7
6
150
2
100
8
4 5
9
1
50
X
0
The method was then translated to a
2.1 mm id BEH C18 column packed with
1.7-µm particles. The flow rate was
reduced to 0.22 mL/min to maintain the
same linear velocity in both columns
while the injection volume was
decreased from 2 µL to 0.8 µL. The
pressure was 380 bar. An initial 0.5 min
hold time on the gradient was introduced to compensate for differences in
delay volume. Under these conditions
the result was very similar (Figure 1B)
but slightly faster compared to the
result obtained by the column with
3.5-µm particles (Figure 1A).
5990-3981EN
0
2
4
6
8
10
Figure 1
Method transfer from an Agilent 1200 Series LC system to an Agilent 1290 Infinity system. Sample:
formulation spiked at 0.5% w/w level with impurities 1 to 9.
Conditions Figure 1 A
B
Column
XBridge C-18,
150 mm × 3.0 mm, 3.5 µm
BEH C18, 150 mm × 2.1 mm, 1.7 µm
Mobile phase
A = 0.25% w/w ammonium acetate in water
B = acetonitrile
Flow rate
0.45 mL/min
0.22 mL/min
Gradient
0-15 min: 5–57.5% B
0-0.5 min: 5% B isocratic
0.5–15.5 min: 5–57.5% B
Temperature
37 °C
37 °C
Injection volume
2 µL
0.8 µL
Detection
DAD, Signal 275/4 nm, Reference 400/60 nm
Maximum pressure 140 bar
26
380 bar
The operating pressure increased from
380 bar to 1020 bar when the flow rate
was changed from 0.22 to 0.66 mL/min.
As expected, at very high pressure, or
high mobile phase velocity in the column, frictional heat is generated [1,2].
Since retention of some of the analytes
is temperature dependent, the selectivity differences at high flow rates were
noted. It can be seen that the resolution
between compound 5 and the main
compound, and of compounds 7 and 8
were especially affected by this temperature change. (Figure 2A).
The heat effect could be counteracted
by reducing the temperature of the column from 37 to 32 °C (Figure 2B). Under
these conditions good separation was
achieved in 4.5–5 min which is about 3
times lower than the original LC
method. However, the pressure
increased to 1070 bar while the
columns are only rated at 1000 bar.
Using the column at higher pressures
than the maximum rated pressure for a
longer time will definitely reduce the
column’s lifetime and the robustness of
the method.
mAU
200
Main
The analysis on the 1.7-µm particle column was performed at 380 bar which is
far below the 1200 bar upper pressure
limit of the 1290 Infinity LC system
pump. Therefore, the flow rate could be
increased to shorten the analysis time.
When doing this, the gradient time
should be reduced in proportion to the
flow rate increase in order to maintain
the same elution profile.
A) 37 °C
3
175
7
150
6
125
100
8
2
4 5
75
9
1
50
25
X
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 min
B) 32 °C
mAU
200
Main
Increasing speed with the
Agilent 1290 Infinity LC system
3
175
7
8
150
6
125
100
4 5
2
75
50
9
1
25
X
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5 min
Figure 2
Influence of frictional heat generation on selectivity and effect of lowering column temperature. Sample:
formulation spiked at 0.5% w/w level with impurities 1 to 9.
Conditions Figure 2 A
Column
BEH C18, 150 mm × 2.1 mm, 1.7 µm
Mobile phase
A = 0.25% w/w ammonium acetate in water
B = acetonitrile
Flow rate
0.66 mL/min
Gradient
0-16 min: 5% B isocratic
0.16-5.16 min: 5–57.5% B
Temperature
37 °C
Injection volume
0.8 µL
Detection
DAD, Signal 275/4 nm, Reference 400/60 nm
Maximum pressure 1020 bar
5990-3981EN
27
B
32 °C
1070 bar
mAU
Main
Therefore, for routine application, the
column length was decreased from
150 mm to 100 mm to provide a maximum backpressure of 880 bar, at
0.66 ml/min. The gradient hold time
and gradient time were reduced further
to preserve the selectivity of the original method. The total analysis time was
3.5 min compared to 15.5 min with the
original 3.5-µm particle column
(Figure 3). The resolution for the initial
peaks is 3.8 which still is higher than
the value obtained with the 1200 Series
LC.
3
120
100
7
6
80
8
60
9
4 5
40
2
1
20
X
0
0
0.5
1
1.5
2
2.5
3 min
Figure 3
Result with the final Agilent 1290 Infinity LC system method. Sample: formulation spiked at 0.5% w/w
level with impurities 1 to 9.
Conditions Figure 3 Final Agilent 1290 Infinity LC system method
Column
BEH C18, 100 mm × 2.1 mm, 1.7 µm
Mobile phase
A = 0.25% w/w ammonium acetate in water
B = acetonitrile
Flow rate
0.66 mL/min
Gradient
0–0.1 min: 5% B isocratic
0.1–3.45 min: 5–57.5% B
Temperature
32 °C
Injection volume
1 µL
Detection
DAD, Signal 275/4 nm, Reference 400/60 nm
Maximum pressure 880 bar
5990-3981EN
28
Method validation
mAU
4
3
2
1
0
X
4
0.5
0.75
1
1.25
1.5
mAU
4
3
2
1
0
0.5
1.75
2
2.25
2.5
6
3
0.5
mAU
4
3
2
1
0
Main
Using the conditions of Figure 3, an
analysis was performed on the parameters of linearity, repeatability of injection, and detection limit (Table 2). It was
determined that good linearity (>0.999
for all compounds) and injection precision were obtained. RSDs at the 0.005%
level, which is close to the limit of
detection (LOD) for some compounds
was below 8% for all and below 3.5%
for most compounds. This is more than
acceptable at this level. The chromatograms for analysis at the LOD are
shown in Figure 4. The LOD varies
between 0.001 and 0.005% w/w relative to the main compound (50–250 pg
on-column). This means that the impurities are detected at levels which are
10 to 50 times lower than the reporting
threshold.
0.75
1
2.75
min
78 9
5
1
1.25
1.5
1.75
2
2.25
2.5
2.75
min
1
1.25
1.5
1.75
2
2.25
2.5
2.75
min
2
0.75
Figure 4
Analysis of formulation (top chromatogram) and spiked formulations with impurities 1 to 9 at LOD level
(middle chromatogram: 0.001% w/w, bottom chromatogram: 0.005% w/w). Conditions: see Figure 3.
Compound Linearity, R²(1)
Repeatability of injection, % RSD(2)
LOD
Area,
0.005%
Area,
0.05%
%
w/w
On-column S/N(3)
(pg)
1
1.0000
4.50
2.65
0.005
250
7.3
2
0.9999
7.54
1.72
0.005
250
8.6
3
1.0000
1.35
0.95
0.001
50
7.9
NA(4)
NA(4)
4
0.9994
1.77
1.74
NA(4)
5
1.0000
2.35
2.24
0.001
50
3.6
6
0.9997
3.32
0.93
0.001
50
4.3
7
1.0000
2.42
0.49
0.001
50
5.3
8
1.0000
1.92
0.81
0.001
50
4.5
9
1.0000
3.61
1.42
0.001
50
3.0
(1)
0.01, 0.02, 0.05, 0.1, 0.2, 0.5%, 1 injection/level
(2) 6 consecutive injections/level
(3) Signal-to-noise ratio, noise was taken from approximately 1–1.25 minutes
(4) No data available, impurity already present in formulation
5990-3981EN
29
Conclusion
References
This application note demonstrates the
feasibility of translating existing HPLC
methods to fast Agilent 1290 Infinity LC
system methods.
1. de Villiers A., Lauer H., Szucs R.,
Goodall S., Sandra P., J. Chromatogr.
A, 1113 84–91. 2006
Initially, the HPLC analysis developed
on a 1200 Series LC was simply transferred to a 1290 Infinity LC System
instrument. The method transfer was
relatively straightforward if some
instrumental characteristics were taken
into account. The original column
(150 mm × 3.0 mm, 3.5-µm particle size)
was then changed to a narrow bore
2.1-mm column with smaller 1.7-µm
particles. This significantly increased
the resolution between the compounds.
2. Gritti F., Guiochon G., J. Chromatogr.
A, 1187 165–179. 2008
The use of particles less than 2 µm
allowed an increase of velocity in the
mobile phase to reduce the analysis
time without hampering resolution.
Frictional heat generation at high pressure and mobile phase velocity changed
the selectivity of the method. The column temperature setting was lowered
in order to maintain the original selectivity.
The final Agilent 1290 Infinity LC system analysis was carried out on a
100-mm column at 880 bar and was
four times faster than the original HPLC
method. This Agilent 1290 Infinity LC
system method was successfully validated. Limit of detection varied
between 0.001 and 0.005% w/w relative to the main compound corresponding to 50–250 pg on-column, which is
10 to 50 times lower than the required
reporting level.
5990-3981EN
30
Software-assisted, high-throughput
identification of main metabolites of
pharmaceutical drugs
Rapid data acquisition by Agilent 1290 Infinity LC, TOF and Q-TOF instrumentation, and subsequent identification of metabolites by Agilent
MassHunter Metabolite Identification software
Application Note
Metabolite identification in drug discovery and drug development
Author
Edgar Naegele
Agilent Technologies
Waldbronn, Germany
Abstract
This Application Note describes:
• Rapid separation of metabolites generated from in-vitro experiments using the
Agilent 1290 Infinity LC, system
• Fast acquisition of TOF mass spectra using Agilent 6530 Accurate-Mass Quadrupole
Time-of-Flight LC/MS systems
• Fast, software-assisted identification of main metabolites from in-vitro
experiments using Agilent MassHunter Metabolite Identification software
• Generation of reports for the identified metabolites using Agilent MassHunter
software
5989-9924EN
31
Introduction
In modern pharmaceutical drug development it is of crucial importance to
analyze the adsorption, distribution,
metabolism and excretion (ADME)
properties of possible new drug candidates as quickly as possible in order to
make decisions about further investments in the development of a special
compound. To find compounds with the
correct properties it is essential to
screen a large number of compounds
for their ADME properties, which
requires to work in an high-throughput
environment. This Application Note
describes the application of the Agilent
1290 Infinity LC system, the Agilent
6530 Q-TOF MS system and the
MassHunter Metabolite Identification
software for fast, high-throughput identification of main metabolites of new
pharmaceutical drug candidate compounds.
Experimental
Equipment
• Agilent 1290 Infinity LC system consisting of 1290 Infinity Binary Pump
with integrated degasser, 1290 High
Performance Autosampler with
thermostat, and 1290 Infinity
Thermostatted Column compartment
• Agilent 6530 Accurate-Mass Q-TOF
LC/MS system
• Agilent MassHunter Metabolite
Identification (MetID) software
• Column: ZORBAX SB-C18,
2.1 × 50 mm, 1.8 µm
Sample preparation
TOF MS method
The following stock solutions were
used:
Source:
Capillary:
Dry gas:
Nebulizer:
Gas temp.:
Skimmer:
Fragmentor:
Mass range:
Acquisition
rate:
Reference
masses:
• 20 mg/mL microsomal S9 preparation
• 0.1 mg/mL buspirone in water
• 1.6 mg NADP in 1.6 mL 0.1 M phosphate buffer, pH 7.4
• 50 mM isocitrate/MgCl2 (203 mg
MgCl2.6H2O + 258.1 mg isocitrate in
20 mL H2O)
• Isocitrate dehydrogenase 0.33 unit/µL
NADPH regeneration system: 1.6 mL
NADP solution + 1.6 mL Isocitrate solution + 100 µL IDH solution.
Incubation mixture: 3.85 µL substrate +
200 µL NADPH regeneration system +
746.15 µL phosphate buffer + 50 µL S9.
Incubation was carried out at 37 °C for
60 minutes. A 100 µL aliquot was taken
at the beginning (t=0) and at t=60 min.
The reaction was stopped by adding
6 µL perchloric acid and 100 µL acetonitrile followed by centrifugation for 15
min at 14,000 rpm. The supernatant
was evaporated to dryness using a
SpeedVac concentrator and reconstituted with water containing 0.1 % formic
acid for LC/MS analysis. The incubation sample stopped at 0 min was used
as control.
LC method
Solvent A:
Solvent B:
Flow:
Gradient
Stop time:
Post time:
Injection:
Column:
Water + 0.1 % formic acid
ACN + 0.1 % formic acid
0.8 mL/min
0 min, 5 %B; 0.10 min,
5 %B; 1.10 min, 75 %B;
1.1.0 min
1 min.
Volume 5 µL, sample
cooler at 4 °C, needle wash
in 50 % methanol for 5 s,
injection loop to bypass
at 0.1 min with flush out
factor 16
Temperature 60 °C
5989-9924EN
32
ESI positive
3500 V
12 L/min
55 psi
350 °C
65 V
200 V
100-1000 m/z
5 spectra/s
121.0508 and 922.0080
Data analysis method in the
MetID software
The first step in the analysis comprised
a comparison between the data file that
contained the metabolite compounds
(metabolite sample) and the data file
that contained only the parent drug
(control sample). All detectable mass
signals were extracted from the MS
level data using the Molecular Feature
Extraction (MFE) algorithm. Related
compound isotope masses and adduct
masses were grouped together into discrete molecular features, and chemical
noise was removed. The compounds
lists of the metabolized sample and the
control were then compared.
All new compounds or those that
increased twofold in the metabolized
sample were considered potential
metabolites and were subjected to further analysis by different algorithms.
The algorithms can identify and qualify
new metabolites, or just qualify
metabolites found by another algorithm.
In this high-throughput experiment all
algorithms’ results were weighted
equally and combined into a final identification relevance score. Metabolites
were qualified when their final score
was above the stringently defined relevance threshold. The results from all
algorithms were collated in a results
table, which could be inspected at-aglance and reported1.
Results and discussion
To achieve fast separation of the
metabolites on a 50 mm, 1.8 µm particle
size column, a 1 minute gradient was
applied by the Agilent 1290 Infinity LC
system. The metabolites were generated from the pharmaceutical test compound buspirone in an in-vitro assay.
For adequate detection with the timeof-flight mass spectrometer the instrument was operated at a data rate of
5 Hz.
After generation the data was loaded
into the MetID software and analyzed
using a common method. The result
was displayed by the MetID software in
an at-a-glance table, in which the result
for each metabolite could be examined
in more detail (figure 1). From the
results table a summary report was
generated, which showed the available
information for each metabolite (figure
2). The more extensive report contained
the detailed results for each metabolite.
As example the result for a monohydroxyl meta-bolite (figures 3 to 5) and
a dihydroxy metabolite (figures 6 to 8)
of buspirone are discussed here.
Figure 1
Result table showing an at-a-glance summary of buspirone metabolite analysis with overall identified
metabolites, extracted ion chromatograms (EIC), extracted compound chromatograms (ECC), isotopic
pattern analysis and calculated formulas.
Name
Mass
RT
Rel.
Qual. User
SC
IPM
EIC
MDF
Form. BioXF
2x Hydroxylation 417.2379 0.59
100.00
✓
✓
✓
✓
✓
✓
✓
✓
Hydroxylation
401.2423 0.63
100.00
✓
✓
✓
✓
✓
✓
✓
✓
Hydroxylation
401.2424 0.66
100.00
✓
✓
✓
✓
✓
✓
✓
✓
2x Hydroxylation 417.2388 0.72
100.00
✓
✓
✓
✓
✓
✓
✓
✓
Hydroxylation
401.2439 0.75
100.00
✓
✓
✓
✓
✓
✓
✓
✓
Hydroxylation
401.2430 0.79
100.00
✓
✓
✓
✓
✓
✓
✓
✓
Buspirone
385.2478 0.82
_
_
_
✓
✓
✓
✓
_
Hydroxylation
401.2429 0.84
75.00
✓
✕
✓
✓
✓
✓
✓
✕
Figure 2
Summary result report, including qualified metabolites sorted by their retention times (RT), with their
metabolite names and relative score, molecular mass and the passed flag for individual algorithm
results. SC=Sample-control comparison, IPM = Isotopic Pattern Matching, EIC = Extracted Ion
Chromatogram, MDF = Mass Defect Filter, Form. = Calculated Formula, BioXF = Assigned
Biotransformation, Qual. = Qualified by Score, User = Qualified by User.
5989-9924EN
33
The extensive report for the monohydroxyl metabolite, which eluted after
0.75 minutes at m/z 402.2511, showed
the detailed information about the
metabolite itself such as measured
accurate mass, calculated formula,
assigned biotransformation and ion
species. Further, the report showed
more detailed information about the
result of each individual algorithm, for
example, Molecular Feature Extraction
(MFE), Extracted Ion Chromatogram
(EIC) compound search and Mass
Defect Filter Result (figure 3). For the
hydroxyl metabolite the possible formula was calculated based not only on a
defined mass error window but also on
the measured isotopic pattern, which
increased the quality of the calculated
formula and limited the possible number of hits significantly. These results
were also displayed in the detailed
metabolite result report for the formula
(figure 4).
Figure 3
Detailed metabolite report for the buspirone hydroxy metabolite at retention time 0.75 min. This part of
the report gives detailed information about the identified metabolite and the identifying algorithms.
Other detailed information about formula (Figure 4), chromatograms and isotopic pattern (Figure 5) are
also available.
Figure 4
Detailed metabolite report about the formula including isotopic pattern, calculated for the buspirone
hydroxy metabolite at retention time 0.75 min.
5989-9924EN
34
0.755
x10 5 EIC (402.2500)
1.6 A
1.2
0.8
C
1.6
0.630
0.4
0
0.787
0.841
0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05
Acquisition Time [min.]
0.754
x10 5 ECC
1.8
1.4
1
0.6
0.2
CIP (C21H32N5O3)
Compound Spectrum (0.739-0.774)
402.2511
x10 5
(M+H)+
B
1.4
1.2
1
0.8
403.2541
(M+H)+
0.6
0.4
404.2567
(M+H)+
0.2
0.05
0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05
Acquisition Time [min.]
0
402.2
402.6
403
403.4 403.8 404.2
Mass-to-Charge [m/z]
404.6
405.2593
(M+H)+
405
405.4
Figure 5
Detailed metabolite report for buspirone hydroxy metabolite at retention time 0.75 min:
A) Extracted Ion Chromatograms (EIC) of compounds with mass 402.25
B) Extracted Compound Chromatogram (ECC) of buspirone hydroxy metabolite at retention time 0.75 min
C) Measured isotopic pattern of buspirone hydroxy metabolite at retention time 0.75 min (blue lines) and caclulated isotopic pattern (CIP, green box).
Finally, the EIC, ECC and isotopic pattern were displayed (figure 5). The EIC
of m/z 402.25 showed 5 peaks for possible hydroxyl metabolites of buspirone
with the selected one at retention time
0.75 minutes (figure 5A). The ECC
showed the extracted MFE compound
for the molecular mass of 401.2439 at
retention time 0.75 minutes identical to
the EIC (figure 5B). The measured isotopic pattern of this compound showed
an excellent fit to the calculated isotopic pattern as a basis for the formula
calculation (figure 5C).
Within the same data analysis the dihydroxy metabolites at a level of two
orders of magnitude below the monohydroxy metabolites were also identified. The extensive report showed
detailed information about the dihydroxy metabolite, which elutes after
0.71 minutes at m/z 418.2461 and
the detailed information about each
algorithm (figure 6).
Figure 6. Detailed metabolite report for dihydroxy metabolite of buspirone at retention time 0.71 min.
This part of the report gives detailed information about the identified metabolite and the identifying
algorithms. Other detailed information about formula (see Figure 7), chromatograms and isotopic
pattern (see Figure 8) are also available.
5989-9924EN
35
The calculation of the formula was
outlined in the detailed formula report
(figure 7).
The EIC of m/z 418.24 showed about
five significant peaks for possible dihydroxylated metabolites of buspirone
with the selected peak at 0.71 minutes
(figure 7A). The ECC showed the
extracted MFE compound for the
molecular mass of 417.2388 at retention
time 0.71 identical to the EIC (figure
7B). The measured and calculated isotopic pattern of this compound is
shown in figure 7C.
Figure 7
Detailed metabolite report about the formula, including isotopic pattern, calculated for dihydroxy
metabolite of buspirone at retention time 0.71 min.
x10 3
3
0.713
EIC (418.2449)
x10 3
3.6
A
2
0.591
0.774
1
CIP (C21H32N5O4)
Compound Spectrum (0.700-0.726)
418.2461
(M +H )+
3.2
2.8
0
0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 2.4
Acquisition Time [min.]
2
x10 3 E CC
0.716
1.6
3.5
C
B
419.2490
(M +H )+
1.2
2.5
0.8
1.5
0.4
0.5
420.2516
(M +H )+
421.2541
(M +H )+
0
0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05
Acquisition Time [min.]
418.2 418.6
419
Figure 8
Detailed metabolite report for dihydroxy metabolite buspirone at retention time 0.71 min:
A) Extracted Ion Chromatograms (EIC) of compounds with mass 418.24
B) Extracted Compound Chromatogram (ECC) of dihydroxy metabolite of buspirone at retention time 0.71 min
C) Measured and calculated isotopic pattern of dihydroxy buspirone metabolite at retention time 0.71 min.
5989-9924EN
36
419.4 419.8 420.2 420.6
M ass-to-Charge [m/z]
421
421.4
Conclusion
This Application Note demonstrated the
use of the Agilent 1290 Infinity LC system with an Agilent Q-TOF LC/MS system for fast separation and accurate
mass measurement of compounds in an
in-vitro metabolite sample under highthroughput conditions. The meta-bolite
compounds were separated in
a run time below one minute and the
width of the peaks extracted by the
Metabolite ID software were below one
second (FWHH). The major metabolites were identified quickly by means
of the Agilent Metabolite Identification
software. A summary report as well as
detailed reports for each metabolite
were generated.
References
1. E. Naegele, F. Wolf, U. Nassal, R.
Jäger, H. Lehmann, F. Kuhlmann, K.
Subramanian, “An interwoven, multialgorithm approach for computerassisted identification of drug metabolites”,
Agilent Technologies Application Note,
publication number 5989-7375EN, 2007.
5989-9924EN
37
5989-9924EN
38
Environmental applications of the
Agilent 1290 Infinity UHPLC System:
The evolution of chromatography
Application Note
Environmental
Authors
Abstract
E. Michael Thurman and Imma Ferrer
This application note presents examples of the use of UHPLC (ultrahigh performance
Center for Environmental Mass
liquid chromatography) for environmental applications using the new Agilent 1290
Spectrometry
Infinity LC. The examples include wastewater analysis of pharmaceuticals with focus
Department of Environmental
on EPA Method 1694 and the way that UHPLC makes this analysis more efficient and
Engineering
reliable. The second example will show that complex mixtures of pesticides may be
University of Colorado
analyzed with fast chromatography, opening the door for many types of UHPLC analy-
Boulder, CO, USA
ses for environmental applications. Finally, an example will show a 60-sec analysis of
pharmaceuticals in wastewater using UHPLC and a ballistic gradient, which is one of
the first examples in environmental analysis. The insights of efficient UHPLC analysis
are discussed and illustrated.
5990-4409EN
39
Introduction
(C-8, C-18 in both StableBond and ZORBAX Eclipse Plus formats). These are useful for difficult water samples, as this
application note will show, including improved peak capacity
for an EPA Method 1694 for pharmaceuticals in wastewater.
They are also useful for rapid resolution of pharmaceuticals
and pesticides using both triple quadrupole mass spectrometry as well as liquid chromatography/time-of-flight mass
spectrometry.
The advent of UHPLC has brought two important innovations
to liquid chromatography/mass spectrometry (LC/MS) analysis. First is the use of 1.8-µm columns, which gives an
increase in plate number from the 5-µm columns of about a
factor of two. This is important for environmental applications
where complex matrices are encountered, such as pharmaceuticals in wastewater analysis. However, the use of 1.8-µm
columns requires higher pressure pumps operating at pressures that exceed 600 bar on a routine basis, which is the
range commonly used for UHPLC. Secondly, the 1.8-µm column allows the use of rapid resolution chromatography and
even ultrafast chromatography, where runs may be less than
1 min in length. The high flow rates required for these analyses create pressures on the order of 800 to 1000 bar, or more,
and are clearly in the range of UHPLC.
Experimental
The work shown here was carried out at the Center for
Environmental Mass Spectrometry by Drs. Imma Ferrer and
Michael Thurman at the University of Colorado in Boulder,
Colorado, USA, using the Agilent 1290 Infinity LC system and
both the Agilent 6430 triple quadrupole LC/MS and the
Agilent 6220 accurate-mass time-of-flight LC/MS system.
The demands of high sample throughput in short timeframes
have given rise to high efficiency and fast liquid chromatography using the 1.8-µm reverse-phase columns. Fast chromatography has become a necessity in those labs that analyze hundreds of samples per day or those labs needing short
turnaround times. Using Rapid Resolution liquid chromatography, results of a sample batch can be reported in a few hours
rather than a few days. In the water quality and the food
industries, regulatory labs produce validated results in less
than an hour so that water treatment may proceed or vegetable shipments can be released the same day they are measured or produced. The end result is greater productivity for
customers and greater cost efficiency for the reporting laboratory. Thus, productivity is improved by shortened analysis
time, which typically requires UHPLC. The definition of Rapid
Resolution liquid chromatography is simple. Liquid chromatographic separations that are less than 10 min are fast, and
separations less than 1 min are popularly known as ultrafast
[1].
Columns
Two different columns were tested for rapid resolution high
throughput (RRHT) analyses including the high pressure (1000
bar) 1.8-µm particle sizes. Table 1 lists the columns tested in
this work and their theoretical plates.
Column
Dimension
(mm)
Particle Theoretical Pressure
(m)
Plates (N) Rated (bar)
ZORBAX Eclipse Plus-C18
2.1 × 100
1.8
21,688
1000
ZORBAX Eclipse Plus-C18
2.1 × 50
1.8
10,392
1000
Table 1
Columns used in this study
Chromatographic and mass spectral conditions
The Agilent 1290 Infinity LC was used for all UHPLC chromatographic separations and the Agilent 1200 Series SL was
used for the standard EPA Method 1694. The conditions were
as follows for each of the figures shown in this application
note.
The other aspect of UHPLC is the increased peak capacity
available when longer columns with 1.8-µm packing are used.
It is now possible to have almost 300 times greater peak
capacity, which is a valuable asset to unknown analysis in
wastewater and other environmental applications such as
pesticide screening. Finally, the UHPLC system should be
robust and capable of both high pressure and high flow
(>1 mL/min at pressures up to 1200 bar) to do both rapid resolution and normal flow chromatography with high peak
capacity. Agilent has 1.8-µm columns specially designed for
pressures to 1200 bar (18,000 psi) and give a variety of phases
Figure 1. Part A
The liquid chromatograph was the Agilent 1200 Series SL. The
gradient was from 10% acetonitrile/water to 100% acetonitrile in 30 min with a 5-min hold time. The flow rate was
0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18,
4.6 mm × 150 mm, 3.5 µm. Peak widths at the base were
15-18 seconds and peak capacity of 100. Maximum pressure
was 75 bar.
5990-4409EN
40
The mass spectrometer was the Agilent 6410 triple
quadrupole LC/MS system in electrospray positive mode with
three time segments in multiple reaction monitoring (MRM)
mode. There were two transitions per compound with 10-ms
dwell time for each transition. The compounds were Group 1
of EPA Method 1694. See our application note for further
detail on compounds and their transitions [2].
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with two
time segments in MRM mode and six compounds per segment. There was one transition per compound with a 5-ms
dwell time for each transition. The compounds were a selected set of 12 compounds from EPA Method 1694.
Figure 3B
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was
1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were
1–3 sec and peak capacity of 60. Maximum pressure was
780 bar.
Figure 1. Part B
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 10 min with a 1-min hold time. The flow rate was
0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were
5–6 sec and peak capacity of 100. Maximum pressure was
375 bar.
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with two
time segments in MRM mode and three compounds per segment. There was one transition per compound with a 5-ms
dwell time for each transition. The compounds were carbamazepine, continine, caffeine, diphenhydramine, thiabendazole, and trimethoprim from EPA Method 1694.
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with one
time segment in MRM mode. There were two transitions per
compound with 10-ms dwell time for each transition. The
compounds were Group 1 of EPA Method 1694. See our application note for further detail on compounds and their transitions [2].
Figures 4 and 5
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was
1.5 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm x 50 mm, 1.8 µm. Peak widths at the base were
1–3 sec and peak capacity of 60. Maximum pressure was
900 bar.
Figure 2
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 20 min with a 2-min hold time. The flow rate was
0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm × 100 mm, 1.8 µm. Peak widths at the base were
5–6 sec and peak capacity of 100. Maximum pressure was
750 bar.
The mass spectrometer was the Agilent 6520 accurate-mass
Q-TOF LC/MS in electrospray positive mode in 2-GHz mode
with 20 scans per second at a mass accuracy of >2 ppm. The
compounds included a mix of 220 pesticides from the list
reported by Thurman et al. 2008.
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with one
time segment in MRM mode. There was one transition per
compound with 10-ms dwell time for each transition. The
compounds were Group 1–4 of EPA Method 1694, plus 15
additional pharmaceuticals. See our application note for further detail on compounds and their transitions [2].
Figure 6
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was
1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm x 50 mm, 1.8 µm. Peak widths at the base varied as a
function of scans per second on the mass spectrometer.
Maximum pressure was 780 bar.
Figure 3A
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time. The flow rate was
1.2 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were
1–3 sec and peak capacity of 60. Maximum pressure was
780 bar.
5990-4409EN
41
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with one
time segment in MRM mode and one compound per segment,
carbamazepine. There was one transition per compound with
dwell times that varied from 1 to 300 ms and were equal to a
range of 3 to greater than 20 scans per second.
Wastewater samples were collected from an effluent site in
Boulder Creek (Boulder, CO) and extracted with polymeric cartridges using a modified EPA protocol. One liter water samples
were extracted directly onto a 500-mg cartridge without pH
adjustment, dried for 10 minutes with air, and eluted with 8
mL of methanol. The methanol was evaporated to 1 mL and
analyzed by LC/MS/MS as described below. "Blank" wastewater extracts were used to prepare the matrix matched standards for validation purposes. The wastewater extracts were
spiked with the mix of pharmaceuticals at different concentrations (ranging from 0.1 to 500 ppb) then analyzed by
LC/MS/MS.
Figure 7
The liquid chromatograph was the Agilent 1290 Infinity LC.
The gradient was from 10% acetonitrile/water to 100% acetonitrile in 2 min without a hold time, 6 min without a hold
time, and 30 min without a hold time. The flow rate was
0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18,
2.1 mm × 100 mm, 1.8 µm. Peak widths at the base were
2–6 sec and peak capacity of 60. Pressure maximum was
750 bar.
Results and Discussion
This application note contains three sections discussing
examples of peak capacity and rapid resolution for pharmaceuticals in wastewater using EPA Method 1694, UHPLC and
LC/TOF-MS of pesticides, and some important chromatographic considerations with UHPLC/MS.
The mass spectrometer was the Agilent 6430 triple
quadrupole LC/MS in electrospray positive mode with one
time segment in MRM mode and one compound per segment,
caffeine. There was one transition per compound with a dwell
time of 5 ms.
Part 1: Environmental pharmaceutical analysis by
LC/MS/MS.
Sample preparation
Pharmaceutical analytical standards were purchased from
Sigma, (St. Louis, MO, USA). Individual pharmaceutical stock
solutions (approximately 1000 g/mL) were prepared in pure
acetonitrile or methanol depending on the solubility of each
individual compound, and stored at –18 ºC. From these solutions, working standard solutions were prepared by dilution
with acetonitrile and water.
The EPA Method 1694 is a standard method requiring 20-min
analysis times or longer to satisfy the method requirements.
However, recent changes published by EPA suggest that other
chromatographic conditions may be used, such as shorter
analysis times and rapid resolution, if sufficient mass spectrometric analysis is used (for example, 2 transitions per compound). Figure 1 shows the use of the new Agilent 1290
Infinity with UHPLC to reduce analysis times from 30 min to
10 min, a 66% decrease in analysis times with a peak capacity
of 100 in both analyses. The original EPA method called for a
ZORBAX Eclipse Plus-C18, 3.5 µm column and the rapid resolution is with the ZORBAX Eclipse Plus-C18
2.1 mm × 50 mm, 1.8 µm column.
Pesticide analytical standards were purchased from Dr.
Ehrenstorfer (Ausburg, Germany). Individual pesticide stock
solutions (1000 g/mL) were prepared in pure acetonitrile or
methanol depending on the solubility of each individual compound, and stored at –18 ºC. From these concentrated solutions, working standard solutions were prepared by dilution
with acetonitrile and water.
5990-4409EN
42
x10 3
+ MRM (436.0000 -> 277.0000) 100 ppb_Group 1_2 transitions.d
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1
12
23
Part A. 30-min analysis with 3.5 µm
ZORBAX Eclipse Plus-C18 for EPA 1694 for 46 compounds
in 30-min gradient at 0.6 mL/min
Peak capacity 100
19.8788
20.001
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Counts vs. acquisition time (min)
×10 5
4
+ESI EIC(243.1359, 244.1387) Scan Frag=190.0V Group1_resolution_50mm_3scans.d
1
3.5
Part B. 10-min analysis with 1.8 µm ZORBAX Eclipse Plus-C18
for EPA 1694 for 46 compounds at 0.6 mL/min
Peak capacity 100
3
2.5
2
1.5
1
0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Counts vs. acquisition time (min)
5
5.5
6
6.5
7
Figure 1
Shows the reduction of time of analysis from a 30-min analysis to a 10-min analysis using the Agilent
1290 Infinity with UHPLC for Group 1 pharmaceuticals in EPA Method 1694.
in order to obtain at least 20 scans across each peak. By
obtaining 20 scans or more, the peak width may be reduced to
1 – 2 sec and the result is ultrafast chromatography. The column is a ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm with a
flow rate of 1.2 mL/min and a 1.5 min gradient. The 12 pharmaceuticals eluted in less than 60 seconds. This, to our
knowledge, is the first example of ultrafast chromatography
applied to an environmental sample. In this case, it is a
wastewater from Boulder, Colorado, that contains the following pharmaceuticals: carbamazepine, continine, caffeine,
diphenhydramine, thiabendazole, and trimethoprim.
Because the pressure is at 375 bar it is possible to easily
increase peak capacity and the number of pharmaceuticals
that may be separated by substituting a longer column
(2.1 mm x 100 mm) and maintaining the same flow rate of
0.6 mL/min. This doubles the pressure from 375 to 750 bar.
The results are shown in Figure 2.
It is also possible to do ultrafast chromatography with the
Agilent 1290 Infinity LC for pharmaceuticals using a triple
quadrupole LC/MS. In the example shown in Figure 3, the
number of compounds have been reduced to 12 compounds
5990-4409EN
43
Cpd 1: 0.764: +ESI EIC(177.1019, 178.1058, 179.1105) Scan Frag=190.0V Groups1-4+Extras_resolution_100mm.d
×10 5
1
4
3.5
3
2.5
2
1.5
1
0.5
0
1
2
3
4
5
6
7
8
9
10
Counts vs. acquisition time (min)
11
12
13
14
15
16
Figure 2
Increased peak capacity showing the separation of the entire list of EPA Method 1694 pharmaceuticals plus 15 new compounds for a total of 90 pharmaceuticals in less than 20 min by using a ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8-?m packing material with UHPLC using the Agilent 1290 Infinity LC. Peaks
are 5 to 6 sec wide and peak capacity is 200.
×10 2
1
+ESI MRM Frag=90.0V CID@35.0 (415.0 -> 159.0) Group1_C18_speed grad_15comp_bis.d
1
12
3
23
0.9
0.8
0.7
Ultrafast Chromatography
ZORBAX Exlipse Plus-C18,
2.1 mm × 50 mm
1.2 mL/min
780 bar
Peak width 0.7 sec
0.6
0.5
0.4
0.3
0.2
0.1
0
4
8
12
16
20
24
28
32
36
40
44
48
52
56
Counts (%) vs. acquisition time (sec)
Figure 3A
The “ultrafast” gradient for 12 pharmaceutical standards.
5990-4409EN
44
60
64
68
72
76
80
84
88
It is important to realize that when using ultrafast gradient
conditions, quality assurance and quality control data are
required. Therefore, all sample purification measures must be
taken during sample preparation to minimize suppression. It
is also necessary to use labeled internal standards for quantitation since the entire sample matrix is eluting in a very narrow window. The reproducibility of the 1290 Infinity was within one second making it easy to obtain reliable data. Also
important is the use of at least two transitions by
LC/MS/MS, one for quantitation and the other as a qualifier
ion. See our application note on the EPA Method for further
examples. [2]
×10 2
1
0.8
+ESI MRM Frag=110.0V CID@25.0 (748.5 -> 158.0) Boulder effluent_C18_speed grad_15comp.d
1
1 2
23
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4 0.45 0.5 0.55 0.6 0.65 0.7
Counts (%) vs. acquisition time (min)
0.75
0.8
0.85
0.9
0.95
1
1.05
Figure 3B
Ultrafast gradient for wastewater from Boulder, CO, USA. The compounds detected included: carbamazepine, continine, caffeine, diphenhydramine,
thiabendazole, and trimethoprim.
5990-4409EN
45
Part 2: UHPLC and LC/TOF/MS
obtain the 20 spectra per second for the TOF-MS instruments.
Figure 4 shows how effective this strategy is when doing
UHPLC with either LC/TOF-MS or LC/Q-TOF-MS. More than
100 pesticides were analyzed in less than 80 sec using the
Agilent 1290 Infinity LC with a ZORBAX Eclipse Plus-C18,
2.1 mm × 50 mm column at a flow rate of 1.5 mL/min. The
UHPLC was required as pressures reached 900 bar. The separation included peak widths at half-height of only 0.7 seconds.
See Figure 5 for the herbicide, terbutryn.
One of the major concerns when doing rapid resolution is
obtaining good sampling across the narrow peaks of 1–2 sec
using mass spectrometry. In the previous examples, we
showed how this is done using triple quadrupole LC/MS and
maintaining 20 scans across each peak. The use of LC/TOFMS and LC/Q-TOF-MS makes this task easy for as many compounds as one would like to monitor. This is because the
TOF-MS instruments are obtaining data in full spectrum mode
at all times. It is merely necessary to set the software to
×10 5
63.0
Cpd 109: Terbutryn
1.1
1.05
1
(2 ppm mass tolerance, MFE)
50 pg on-column
ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm
1.8 µm
Flow rate 1.5 mL/min
900 bar
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
4
8
12
16
20
24
28
32
36
40
44
48
52
56
Counts (%) vs. acquisition time (sec)
Figure 4
Pesticide analysis of over 100 compounds by LC/TOF-MS in less than 2 min.
5990-4409EN
46
60
64
68
72
76
80
84
88
The chromatographic speed, separation, and power of
LC/TOF-MS makes it possible to analyze complex mixtures of
pesticides from food or water matrices in a few minutes. This
allows the rapid analysis used in the food monitoring industry
in Europe, where shipments of vegetables and fruits are not
unloaded until the analysis of pesticides are complete.
Therefore, rapid analysis is important in these cases.
63.0
Cpd 109: Terbutryn
×10 5
1.06
1.02
0.98
0.94
0.9
0.86
0.82
0.78
0.74
0.7
0.66
20 Data points
across peak
0.7 second
Peak width
at half height
0.62
0.58
0.54
0.5
0.46
0.42
0.38
0.34
0.3
0.26
0.22
0.18
0.14
0.1
0.06
4
8
12
16
20
24
28
32
36 40 44 48 52 56
Counts vs. acquisition time (sec)
60
64
68
72
76
80
84
88
Figure 5
Peak width of 0.7 sec at half-height using the Agilent 1290 Infinity with a ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm with a flow rate of 1.5 mL/min at a
pressure of 900 bar.
5990-4409EN
47
Part 3: Chromatographic considerations with
UHPLC/MS
Optimization of chromatographic and mass spectrometry conditions for the best use of UHPLC includes the following
ideas. First, it is important to have at least 20 MS data points
across each peak in order to obtain the 1–2 sec peak widths
of ultrafast chromatography. An illustration of the importance
of data cycles per second is shown in Figure 6.
The peak broadening that appears at the lower cycle rates is
caused by a smoothing routine meant to shape peaks for
good integration and quantitation and is a common procedure
in all chromatographic software. Therefore, when using triple
quadrupole LC/MS instruments, it is recommended to use a
short dwell time of 5 ms and the dynamic MRM procedures to
insure that 20 cycles per peak are obtained. In the case of
LC/TOF-MS and LC/Q-TOF-MS instruments it is only necessary to set the software to obtain 20 spectra per second
across the mass range that is acquired.
Secondly, quantitative aspects of analysis are a consideration
in good UHPLC practice. Here it is important that
maximum peak sensitivity be obtained. Figure 7 shows an
example where caffeine is maximized for peak intensity and
peak area by adjusting the gradient and flow rate until the
maximum signal is obtained. In this case, a 6-min gradient
resulted in the optimum signal-to-noise (S/N) ratio of 180 and
an area count of 55,000 counts at a retention time of 1.7 min.
Note that the signal-to-noise drops to half at a value of 91 with
the longer gradient of 30 min but a retention time increase of
only 0.2 min. Thus, it is important to test various flow rates and
retention times to optimize signal strength; especially of the
polar and early eluting compounds in a chromatographic analysis.
A final consideration in good UHPLC chromatography is the
suppression of LC/MS signal. It was mentioned earlier and
must be emphasized that standards in fast and ultrafast analysis will often show little or no suppression because of the purity of the standard mixture. However, real samples may show
suppression; therefore, it is important to dilute samples or to
purify them in extraction procedures to limit the amount of
matrix that is present. Finally, it is valuable to use deuterated
or C-13 labeled standards when measuring pharmaceuticals in
wastewater and other complex matrices. This is the recommended procedure of EPA Method 1694 and the readers are
referred to our application note on this topic [2].
>20 cps
10 cps
5 cps
3 cps
Figure 6
This figure shows the effect of dwell time and data points per peak and how it affects the peak shape for a single compound, carbamazepine, from a 1-sec peak
at > 20 cycles per second (cps) to 3–4 sec peak at 3 cycles per second (cps).
5990-4409EN
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×10 2
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
+ESI TIC MRM Frag=110.0V CID@** (** -> **) Caffeine_C18_long grad_5uL.d
1
1.5 min gradient
s/n 190
Area: 43K
6 min gradient
s/n 180
Area: 55K
30 min gradient
s/n 91
Area: 46K
0.4 0.6 0.8
1
1.2 1.4
1.6
1.8
2
ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm
10% ACN to 100% ACN
750 Bar
0.6 mL/min
2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8
Counts (%) vs. acquisition time (min)
4
4.2 4.4 4.6 4.8
5
5.2
Figure 7
The effect of retention time on the signal to noise (S/N) and the area counts for caffeine in LC/MS analysis.
Conclusions
References
In conclusion, we recommend the ZORBAX Eclipse Plus-C18,
2.1 mm × 50 mm columns for rapid resolution and ultrafast
chromatographic separations, while for maximum peak
capacity the ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm is a
better choice. Our results show that fast flow rates greater
than 1.5 mL/min may be used and pressures greater than
1000 bar are possible with confidence and reliability. Finally,
we see the new Agilent 1290 Infinity LC as the best example
of the evolution of chromatography from the gravity columns
of Tsweet to the UHPLC realm of ultrafast high pressure liquid
chromatography "made-easy."
1.
EPA Method 1694: Pharmaceuticals and personal care
products in water, soil, sediment, and biosolids by
HPLC/MS/MS, December 2007, EPA-821-R-08-002.
2.
I. Ferrer, E. M. Thurman, J. A. Zweigenbaum, “EPA
Method 1694: Agilent's 6410A LC/MS/MS Solution for
Pharmaceuticals and Personal Care Products in Water,
Soil, Sediment, and Biosolids by HPLC/MS/MS,” 2008,
Application Technologies publication 5989-9665EN,
3.
E. M. Thurman, I. Ferrer, J. A. Zweigenbaum, “MultiResidue Analysis of 301 Pesticides in Food Samples by
LC/Triple Quadrupole Mass Spectrometry,” 2008,
Application Technologies publication 5989-6414.
Acknowledgements
For More Information
The Center acknowledges the help and advice of Drs. Jerry
Zweigenbaum, Michael Woodman, and Peter Stone of Agilent
Technologies, Inc.
For more information on our products and services, visit our
Web site at www.agilent.com/chem.
5990-4409EN
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5990-4409EN
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Screening impurities in fine
chemicals using the Agilent 1290
Infinity LC System
Application Note
Fine Chemical
Authors
Abstract
Michael Woodman
The Agilent 1290 Infinity LC System with ultra violet/visible (UV/VIS) Diode Array
Agilent Technologies, Inc.
detection (DAD) is used to analyze octyl-dimethyl-p-aminobenzoic acid for the pres-
2850 Centerville Road
ence of impurities. The system is used for the chromatographic separation of the
Wilmington, DE 19808
compound from its impurities on 3.0 and 2.1 mm id C18 columns, of various lengths,
USA
with 1.8-um packing materials prepared in 600-bar (9000 psi) or special 1200-bar
(18,000 psi) configurations. The ability of the 1290 Infinity LC System to operate with
long, high resolution columns under conditions of rapid analysis is demonstrated with
low viscosity acetonitrile (ACN) and higher viscosity methanol (MeOH) solvent conditions.
5990-4293EN
51
Introduction
Experimental
The analysis of impurities in starting materials, intermediates
and finished products intended for a wide range of final uses
is essential for ensuring product quality, performance, and
consumer safety. The general conditions for successful analysis of impurities by high-performance liquid chromatography
(HPLC) include gradient elution and multi-wavelength monitoring of the overall separation and may benefit from other
detectors including evaporative light scattering (ELSD) and
mass spectrometers (MS). Because impurity determination is
the primary goal, one needs to ensure that mobile phase,
vials, and HPLC components are free of minor impurities that
might lead to confusing results during the analysis. Careful
preparation of diluent blanks and blanks that might represent
contamination sources due to additional sample preparation,
such as filtration, are also appropriate. The analysis sequence
is likely to include runs of the production material, solvent or
diluent blank runs. It is also typical to include limit standards
prepared by diluting the primary component to the lowest
level where detection of impurities might be required. Finally,
it is generally essential to include an authentic high purity reference standard.
Sample Preparation
The primary OD-PABA solution was prepared at a concentration of 1 mg/mL in 2-propanol and subsequently diluted to
lower concentrations as needed. Injection volumes of
0.2–2 µL were made into the LC/DAD system.
LC Method Details
LC Conditions
Agilent 1290 Infinity LC system binary pump G4220A,
Agilent 1290 Infinity LC system autosampler G4226A
Agilent Thermostatted Column Compartment G1316C with switching valve
Agilent 1290 Infinity system diode array UV/VIS detector G4212A with 10 mm
path fiber optic flow cell
Para-aminobenzoic acid (PABA) has historically been used as
an ultra-violet filter ingredient in sunscreen formulations. As
its use can increase the risk of skin cancer a derivative in the
form of octyl-dimethyl-p-aminobenzoic acid (OD-PABA), is
currently and more commonly used. However, PABA may be
formed as a degradate of OD-PABA, so it is important to monitor its potential presence in samples of OD-PABA. As a commercial product, the purity of OD-PABA is important to manufacturers, for the purposes of safety and economics. In this
work we investigate the capability of the Agilent 1290 Infinity
LC system (UHPLC system with 1200 bar pressure limit) to
detect impurities in OD-PABA samples with UV/VIS Diode
Array detection.
(CH 2 ) 3
CH3
H3C
CH3
Column temp:
40 °C
Mobile phase:
A = HPLC grade water
B = Acetonitrile (ACN) or methanol (MeOH)
(See individual figures)
Flow rate:
See individual figures
Gradient:
Gradient: the gradient conditions were either 40% to 90%
ACN or 50% to 100% MeOH. The gradient slope was maintained at 3.5% organic phase increase per column volume,
altering gradient time and flow rate accordingly. This is
based on calculations using a modification of the Agilent
Method Translator. [1]
Monitoring 210, 254, 280 and 320 nm, bandwidth 4 nm, reference wavelength
off
O
N
(See individual figures for specific usage)
Agilent ZORBAX SB-C18 RRHT, 3 mm × 50 mm, 1.8 µm
600 bar p/n 827975-302
Agilent ZORBAX SB-C18 RRHD, 2.1 mm × 100 mm, 1.8 µm
1200 bar, p/n 858700-902
Agilent ZORBAX SB-C18 RRHD, 2.1 mm × 150 mm, 1.8 µm
1200 bar, p/n 859700-902
UV Conditions
The structure of the OD-PABA compound analyzed in this
work is shown in Figure 1.
H3C
Columns:
C 17 H27NO2
O
Figure 1
Octyl-dimethyl-p-aminobenzoic acid (OD-PABA).
5990-4293EN
52
Results and Discussion
The UV response of OD-PABA, with four wavelengths monitored, is shown at a retention time of 2 min in Figure 2. Multiwavelength monitoring of the separation provides a simple
way to account for multiple impurities and assist in the selection of a final wavelength condition that can maximize sensitivity for all detected analytes.
Octyl dimethyl para-aminobenzoic acid, 40 °C, 1.5 mL/min, 40% to 90% ACN/water
over 2 minutes. Up to 460 bar on ZORBAX StableBond C18, 3 mm × 50 mm
Figure 2
Multi-wavelength UV chromatogram of OD-PABA production material on a 3 mm × 50 mm ZORBAX Rapid Resolution High Throughput (RRHT) column. The
chromatogram demonstrates the typical difficulties encountered with this type of separation, which are a need for wide dynamic range detection and sensitive
impurity measurement. The peak at 0.75 minutes is confirmed by retention time matching and UV spectra to be PABA, the primary impurity in the mixture.
5990-4293EN
53
Figure 3
An expanded presentation of the chromatogram shown in Figure 2 based on the 3 mm × 50 mm gradient separation.
In Figure 3 the expanded multi-wavelength chromatogram
allows us to see close detail and shows the number of impurities, as well as several areas where chromatographic resolution is clearly inadequate for individual component measurement. Despite the small particle size used in this column, the
relatively short length limits the total resolution. As we move
to longer column dimensions we will often reduce column
diameter to reduce overall solvent consumption at the same
time.
5990-4293EN
54
Figure 4
Analysis of the standard material on a 2.1 mm × 100 mm Agilent ZORBAX StableBond C18 column prepared for operation at 1200 bar pressure limit. Acetonitrile
water gradient, 0.74 mL per minute, gradient time 4.0 minutes.
In Figure 4, we see that increasing the length of the column
has resulted in a significant increase in the resolution of some
of the observed components. To further increase resolution it
would be practical to explore longer columns or explore
alternative mobile phase or column chemistries.
5990-4293EN
55
Figure 5
An expanded view of the acetonitrile separation using the same gradient slope on a 2.1 mm × 150 mm column rated for 1200 bar operating pressure.
Agilent ZORBAX StableBond C18, 1.8 µm.
The increased column length clearly gives more resolution,
however the increased back pressure also limits the flow rate
if one is to operate in a conservative range of operating pressure. The Agilent 1290 Infinity LC system and associated
ZORBAX Rapid Resolution High Definition (RRHD) chemistries
are capable of operating pressures up to 1200 bar, approximately 18,000 psi. To ensure robust and rugged system operation many users typically specify the upper pressure limit for a
method at a value less than 80% of the rated operating
pressure.
5990-4293EN
56
Octyl dimethyl para-aminobenzoic acid, 0.52 mL/min, 50% to 100% MeOH/water over 5.7 minutes.
Up to 845 bar on Agilent ZORBAX StableBond C18, 2.1 mm × 100 mm, 1.8 µm.
Figure 6
Separation of the sample mixture on a 2.1 mm × 100 mm Agilent ZORBAX StableBond C18, using methanol as the organic phase. Flow rate
0.52 mL/min gradient time 5.7 min, for a gradient of 5% to 100% methanol
When considering the fundamental components of the resolution equation we are all quite familiar with the concepts of
capacity, selectivity, and efficiency. Increasing the column
length, like decreasing the particle size of the packing material, will increase the efficiency of the overall separation.
Because the increase in efficiency yields a relatively low
return in terms of resolution, users often need to ensure that
the capacity factor is optimized by exploring alternative chemical variables that could promote increased selectivity in the
separation.
to be problematic. In this example, however, the highly conjugated structures of the parents and related impurity structures allow sensitive detection at wavelengths well above
the UV cutoff of common organic solvents used in reversed
phase chromatography. In about the same amount of analysis time as the example in Figure 5, we achieve significantly
higher selectivity leading to more resolved impurities while
reducing overall solvent consumption and eliminating the
need for expensive acetonitrile as the organic phase.
In Figure 6 we see the dramatic results achieved by changing
the separation conditions from using acetonitrile as the
organic phase to methanol. If this separation was highly
dependent on monitoring the separation at very low wavelengths one might find the UV cutoff of the methanol, 205 nm,
5990-4293EN
57
Octyl dimethyl para-aminobenzoic acid, 0.52 mL/min, 50% to 100% MeOH/water over 5.7 minutes.
Up to 845 bar on Agilent ZORBAX StableBond C18, 2.1 mm × 100 mm, 1.8 µm, 40 °C.
Figure 7
An expanded view of the small region of the chromatogram in Figure 6 showing a significant number of low concentration impurities. Conditions as in Figure 6.
Estimated impurity concentrations for the smallest peaks in this figure are less than 0.02%.
Conclusions
The detection of low-level impurities in
synthetic materials and highly refined
natural products is of critical importance to the ultimate utility of these
substances. Rapid analysis by HPLC
using high-resolution columns and
appropriately chosen organic phases
ensures consistent results with rapid
analysis turnaround time. Using the
Agilent 1290 Infinity LC system, we
were able to easily demonstrate UHPLC
capabilities well within the operating
range of the designed system. Higher
throughput could still be achieved with
this system by increasing flow rate and
simultaneously reducing the gradient
segment time to reproduce the gradient slope in a shorter total analysis
time.
For More Information
For more information on our products
and services, visit our Web site at
www.agilent.com/chem.
5990-4293EN
58
References
1.
http://www.chem.agilent.com/
en-US/products/instruments/
lc/pages/gp60931.aspx
High-resolution analysis of intact
triglycerides by reversed phase
HPLC using the Agilent 1290 Infinity
LC UHPLC System
Application Note
Food, Hydrocarbon Processing
Authors
Abstract
Michael Woodman
The Agilent 1290 Infinity LC System with ultraviolet/visible (UV/VIS) Diode Array
Agilent Technologies, Inc.
detection (DAD) is used to analyze triglycerides in soybean oil under non-aqueous
2850 Centerville Road
reversed phase gradient conditions. The Agilent 1290 Infinity LC System was used for
Wilmington, DE 19808
the chromatographic separation of the sample on 3.0 and 2.1 mm id C18 columns, of
USA
various lengths, with 1.8-µm packing materials prepared in 600 bar (9000 psi) or special 1200 bar (18,000 psi) configurations. The ability of the Agilent 1290 Infinity LC
System to operate with long, high resolution columns is demonstrated with isopropanol (IPA) or methyl tert butyl ether (MTBE) as the strong solvent and acetonitrile
as the weak component of the mobile phase mixture.
5990-4292EN
59
Introduction
In this example, from top to bottom, palmitic acid (C16:0),
oleic acid (C18:1), alpha-linolenic acid (C18:3) are shown with
respect to chain length and degree of unsaturation. The
chemical formula is C55H98O6.
The analysis of intact triglycerides from animal or vegetable
sources has many practical uses including understanding the
chemical composition of the triglyceride, assessing fuel
potential, and understanding lipid metabolism and behavior in
living systems. The general conditions for successful analysis
of these components by high-performance liquid chromatograph (HPLC) include gradient elution and low-wavelength
monitoring of the overall separation. Because triglycerides
have relatively few chromophores it is also beneficial to use
evaporative light scattering detectors (ELSD) or mass spectrometers to facilitate other views of the separation.
Experimental
Sample Preparation
The primary solution was prepared at a concentration of
10 mg/mL, in 2-propanol or 2:1 volume to volume
MeOH/MTBE, and subsequently diluted to lower concentrations as needed. Injection volumes of 0.2-2 µL were made
into the LC/DAD system.
During the development of this application, we analyzed a
number of vegetable oils from various sources including soy,
corn, rice bran, safflower, grape seed, olive, and palm oil.
Because of the wide abundance of soybean oil in the United
States and its growing significance in the production of biofuels, most of this work was standardized on maximizing the
resolution of soybean oil triglycerides. These general conditions, however, are also suitable for a wide variety of samples
including samples from animal lipid sources.
O
H2 C
O O
*HC
O O
H2 C
O
9
12
15
α
ω
Figure 1
Typical triglyceride structure.
Intact triglycerides generally have very low water solubility
and as such are commonly separated by normal phase chromatography, which separates species largely based on differences in polar functional groups, or by reversed phase chromatography operating in a non-aqueous mode of separation,
which has more selectivity for small differences on carbon
character such as chain length or unsaturation.
LC Method Details
LC Conditions
Agilent 1290 Infinity LC System binary pump G4220A,
Agilent 1290 Infinity LC System autosampler G4226A
Agilent Thermostatted Column Compartment G1316C with switching valve
Agilent 1290 Infinity LC System diode array UV/VIS detector G4212A with
10 mm path fiber optic flow cell
According to information published by Perkins [1] the predominant fatty acids, which are the building blocks of triglycerides on a glycerol backbone, found in soybean oil are myristic (14:0), palmitic (16:0), oleic (18:1), linoleic (18:2) and
linolenic (18:3). Many other minor fatty acids are also present
and because all of the fatty acids are randomly constructed
into triglycerides, an extensive permutation of fatty acid substructure is obviously possible. Because the predominant difference between fatty acids consists of carbon chain length
and number of double bonds, most of the diversity in triglycerides is found in the rather non-polar organic structural features. As a result, reversed phase chromatography is most
useful for this application. Triglycerides have extremely poor
solubility in water so one normally chooses either a high
organic starting position, with respect to the aqueous content or, as in this work, a completely non-aqueous separation
environment.
Columns:
(See individual figures for specific usage)
Agilent ZORBAX SB-C18 RRHT, 3 mm × 150 mm, 1.8 µm
600 bar, p/n 829975-302
Agilent ZORBAX SB-C18 RRHD, 2.1 mm × 100 mm, 1.8 µm
1200 bar, p/n 858700-902
Agilent ZORBAX SB-C18 RRHD, 2.1 mm × 150 mm, 1.8 µm
1200 bar, p/n 859700-902
In some cases, columns were coupled to extend the
length and resolution.
Column temp:
20 °C or 30 °C
Mobile phase:
A = acetonitrile
B = isopropanol (IPA) or tert butyl methyl ether
(MTBE) (See individual figures)
Flow rate:
See individual figures
Gradient:
The gradient conditions were either 20% to 60% IPA or 10%
to 40% MTBE, based on the strong eluting strength of
MTBE when compared to IPA. The gradient slope was
maintained at 2.6% organic phase increase per column
volume for IPA gradients and 2.0% with MTBE, altering
gradient time and flow rate accordingly. This was determined by calculations using a modification of the Agilent
Method Translator. [3]
The typical structure of a triglyceride is shown in Figure 1. [2]
UV Conditions
Monitoring 210, 220 and 230 nm, bandwidth 4 nm, reference wavelength off
5990-4292EN
60
methanol has consistently shown a dramatic reduction in the
overall resolution of the triglycerides. The significant increase
in operating pressure, when running the gradient from acetonitrile to IPA, is clearly limiting and undesirable. Increasing
the operating temperature of the column as a means of reducing solvent viscosity has proven to be undesirable because the
chromatographic resolution tends to collapse as temperature
increases.
Results and Discussion
A typical gradient separation of triglycerides using acetonitrile
IPA gradient is shown in Figure 2.
Some general comments are appropriate about the conditions
and chromatographic profile shown in Figure 2. While it would
be ideal to consider less expensive methanol as the weak eluent, introduction of methanol or denatured ethanol containing
Figure 2
A 210-nm UV chromatogram of soybean oil sample on a 3 mm × 150 mm ZORBAX Rapid Resolution High Throughput (RRHT), 1.8 ?m column, upper panel.
System pressure trace showing the general progress of the gradient elution, lower panel. Flow rate 0.6 mL/min, gradient time 24 min. Strong solvent,
isopropanol. The chromatogram demonstrates the typical difficulty encountered with this type of separation, which is small clusters of chromatographically
similar triglycerides. These clusters are not positional isomers of the same carbon number and degree of unsaturation, rather a mixture of various chain lengths
and number of double bonds as shown by mass spectrometric evaluation
5990-4292EN
61
Soybean Oil, 10 µg, 0.6 mL/min, 20% to 60% IPA/ACN over 24 minutes.
Up to 370 bar on ZORBAX StableBond C18, 3 mm × 150 mm 1.8 µm, 30 °C
Soybean Oil, 10 µg, 0.6 mL/min, 20% to 60% IPA/ACN over 24 minutes.
Up to 400 bar on ZORBAX StableBond C18, 3 mm × 150 mm 1.8 µm, 20 °C
Figure 3
An expanded presentation of the chromatogram shown in Figure 2 at 30 °C, upper panel, compared with the same conditions in Figure 2 operating the column
at 20 °C.
the availability of the Agilent 1290 Infinity LC System with
increased operating pressure capability has allowed us to
reduce the temperature to 20 °C and demonstrate a usable
improvement in separation.
In Figure 3 we see the improvement achieved by operating
the separation at 20 °C rather than 30 °C. The operating pressure increase is approximately 10% at the lower temperature.
While many of our separations have been performed at 30 °C
as a compromise between separation and operating pressure,
5990-4292EN
62
Soybean Oil, 10 µg, 0.29 mL/min, 20% to 60% IPA/ACN over 27 minutes.
Up to 760 bar on ZORBAX StableBond C18, 2.1 mm × 250 mm 1.8 µm, 20 °C
Figure 4
Analysis of the soybean oil sample on an Agilent ZORBAX StableBond C18 column, 2.1 mm × 250 mm, 1.8 ?m, (150 mm in series with 100 mm) prepared for
operation at 1200 bar pressure limit. Flow rate 0.29 mL/min, gradient time 27 min. Maximum observed pressure 760 bar.
In Figure 4, we see that increasing the length of the column
has resulted in a significant increase in the resolution of some
of the observed components. To further increase resolution, it
would be practical to explore longer columns or explore alternative mobile phase or column chemistries. As with most very
high performance separations, rate-limiting features tend to
include operating pressure, operating temperature, and maximum flow rate. The triglyceride separations evaluated thus far
have not been receptive to operation at higher column temperatures or higher flow rates, presumably because of their
relatively high molecular weight and flexible organic structure.
Even when gradient slope translations are carefully made to
ensure organic strength consistency from method to method,
operating at higher flow rates has consistently shown degradation of the overall separation. Because the isopropanol has
significantly high viscosity and high pressure, it seemed appropriate to consider other nonpolar solvents that are miscible
with acetonitrile and friendly to low UV detection, as a substitute for isopropanol.
5990-4292EN
63
Soybean Oil, 10 µg, 0.29 mL/min, 10% to 40% IPA/ACN over 27 minutes.
Up to 440 bar on ZORBAX StableBond C18, 2.1 mm × 250 mm 1.8 µm, 20 °C
Figure 5
By substituting MTBE for isopropanol with otherwise the same conditions as Figure 4, and then re-optimizing the gradient for the significant increase in eluting
strength of MTBE, we arrive at a new set of operating conditions where there is only a small difference in operating pressure over the gradient run. Flow rate
0.29 mL/min, gradient 27 min for 10% to 40% MTBE, maximum observed pressure 440 bar.
In Figure 5, the change to MTBE and subsequent readjustment of the gradient resulted in a separation that was very
comparable to the original isopropanol separation, however
at a much lower maximum operating pressure. In view of the
prior evidence and comments regarding increased temperature or flow rate resulting in degraded separation, it seemed
that the most appropriate way to take advantage of the new
operating pressure capability of the Agilent 1290 Infinity LC
System was to continue to increase the column length. The
Agilent 1290 Infinity LC System and associated ZORBAX
chemistries are capable of operating pressures up to 1200 bar,
or approximately 18,000 psi. To ensure robust and rugged system operation many users typically specify the upper pressure
limit for a method at a value less than 80% of the rated
operating pressure.
5990-4292EN
64
Soybean Oil, 30 µg, 0.29 mL/min, 10% to 40% MTBE/ACN over 43 minutes.
Up to 730 bar on ZORBAX StableBond C18, 2.1 mm × 400 mm, 1.8 µm, 20 °C
Figure 6 Separation of the soybean oil sample on a 2.1 mm × 400 mm ZORBAX StableBond C18, 1.8 ?m 1200 bar columns (150 mm + 150 mm + 100 mm in
series). Flow rate 0.29 mL/min gradient time 43 min, for a gradient of 10% to 40% MTBE. Maximum operating pressure 730 bar at 20 °C.
As shown in Figure 6, having previously optimized the column
temperature, operating flow rate and gradient slope for the
best possible balance between resolution and analysis time,
and after investigating a variety of solvents as candidates for
both the weak solvent and strong solvent choice, we are left
with an ultimate opportunity to operate on a very long column
set of 1.8 ?m particle size columns under conditions ideal for
the separation of this group of triglycerides. With an operating
pressure of only 730 bar, which is about 60% of the rated
capability of the Agilent 1290 Infinity LC System, it is clearly
possible to consider even longer column lengths or a further
reduction in the operating temperature as both of these seem
promising in terms of delivering even higher resolution out of
the mixture. The separation with MTBE or isopropanol can be
adapted for use with a mass spectrometer as one of the detectors. In previous studies (see www.Agilent.com/chem ASMS
2009 for a poster on this subject) we have been able to demonstrate the capability of quickly and confidently identifying the
composition of many of the triglycerides found in this and
other samples. For optimum electrospray performance in the
non-aqueous, non-buffered environment it was useful to do
post UV detector addition of a mixture of methanol and water
with ammonium formate buffer to enhance ionization and to
ensure a consistent ability to preserve the molecular ion into
the mass spectrometer. It has been shown by McIntyre [4] that
the presence of ammonium formate in the mobile phase significantly improves the probability that a molecular ion will be
formed and preserved in the mass analyzer portion of a mass
spectrometer.
5990-4292EN
65
Conclusions
Using the Agilent 1290 Infinity LC System, we were able to
easily demonstrate UHPLC capabilities well within the
operating range of the instrument. The significantly
enhanced resolution afforded by long sub-2 micron particle
size columns in the sub-ambient column compartment
environment will contribute significantly to our understanding of the major and minor composition of this sample and
other similar materials. This should significantly enhance
the contribution of liquid chromatography to the understanding of seed oil composition, the role of triglycerides in
metabolism, and the area of lipidomics where great interest has been directed on the LC separation coupled to
time-of-flight high-resolution mass spectrometry (LC/TOF).
References
1. E. G. Perkins, "Analyses of Fats, Oils, and Derivatives,"
AOCS Press, 1993
2.
http://en.wikipedia.org/wiki/File:Fat_triglyceride_shorth
and_formula.PNG
3. (http://www.chem.agilent.com/en-US/products/instruments/lc/pages/gp60931.aspx)
4. D. McIntyre, “The Analysis of Triglycerides in Edible Oils
by APCI LC/MS”, May, 2000 Agilent Technologies
5990-4292EN
66
Increasing resolution and speed by
operating UHPLC columns up to
1200 bar
Technical Note
mAU
200
ZORBAX RRHD SB-C18, 2.1 x 100 mm, 1.8 µm
Maximum pressure = 595 bar
nc = 485
Rs: 1.37
150
100
50
0
0
2
4
6
mAU
200
8
10
12
14
16
18
min
ZORBAX RRHD SB-C18, 2.1 x 150 mm, 1.8 µm
Maximum pressure = 768 bar
nc = 589
Rs: 2.40
150
100
50
0
0
2
4
6
8
10
12
14
16
18
min
Abstract
LC columns with sub-2-µm particles have gained popularity because they deliver high
productivity and resolving power. In the past, some of the efficiency and throughput
benefits of these columns could not be realized because LCs and columns were limited to backpressures of 400 to 600 bar. That changed when Agilent ZORBAX Rapid
Resolution High Definition (RRHD) columns with 1.8 µm particles were designed to
take advantage of the unique 1200-bar pressure limit of the Agilent 1290 Infinity LC
System. Operation in this high-pressure domain allows chromatographers to achieve
even more speed and resolution for complex separations.
5990-4552EN
67
Introduction
particles are designed for optimal performance over the entire
operating range of this LC, and are the only commercially
available columns that can be used up to the 1200-bar limit.
This combination of the 1290 Infinity and ZORBAX RRHD
(1.8 µm) columns forms a total solution that provides new levels of LC performance and flexibility.
The productivity and resolution benefits of ultra high performance liquid chromatography (UHPLC) and columns with sub2-µm particles are well recognized in the scientific community. Shorter LC columns with 1.8 µm particles allow analysts to
achieve faster runtimes while maintaining column efficiency
and resolution. Longer columns with these small particles
enable additional resolution for complex mixtures. However,
smaller particles produce higher backpressure. When the
backpressure limit of the column or the LC is reached, it is
impossible to further increase the flow rate for a faster analysis, or to use a longer column to achieve greater resolution.
The ZORBAX RRHD (1.8 µm) columns exceed the capabilities
of the Agilent ZORBAX Rapid Resolution High Throughput
(RRHT) columns with 1.8 µm particles, which have a 600-bar
limit. New hardware and an improved packing process extend
the stability of the ZORBAX RRHD (1.8 µm) columns to higher
pressures. The ZORBAX RRHD (1.8 µm) columns are available
in short and long lengths, and can be used up to 1200 bar to
achieve the chromatographic definition needed to separate
complex mixtures completely and reliably.
The Agilent 1290 Infinity LC System uniquely addresses the
need for a more flexible LC that operates under higher backpressures up to 1200 bar. (See Figure 1.) Agilent ZORBAX
Rapid Resolution High Definition (RRHD) columns with 1.8 µm
bar
1200
Agilent 1290 Infinity
High resolution in the power range
High speed in the power range
1000
800
System D
600
Agilent RRLC
System C
400
200
System B
System A
Standard LC
0
0
1
2
3
4
5
mL/min
Figure 1
ZORBAX RRHD (1.8 µm) columns use the full pressure and flow range of the 1290 Infinity LC, for separations
with higher speed and definition.
5990-4552EN
68
Results and discussion
component mixture of antioxidants. This was a complex separation, but an analysis time of less than two minutes was
achieved by using a fast flow rate of 1.6 mL/min with an
Agilent ZORBAX RRHD Eclipse Plus C18 column, 2.1 x 50 mm,
1.8 µm. Note that a gradient can produce a pressure change
of more than 300 bar during an analysis, especially if it covers
a very wide range of organic mobile phase. The gradient program at this 1.6 mL/min flow rate generated a maximum pressure of 1070 bar – exceeding the limit on other UHPLC systems and columns. The extended pressure range of the 1290
Infinity LC with the ZORBAX RRHD (1.8 µm) columns enabled
this fast separation.
As the following examples show, the combination of these
columns with the 1290 Infinity LC System makes it possible to
increase analysis speed for complex samples, or to achieve
greater resolution in a limited amount of time.
Example 1: maximize speed with higher flow rates
Many labs need to decrease LC runtimes to increase sample
throughput. In the example shown in Figure 2, the goal was to
achieve maximum speed in a gradient separation of a 10-
mAU
THBP
600
ZORBAX RRHD Eclipse Plus C18, 2.1 x 50 mm, 1.8 µm
Maximum pressure = 1070 bar
Chromatographic conditions for Figure 2
500
Column
Mobile phase
400
300
Data courtesy of: Gerd Vanhoenacker
Research Institute for Chromatography
Kennedypark 26 8500 Kortrijk Belgium
PG
Flow rate
Gradient
Injection volume
Detector
OG
DG
200
NDGA
TBHQ
BHA
BHT
100
AP
Temperature
Sample
Ionox-100
ZORBAX RRHD Eclipse Plus C18,
2.1 x 50 mm, 1.8 µm
A = 0.02% H3PO4,
B = acetonitrile:methanol 3:1
1.6 mL/min
0 – 1.85 min 30 – 100% B
1 µL
Sig = 280/10 nm,
Ref = 400/50, 80 Hz
Sig = 255/10 nm,
Ref = 400/50, 80 Hz
45 °C
Antioxidant mixture, ~ 100 ppm
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
min
Figure 2
By operating at greater than 1000 bar, the ZORBAX RRHD (1.8 µm) column produced this gradient separation of 10 antioxidants in less than two minutes.
Agilent 1290 Infinity LC –
technology for more resolution,
speed and sensitivity
5990-4552EN
69
Example 2: maximize resolution with tandem
columns
a group of alkylphenones on a 150 mm ZORBAX RRHD
(1.8 µm) column versus two RRHD columns in series. In this
example, tandem columns with a total length of 250 mm
increased resolution by 36% and efficiency by the expected
60%. These columns in series generated very high resolution,
but required close to 1100 bar pressure for this gradient separation. This high pressure was well within the limit for the
Agilent LC and column.
Longer LC columns deliver more resolving power for mixtures
with a large number of components. While columns are available in standard lengths of 50, 100, and 150 mm, analysts may
need additional options to increase resolution. As this example shows, they can combine columns in series and achieve
even better separations. Figure 3 compares the separation of
ZORBAX RRHD SB-C18,1.8 µm
150 mm (Maximum pressure = 582 bar)
mAU
300
a3,2: 1.19
Rs3,2: 6.42
N6 : 31,400
Pw2: 0.016 min
Pw6: 0.029 min
250
200
150
100
50
Chromatographic conditions for Figure 3
0
0
0.5
1
mAU
300
1.5
2
2.5
3
3.5
a3,2: 1.20
Rs3,2: 8.76
N6 : 50,200
Pw2: 0.018 min
Pw6: 0.037 min
150 + 100 mm (Maximum pressure = 1095 bar)
1.
2.
3.
4.
5.
6.
250
200
150
100
50
min
Uracil
Acetophenone
Propiophenone
Butyrophenone
Valerophenone
Hexanophenone
Columns
Mobile phase
Flow rate
Temperature
Sample
Abbreviations
0
0
0.5
1
1.5
2
2.5
3
3.5
ZORBAX RRHD SB-C18, 1.8 µm
25:75 water:acetonitrile
0.5 mL/min
Ambient
Alkylphenones
a = separation factor
Rs = resolution
N = number of theoretical plates
Pw = peak width
min
Figure 3
Because they operate at pressures up to 1200 bar, ZORBAX RRHD (1.8 µm) columns can be used in tandem to maximize efficiency and resolution.
Example 3: increase peak capacity with longer
columns
the 150 mm column, and some minor components were completely resolved. This gradient separation used a maximum
pressure of almost 800 bar, representing a more typical
UHPLC separation, and the sample was well-resolved with the
ZORBAX RRHD (1.8 µm) column.
A third example (Figure 4) shows a separation of a complex
licorice root extract. As the column length increased, the peak
capacity increased from 486 on the 100 mm column to 589 on
ZORBAX RRHD SB-C18, 2.1 x 100 mm, 1.8 µm
Maximum pressure = 595 bar
nc = 485
mAU
Rs: 1.37
200
150
Chromatographic conditions for Figure 4
Method
100
Columns
Mobile phase
50
0
0
2
4
6
mAU
200
8
10
12
14
16
18
min
ZORBAX RRHD SB-C18, 2.1 x 150 mm, 1.8 µm
Maximum pressure = 768 bar
nc = 589
Rs: 2.40
150
100
50
0
0
2
4
6
8
10
12
14
16
18
min
Gradient
Flow rate
Detector
Temperature
Sample
Abbreviation
ZORBAX RRHD SB-C18, 2.1 mm, 1.8 µm
A = 0.1% formic acid
B = acetonitrile with 0.1% formic acid
10 – 100% B in 30 min
0.4 mL/min
UV, 280 nm
Ambient
Licorice root extract
Rs = resolution
nc = peak capacity
Figure 4
A longer 150 mm ZORBAX RRHD (1.8 µm) column enabled greater peak capacity and a better separation for this sample of licorice root.
5990-4552EN
70
Conclusion
Agilent ZORBAX Rapid Resolution High Definition (RRHD)
columns with 1.8 µm particles are designed and manufactured for reliable operation at 1200 bar – the highest pressure
in the industry. High-pressure operation enables use of longer
columns, tandem columns, and/or higher flow rates.
Chromatographers can use these columns over the full operating range of the Agilent 1290 Infinity LC System, providing
greater flexibility to achieve maximum speed and resolution
for complex samples.
5990-4552EN
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72
Metabolic stability study using
cassette analysis and polarity
switching in an ultra high performance liquid chromatography
(UHPLC)-triple quadrupole LC/MS
System
Application Note
Authors
Abstract
Anabel S. Fandiño, Edgar Naegele
This Application Note demonstrates the performance of the Agilent 1290 Infinity LC
Agilent Technologies, Inc.
System coupled to an Agilent 6460A Triple Quadrupole LC/MS for the evaluation of
Santa Clara, CA USA
metabolic stability using the cassette analysis approach combined with fast polarity
switching.
We present a UHPLC/MS/MS method that demonstrates:
• High throughput analysis (retention times less than 0.8 min at a mobile phase flow
rate of 1.5 mL/min and binary pump pressure 1,100 bar)
• High speed MS/MS analysis with cycle time as low as 114 ms to monitor
6 transitions and switch polarity (polarity switching time 30 ms).
• Good precision with area RSD [%] or relative area RSD [%] less than 10%, enabled
by collecting enough data points (at least 9 points) across the chromatographic
peaks (as narrow as 0.37 sec at half height).
• Good accuracy over the expected concentration range using either external or
internal standard calibration (within 90.7 – 107.1%)
• Comparable results (% parent drug remaining) when using polarity switching or
non-switched analysis
• Excellent chromatographic resolution to avoid overestimation in quantitation due
to in-source CID conversion of a metabolite into its parent drug
5990-4469EN
73
Introduction
In vitro ADME assays like metabolic
stability play an important role in the
early understanding of in vivo pharmacokinetic characteristics and help to
discard nondrug-like compounds that
would fail later stages of development.
Thus, fast screening methods are needed to screen the large collection of new
chemical entities that need characterization in the early stages of drug discovery. Typically, metabolic stability
assessment is performed using the cassette approach and fast LC/MS/MS
methods. In the cassette approach, a
cocktail of substrates ionizing either in
positive or in negative mode is created
post-incubation in order to reduce the
number of samples to analyze. Fast LC
methods, which use high mobile phase
flow rates and short sub-2 ìm columns,
allow reducing the analysis time and
therefore increasing sample throughput
[1, 2]. Detection is usually performed
using a triple quadrupole mass spectrometer working in MRM scan mode.
Cassettes are typically designed so that
substrates ionizing either in positive or
negative mode are pooled together for
LC/MS/MS analysis. The reason for
that is because MS/MS analysis may
show challenges with quantitative data
quality if the MS detector is not able to
acquire data fast enough in order to collect at least 9 to 10 data points across
the extremely narrow peaks generated
using fast chromatography.
Nevertheless, pooling of substrates ionizing in positive and negative mode
would present a pronounced flexibility
advantage.
This work describes the advantage of
combining not only cassette analysis
and fast LC but also using fast polarity
switching MS/MS analysis to increase
throughput and flexibility in a metabolic
stability assay, while maintaining good
precision and accuracy. Moreover, the
Agilent 1290 Infinity LC system flow
range of up to 2 mL/min at 1,200 bar
allowed excellent chromatographic resolution between critical peaks.
Experimental
Reagents and supplies Buspirone
hydrochloride and verapamil hydrochloride were purchased from SigmaAldrich (St. Louis, MO USA).
Dextromethorphan, diclofenac, and dextrorphan-d3 (ISTD) were purchased
from Cerilliant (Round Rock, TX USA).
Diclofenac-d4 (phenyl-d4) was purchased from CDN Isotopes (Quebec,
Canada). NADPH regeneration system,
solution A (beta-nicotinamide adenine
dinucleotide phosphate sodium salt
(NADP), glucose-6-phosphate and
MgCl2 in water) and solution B (glucose-6-phosphate dehydrogenase in
5 mM sodium citrate) as well as 0.5 M
potassium phosphate buffer, pH 7.4
were purchased from BD Biosciences
(Woburn, MA USA). Male Sprague
Dawley rat liver S9 homogenate was
purchased from In vitro Technologies
(Baltimore, MD). All other reagents and
organic solvents were of analytical
grade and from VWR (Darmstadt,
Germany).
Incubation of substrates with rat liver
S9 fractions The incubation mixtures for
phase I metabolism consisted of an
amount of S9 preparation equivalent to
0.3 mg protein, 1 ìM substrate (buspirone, verapamil, dextromethorphan or
diclofenac from a 100 ìM stock solution
in 70% water / 30% acetonitrile),
1.3 mM NADP, 3.3 mM glucose-6-phosphate, 3.3 mM magnesium chloride and
0.4 U/mL glucose-6-phosphate dehydrogenase in 0.1 M phosphate buffer
5990-4469EN
74
(pH 7.4) made up to a total volume of
300 µL. Incubation was carried out at
37°C. A cocktail of the 4 substrates was
created post-incubation to reduce analysis time. A 25 µL aliquot was taken at
0, 5, 10, 15, 25 and 35 min from each
incubate and the reaction was stopped
by adding 300 µL acetonitrile containing
the internal standards (dextrorphan-d3
and diclofenac-d4) followed by centrifugation for 10 min at 14,000 g. The
supernatant was evaporated to dryness
using a gentle stream of nitrogen and
reconstituted with water/acetonitrile
80/20 v/v containing 0.1% formic acid
for UHPLC/MS/MS analysis.
Equipment
• Agilent 1290 Infinity LC System comprising 1290 Infinity Binary Pump with
integrated degasser, 1290 High
Performance Autosampler with
Thermostat and 1290 Infinity
Thermostatted Column Compartment
• Agilent 6460A Triple Quadrupole
LC/MS System with Agilent Jet
Stream technology
• Agilent MassHunter Workstation software for instrument control, data
acquisition and data processing
• Agilent MassHunter Optimizer
Software
• Agilent Rapid Resolution High
Definition (RRHD) Zorbax SB-C18,
2.1 x 50 mm, 1.8 µm column
Agilent 1290 Infinity Methods
The mobile phase consisted of:
Solvent A: water with 0.1% formic acid
Solvent B: acetonitrile with 0.1% formic
acid
Injection volume: 1 µL
Compound
Verapamil
Buspirone
Diclofenac -d4
Diclofenac
Dextromethorphan
Dextrorphan -d3
ISTD
X
X
Precursor
Ion
455.3
386
298
294
272.2
261.2
MS1
Res
Unit
Unit
Unit
Unit
Unit
Unit
Product
Ion
165.1
122.1
254
250
215.2
157.1
Needle wash: 20 sec in flushport with
methanol/water 50/50 v/v (0.1%
formic acid)
Table 1
MRM acquisition with positive/negative switching.
UHPLC Method 1:
Compound
Column temperature: 25°C or 40°C
Verapamil
Buspirone
Diclofenac
Dextromethorphan
Dextrorphan -d3
Flow rate: 1.0 mL/min
Gradient: 25% B during 0.2 min, 80% B
at 1 min, 80% B at 1.25 min, 25% B at
1.26 min, stop time at 1.8 min.
UHPLC Method 2:
ISTD
X
Precursor
Ion
455.3
386
296
272.2
261.2
MS2
Res
Unit
Unit
Unit
Unit
Unit
Unit
Dwell time
[ms]
5
5
5
5
5
5
Frag V
[V]
185
185
80
95
190
125
CE
[V]
26
28
4
5
22
40
Polarity
Positive
Positive
Negative
Negative
Positive
Positive
MS1
Res
Unit
Unit
Unit
Unit
Unit
Product
Ion
165.1
122.1
250
215.2
157.1
MS2
Res
Unit
Unit
Unit
Unit
Unit
Dwell time
[ms]
22
22
22
22
22
Frag V
[V]
185
185
80
190
125
CE
[V]
26
28
8
22
40
Polarity
Precursor
Ion
455.3
MS1
Res
Unit
Product
Ion
165.1
MS2
Res
Unit
Dwell time
[ms]
22
Frag V
[V]
185
CE
[V]
26
Polarity
Negative
386
Unit
122.1
Unit
22
185
28
Negative
294
272.2
Unit
Unit
250
215.2
Unit
Unit
22
22
95
190
5
22
Negative
Negative
298
Unit
254
Unit
22
80
4
Negative
Positive
Positive
Positive
Positive
Positive
Table 2
MRM acquisition in positive polarity.
Column temperature: 60 °C
Flow rate: 1.5 mL/min
Compound
Gradient: 25% B during 0.2 min, 80% B
at 0.73 min, 80% B at 1.00 min, 25% B
at 1.01 min, stop time at 1.5 min.
Arbitrary
transition 1
Arbitrary
transition 2
Diclofenac
Arbitrary
transition 3
Diclofenac -d4
Agilent 6460A triple quadrupole
conditions
Scan type: MRM (MassHunter optimizer software allows to quickly determine
the optimal fragmentor voltage, MRM
transitions and collision energy for the
selected transitions. Optimized parameters are shown in Tables 1, 2 and 3)
ISTD
X
Table 3
MRM acquisition in negative polarity.
Polarity: positive/negative, positive
only or negative only
Parameters: drying gas temperature:
350°C, drying gas flow: 10 L/min,
sheath gas temperature: 400°C, sheath
gas flow: 12 L/min, nebulizer pressure:
35 psig, nozzle voltage: 0 V (+)
1000 V (-), capillary voltage:
4,000 V (+/-)
Polarity switching time: 30 ms
5990-4469EN
75
Results and Discussion
Speed and data quality using fast
polarity switching
Operating the UHPLC system at high flow
rates (1.0 mL/min or 1.5 mL/min) and pressures up to 1,100 bar enabled a run time of
only 1.5 min and generated peak widths
less than a second at half height (typically
0.4 to 1.0 sec). Sufficient data points across
the peak (> 9 points) could be collected due
to low MS cycle times, ensuring high precision quantitation (area RSD [%] and relative
area RSD [%] = area target / area internal
standard < 10). Figure 1 shows in comparison the MRM chromatograms, data quality
and analysis time achieved using flow rates
of 1.5 and 1.0 mL/min.
Good quantitation requires 9 – 10 data
points across a peak. As peaks get
narrower, the MS detector must be able to
Flow rate 1.5 mL/min, 1070 bar at 25%B, column: 60 °C
x105
8
+ MRM (455.3 -> 165.1)
Flow rate 1.0 mL/min, 850 bar at 25%B, column: 40°C
x105
Verapamil, 0.54 min
4
Avg W1/2 = 0.71 sec
15 points across W
Rel. Area RSD [%] = 2.9
Area RSD [%] = 6.9
2
+ MRM (386.0 -> 122.1)
3
2
Buspirone, 0.22 min
x105
Avg W1/2 = 0.70 sec
20 points across W
Rel. Area RSD [%] = 4.2
Area RSD [%] = 2.9
1.5
+ MRM (386.0 -> 122.1)
Buspirone, 0.40 min
Avg W1/2 = 1.3 sec
35 points across W
Rel. Area RSD [%] = 1.8
Area RSD [%] = 3.4
1
1
x104
Verapamil, 0.75 min
4
2
x105
+ MRM (455.3 -> 165.1)
6
Avg W1/2 = 0.44 sec
11 points across W
Rel. Area RSD [%] = 4.0
Area RSD [%] = 4.9
6
acquire faster. MS cycle times are reduced.
During each cycle, the MS system must
monitor 6 transitions and switch polarity.
This is enabled by 30 ms polarity switching
time and a total cycle time of only 114 ms
as shown in Figure 2 for the substrate
diclofenac. The graphic shows that even for
peaks exhibiting a width of 0.37 sec at half
height, the mass spectrometer was able to
collect a total of 9 points, ensuring good
area and relative area precision of 4.8 and
7.9%, respectively.
0.5
+ MRM (272.2 -> 215.2)
x104
Dextromethorphan, 0.30 min
Avg W 1/2 = 0.98 sec
28 points across W
Rel. Area RSD [%] = 2.2
Area RSD [%] = 6.2
2
1.5
1
+ MRM (272.2 -> 215.2)
Dextromethrophan, 0.53 min
Avg W1/2 = 1.3 sec
35 points across W
Rel. Area RSD [%] = 2.7
Area RSD [%] = 4.9
3
2
1
0.5
x103
4
3
2
-
MRM (294.0 -> 250.0)
x103
Analysis time < 0.8 min
Diclofenac, 0.73 min
-
MRM (294.0 -> 250.0)
Analysis time < 1.1 min
Diclofenac, 1.04 min
4
Avg W 1/2 = 0.98 sec
9 points across W
Rel. Area RSD [%] = 7.9
Area RSD [%] = 4.8
3
2
1
Avg W1/2 = 0.52 sec
11 points across W
Rel. Area RSD [%] = 6.5
Area RSD [%] = 10
1
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
Counts vs. Acquisition Time (min)
0.1
0.2
0.3
Figure 1
MRM chromatograms, data quality and analysis time achieved using flow rates of 1.5 and 1.0 mL/min.
5990-4469EN
76
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
Counts vs. Acquisition Time (min)
Evaluation of linearity, precision and
accuracy using fast polarity switching
x103
3.8
-MRM (294.0 -> 250.0)
3.4
Diclofenac, narrowest peak
3
Average W1/2 = 0.37 sec
2.6
2.2
9 points across W
Rel. Area RSD [%] = 7.9
Area RSD [%] = 4.8
1.8
1.4
1
0.6
0.2
0.7
0.71
0.72
0.73
0.74
W = 1.5 sec
0.75
0.76
0.77
Counts vs. Acquisition Time (min)
Cycle time = 114 ms
Figure 2
MRM chromatogram of diclofenac obtained using a flow rate of 1.5 mL/min. The graphic shows the
peak width at half height (0.37 sec) and the number of data points collected across the peak (9 data
points).
Type
r2
Accuracy range [%]
Area RSD [%]
Rel. area
Buspirone
external
isotope
calibration
dilution
Linear
Linear
0.9989
0.9994
97.4 – 102.0
94.4 – 107.1
2.4 – 6.1
n.a
1.7 – 4.9
Verapamil
external
isotope
calibration
dilution
Linear
Linear
0.9965
0.9989
90.7 – 105.1
96.2 – 103.2
2.3 – 6.9
n.a.
1.4 – 6.0
Linearity, precision and accuracy were tested
using standards at a flow rate of 1.0 mL/min
and column temperature 25°C (UHPLC
method 1). The % of remaining parent was
simulated over the expected range. The
results obtained from the linearity test using
either external or internal calibration demonstrate that the cassette analysis with polarity
switching approach can provide linear results
over the desired range. Moreover, good precision and accuracy values were achieved at
all levels as shown in Table 4. When using
internal standard calibration, a conflict
between 35Cl2-Diclofenac-d4 (m/z 298) and
37Cl -Diclofenac (m/z 298) was observed.
2
The response of the transition 298 & 254
arising from 37Cl2-Diclofenac contributes to
the response of the internal standard
35Cl -Diclofenac-d . This led to a quadratic
2
4
curve due to artificially “increasing” internal
standard. To solve this, either external calibration or a unique transition to diclofenac-d4
like 298 & 217 (CE =17) can be used.
Dextromethorphan
external
isotope
calibration
dilution
Linear
Linear
0.9978
0.9980
97.6 – 102.4
95.5 – 106.1
3.7 – 5.1
n.a.
2.6 – 5.9
Diclofenac
external
isotope
calibration
dilution
Linear
Quadratic
0.9990
0.9991
96.2 – 103.1
99,9 – 100.4
1.7 – 8.0
n.a.
0.9 – 4.1
Table 4
Calibration curve type, Correlation coefficient (r2), accuracy range [%], area RSD [%] and relative area RSD range [%] obtained using cassette analysis and
fast polarity switching.
References: n.a.: not applicable
5990-4469EN
77
Switched versus non-switched
analysis – Area response and
precision for pooled incubates
Average area responses obtained using
switched analysis (fast polarity switching)
or non-switched analysis (positive only or
chromatograms, average area response and
relative area RSD [%] obtained for the
pooled incubates analyzed using a flow rate
of 1.0 mL/min and column temperature
25°C (UHPLC method 1).
negative only analysis) were similar. Area
RSD and relative area RSD values were less
than 10.4% using polarity switching and
less than 8.6% using non-switched analysis.
Figure 3 shows in comparison the MRM
Flow rate 1.0 mL/min, 870 bar at 25%B, column: 25°C
x104
+ MRM (455.3 -> 165.1)
x104
Verapamil
6
4
Avg. Switched +/- to +
4
2
only response = 114%
2
6
x102
x102
+ MRM (386.0 -> 122.1)
6
Buspirone
6
4
Avg. Switched +/- to +
4
2
only response = 110%
2
x103
x103
5
4
+ MRM (272.2 -> 215.2)
4
Dextromethorphan
2
Avg. Switched +/- to +
3
2
only response = 109%
1
x103
3
2
1
x103
- MRM (294.0 -> 250.0)
0.3
+ MRM (272.2 -> 215.2)
- MRM (294.0 -> 250.0)
2
only response = 70%
0.2
+ MRM (386.0 -> 122.1)
4
3
Diclofenac
Avg. Switched +/- to +
0.1
+ MRM (455.3 -> 165.1)
1
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
Counts vs. Acquisition Time (min)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Switched Analysis
Non-Switched Analysis
6 MRM transitions (4 targets + 2 internal standards)
5 MRM transitions (4 targets + 1 internal standard)
Dwell time = 5 ms
Dwell time = 22 ms
Cycle time = 114 ms
Cycle time = 115 ms
Time [min]
0
5
10
15
25
35
Buspirone
Rel. Area RSD [%]
pos./neg. pos. only
2.7
0.5
5.2
3.3
7.2
4.9
4.0
3.9
3.2
4.3
9.2
3.8
Verapamil
Rel. Area RSD [%]
pos./neg. pos. only
8.7
1.0
8.1
2.8
6.5
5.5
4.8
2.0
5.6
1.7
5.4
5.8
Dextromethorphan
Rel. Area RSD [%]
pos./neg. pos. only
3.0
2.7
5.7
2.9
5.0
3.7
4.1
2.4
10.4
1.7
8.9
8.6
0.8
0.9
1
1.1
1.2
Counts vs. Acquisition Time (min)
Diclofenac
Rel. Area RSD [%]
Area RSD [%]
pos./neg. neg. only
pos./neg. neg. only
3.2
1.5
5.3
3.6
6.4
1.4
3.9
7.0
8.1
2.5
2.4
4.1
8.9
3.0
10.2
5.2
6.8
2.4
4.3
4.8
6.3
2.9
6.9
8.6
Figure 3
MRM chromatograms showing average area response obtained using fast polarity switching (on the left) in comparison to non-switched analysis (on the
right). The table at the bottom shows the precision obtained using fast polarity switching or non-switched analysis for all incubation times.
5990-4469EN
78
Switched versus non-switched
analysis – Metabolic stability
(% parent drug remaining)
the 5-, 10-, 15-, 25- and 35-min samples to
that in the 0-min sample and was calculated as follows:
The % parent drug remaining was determined by comparing the average relative
area of the parent compound measured in
% parent remaining = (avg. rel. area at tx /
avg. rel. area at t0) x 100
% Parent remaining
(Switched vs. nonswitched analysis
Buspirone
Linear Equation
y = 0.9911 x + 0.2671
r2
0.9999
Selected example:
% Verapamil remaining
Verapamil
y = 0.9593 x + 0.2026
0.9914
Fig. 4 shows the correlation of results
obtained for verapamil using fast polarity
switching and non-switched analysis. The
results of the switched and non-switched
analysis were comparable for all substrates
with r2 > 0.9848.
Dextromethorphan
y = 0.9430 x - 1.88
0.9848
Diclofenac
y = 1.1473 x - 15.16
0.9946
100
90
Non-switched analysis
80
t5
70
60
t10
50
y = 0.9593x + 0.2026
R2 = 0.9914
40
30
t15
20
t35
10
0
0
t25
10
20
30
40
50
60
70
80
90
100
Pos./Neg. switching analysis
Figure 4
Comparison of % parent drug remaining from fast polarity switching with non-switched analysis using UHPLC method 1 (flow rate = 1.0 mL/min,
column temperature = 25°C).
Chromatographic resolution –
Advantage for cassette analysis
+ MRM (402.0 -> 122.1)
Buspirone N-oxide (metabolite)
C21H31N5O3 (402 & 122.1)
2.5
O
N
N
N
2
N
O
N
1.5
In-source deoxygenation
decomposition
O
0.57 min
1
0.5
x105
3
+ MRM (386.0 -> 122.1)
0.46 min
Buspirone
incubated parent drug
N
O
2.5
N
N
C21H31N5O2
(386 & 122.1)
2
N
N
O
1.5
Buspirone (In-source
CID artifact build from
Buspirone N-oxide)
1
0.5
0.57 min
0
0.35
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
Counts vs. Acquisition Time (min)
Figure 5
Excellent chromatographic resolution achieved between the incubated parent drug buspirone and one
of its metabolites (buspirone N-oxide) which builds buspirone by in-source deoxygenation.
5990-4469EN
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Excellent resolution between critical
peaks was demonstrated. During the
determination of metabolic stability, it is
important to achieve high chromatographic resolution to ensure that the
metabolites produced by the hepatic S9
fraction are resolved from the parent
compounds. Coelution may result in
overestimation of the amount of parent
compound present if the metabolite is
thermally unstable and can convert into
the parent compound by in-source CID;
Fig. 5 shows as example the conversion
of buspirone N-oxide to buspirone (parent drug) by deoxygenation. Excellent
resolution between buspirone and buspirone N-oxide allowed to distinguish
between the parent drug buspirone
(originally present in the incubation
sample, retention time = 0.46 min) and
the artifactually build buspirone (build
by insource CID from buspirone Noxide, retention time = 0.56 min).
Conclusions
References
The combination of cassette analysis
and fast polarity switching in the
Agilent UHPLC/MS/MS system provided:
1.
Timothy J. Carlson and Michael B.
Fisher, “Recent Advances in High
Throughput Screening for ADME
Properties,” Combinatorial
Chemistry and High Throughput
Screening, Vol 11, No 3, March
2008, 258-264.
2.
Walter A. Korfmacher, “Principles
and applications of LC-MS in drug
discovery”, Drug Discovery Today,
Vol 10, No 20, October 2005, 13571367.
• Flexibility for cassette design:
Compounds ionizing in positive and
negative mode can be pooled
together.
• High throughput analysis (retention
times less than 0.8 min at a mobile
phase flow rate of 1.5 mL/min and
binary pump pressure of 1,100 bar)
• High speed MS/MS analysis with
cycle time as low as 114 ms to mon
itor 6 transitions and switch polarity
(polarity switching time 30 ms).
• Good precision with area RSD [%]
and relative area RSD [%] less than
10%, enabled by collecting enough
data points (at least 9 points) across
the chromatographic peaks (as nar
row as 0.37 sec at half height).
• Good accuracy over the expected
concentration range using either
external or internal standard calibra
tion (accuracy within 90.7 - 107.1%)
• Comparable results (% parent drug
remaining) when using polarity
switching or non-switched analysis
• Excellent chromatographic resolu
tion to avoid overestimation in quan
titation due to in-source CID conver
sion of a metabolite into its parent
drug
5990-4469EN
80
Extended ionization capability of
thermal gradient focusing ESI in
high-throughput in-vitro ADME
assays
Application Note
Drug Discovery
+ MRM (386.00000 -> 122.09961)
+ MRM (455.29999 -> 165.09961)
x10 4 1
Verapamil
6
C 27H 38N 2O 4
5.5
H3C
5 CH3
O
HCl
4.5 O
4
O
N
3.5 CH3
CH3
CH3
N H3C
3
2.5
2
Conventional ESI
1.5
Area RSD [%] = 7.2
1
0.5
x10 3
6.75 1
0.795
O
Agilent Jet Stream
Area RSD [%] = 4.9
Area: 12 x
S/N: 11 x
N
N
O
4.75
Agilent Jet Stream
3.75
Area RSD [%] = 3.7
2.75
Area: 23 x
S/N: 22 x
1.75
Conventional ESI
Area RSD [%] = 9.1
0.75
0
_
+ MRM (272.20001 -> 215.19922)
x10 4 1
1.3
Dextromethorphan
1.2
C18H 25NO
1.1
1
CH3
0.9
O
0.8
0.7
H
0.6
N CH3
0.5
0.4
0.3
0.2
0.1
x10 3
0.606
MRM (294.00000 -> 250.00000)
1
1.071
3.4
Diclofenac
C14H11Cl 2NO 2
3
Agilent Jet Stream
Area RSD [%] = 5.1
Area: 25 x
S/N: 12 x
2.6
Area RSD [%] = 3.6
1.8
Area: 6 x
S/N: 5 x
1
Conventional ESI
Area RSD [%] = 9.2
Agilent Jet Stream
2.2
1.4
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5
Counts vs. acquisition time (min)
Anabel S. Fandiño
N
N
C 21H 31N 5O 2
CH3
0
Author
N
O
Buspirone
5.75
0.6
Cl
HO
O
HN
Cl
Conventional ESI
Area RSD [%] = 13
0.2
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5
Counts vs. acquisition time (min)
Agilent Technologies, Inc.
5301 Stevens Creek Blvd.
Santa Clara, CA 95051
USA
Abstract
Several factors cause fast LC/MS/MS method development in the drug discovery
area to be an arduous task. Combination of ESI/APCI sources offers broad response
with multiple ionization modes, but optimization can be difficult and some sources
limit flow rates to 1 mL/min, while others compromise chromatographic performance. The scan speed of the mass spectrometer needs to be fast enough to
acquire an adequate number of data points across the narrow peaks generated
using sub-2 µm columns. At typical fast LC conditions, current HPLC systems (pressure limit ~ 400 bar) would yield back pressures greater than the threshold limit. In
this application example, we utilized the Agilent 1290 Infinity UHPLC system coupled with an Agilent 6460 Triple Quadrupole mass spectrometer comprising thermal
gradient focusing ESI (Agilent Jet Stream technology, AJS) to streamline highthroughput bioanalytical method development using in-vitro metabolic stability samples. Incubations of the substrates buspirone, verapamil, dextromethorphan or
diclofenac were carried out separately. An aliquot was taken at increasing time
points from each incubate and then pooled together for analysis. AJS technology
was compared to conventional orthogonal ESI using generic source values. The
Agilent 1290 Infinity LC Triple Quadrupole MS/MS system, which allows flow rates
up to 2 mL/min, pressures up to 1200 bar, dwell times as low as 1-2 ms, and polarity switching time of 30 ms, achieved an analysis time of less than 1.1 min without
sacrificing quantitative data quality. Due to the high data acquisition rate provided
by the Agilent 6460A Triple Quadrupole mass spectrometer, compounds ionizing in
positive and negative modes were analyzed in a single run. An adequate number of
data points (>10) could be collected across the extremely narrow peaks (Average
full width half maximum (FWHM) < 1.3 sec) generated by the Agilent 1290 Infinity
LC system. AJS showed enhanced area response and signal-to-noise in comparison
to conventional orthogonal ESI.
5990-4932EN
81
Instrument Conditions
Agilent 1290 infinity LC MS/MS system: Agilent 1290 Infinity UHPLC
System comprising binary pump/integrated degasser, high performance
autosampler with thermostat and thermostatted column compartment,
Agilent 6460A Triple Quadrupole LC/MS
with AJS or with conventional orthogonal ESI.
Conditions
Column:
Mobile phase:
Injection volume:
Method:
Column temperature:
Flow rate:
Gradient:
Scan type:
Polarity:
RRHD ZORBAX SB-C18, 2.1 mm × 50 mm, 1.8 µm
A= 0.1% FA in H2O, B= 0.1% FA in ACN
1 µL
25 °C
1.0 mL/min
25% to 80% B in 1 min, 1.25 min 80% B, 1.26 min 25% B, stop 1.8 min
MRM
Pos/Neg
Parameters
Drying gas temperature:
Drying gas flow:
Sheath gas:
Nebulizer:
Nozzle:
Capillary:
Dwell time:
350 °C (ESI / ESI + AJS)
10 L/min (ESI + AJS), 13 L/min (ESI)
400 °C and 12 L/min (ESI + AJS)
35 psig (ESI + AJS), 60 psig (ESI)
0 (+) 1500 V (–) (ESI + AJS)
3500 V (±) (ESI / ESI + AJS)
5 ms
Chromatograms
+ MRM (386.00000 -> 122.09961)
+ MRM (455.29999 -> 165.09961)
x10
4
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
x10 3
6.75 1
0.795
1 Verapamil
5.75
H3C
CH3
HCl
O
Agilent Jet Stream
O
N
CH3
CH3
N H3C
Area RSD [%] = 4.9
CH3
Area: 12 x
S/N: 11 x
N
N
C 21H 31N 5O 2
CH3
O
N
N
Buspirone
C 27H 38N 2O 4
O
N
O
O
4.75
Agilent Jet Stream
3.75
Area RSD [%] = 3.7
2.75
Area: 23 x
S/N: 22 x
1.75
Conventional ESI
Conventional ESI
Area RSD [%] = 7.2
Area RSD [%] = 9.1
0.75
0
0
_
+ MRM (272.20001 -> 215.19922)
x10 4 1
1.3
Dextromethorphan
1.2
C18H 25NO
1.1
1
CH3
0.9
O
0.8
0.7
H
0.6
N CH3
0.5
0.4
0.3
0.2
0.1
x10 3
0.606
MRM (294.00000 -> 250.00000)
1
1.071
3.4
Diclofenac
C14H11Cl 2NO 2
3
Agilent Jet Stream
Area RSD [%] = 5.1
Area: 25 x
S/N: 12 x
2.6
Area RSD [%] = 3.6
1.8
Area: 6 x
S/N: 5 x
1.4
1
Conventional ESI
Area RSD [%] = 9.2
Agilent Jet Stream
2.2
0.6
Cl
HO
O
HN
Cl
Conventional ESI
Area RSD [%] = 13
0.2
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5
Counts vs. acquisition time (min)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5
Counts vs. acquisition time (min)
Figure 1.
Overlaid MRM chromatograms obtained using AJS in comparison to conventional orthogonal ESI for the metabolic stability substrates after 35 minutes of
incubation with rat liver S9 fraction.
5990-4932EN
82
High-throughput bioanalytical
method development using
UHPLC/triple quadrupole mass
spectrometry
Application Note
Drug Discovery, Drug Development
Author
Anabel S. Fandiño
x10 1
8
7.8
7.6
7.4
7.2
7
6.8
6.6
6.4
6.2
6
5.8
5.6
5.4
5.2
5
4.8
4.6
4.4
4.2
1
1
B1) Agilent Jet Stream, 1.0 mL/min,
max pressure = 570 bar
Analysis time < 0.6 min
LLOQ in human plasma = 0.06 ng/mL
(19.3 fg on column)
Area RSD [%] = 8.5
Accuracy [%] = 88.4
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95
Counts vs. acquisition time (min)
1
Agilent Technologies, Inc.
5301 Stevens Creek Blvd.
Santa Clara, CA 95051
USA
Abstract
Several factors cause fast LC/MS/MS method development in the bioanalytical
area to be an arduous task. In order to maintain sensitivity while speeding up analysis time, target analytes should not elute in the chromatographic region affected by
ion suppression. The scan speed of the mass spectrometer must be fast enough to
acquire an adequate number of data points to define the narrow peaks generated
using sub-2 µm columns. At typical fast LC conditions, current HPLC systems (pressure limit ~ 400 bar (5800 psi)) would yield back pressures close to, or greater than
the threshold limit. In this application example, we utilized the Agilent 1290 Infinity
LC system coupled to an Agilent 6460 Triple Quadrupole mass spectrometer comprising thermal gradient focusing ESI (Agilent Jet Stream technology, AJS) to
streamline high-throughput bioanalytical method development using alprazolam
spiked in human plasma (concentration range: 2 nM to 5000 nM, corresponding to
0.06 ng/mL to 1544 ng/mL). A 100-µL sample of spiked plasma was precipitated
with three parts of ACN and centrifuged. A 200-µL amount of the supernatant was
diluted with three parts of H2O containing 0.1% formic acid (FA). Each sample was
injected three times. AJS technology was compared to conventional orthogonal ESI
using generic source values.
The Agilent 1290 Infinity LC Triple Quadrupole MS/MS system, which allows flow
rates up to 2 mL/min, pressures up to 1200 bar, and dwell times as low as 1-2 ms
achieved an analysis time of less than 0.5 min without sacrificing quantitative data
quality. The greater column efficiency of the Agilent rapid resolution high definition
columns (RRHD) resulted in narrow peaks, increased analyte peak height, excellent
resolution from matrix components, and improved analyte response (sensitivity).
5990-4933EN
83
Instrument Conditions
Conditions
Column:
Mobile phase:
Injection volume:
Method 1
Column temperature:
Flow rate:
Gradient:
Method 2
Column temperature:
Flow rate:
Gradient:
MS Scan type:
Polarity:
Parameters
Drying gas temperature:
Drying gas flow:
Sheath gas temperature:
Sheath gas flow:
Nebulizer pressure:
Nozzle:
Capillary:
Transition:
Fragmentor:
Dwell time:
Agilent 1290 Infinity LC MS/MS system: Agilent 1290 Infinity LC System
comprising binary pump with integrated
degasser, high performance autosampler with thermostat and thermostatted
column compartment, Agilent 6460A
Triple Quadrupole LC/MS with AJS
Technology or with conventional orthogonal ESI.
RRHD ZORBAX Eclipse Plus C18, 2.1 mm × 50 mm, 1.8 µm
A= 0.1% FA in H2O, B= 0.1% FA in ACN
5 µL
50 °C
1.0 mL/min
0 min 25% B, 0.8 min 90% B, 0.81 min 25% B, stop time 1.5 min
50 °C
1.2 mL/min
0 min 25% B, 0.67 min 90% B, 0.68 min 25% B, stop time 1.37 min.
MRM
Positive
350 °C (ESI and ESI + AJS)
10 L/min (ESI + AJS), 13 L/min (ESI)
400 °C (ESI + AJS)
12 L/min (ESI + AJS)
35 psig (ESI + AJS), 60 psig (ESI)
0 V (ESI + AJS)
3500 V (ESI and ESI + AJS)
309.2&281.1
145 V, CE: 24 V
50 ms
Chromatograms
Alprazolam (C17H13CIN 4) spiked in human plasma, RRHD ZORBAX Ecilpse Plus C18
Agilent 1290 Infinity LC
method 1: 1.0 mL/min
x10 2
3.8
3.6
A1) 1.5 ng/mL (482.5 fg on column)
1
1
3.4
Agilent Jet Stream
3.2
Area RSD [%] = 2.5
H3C
2.6
2.4
2.2
Area: 10 x
S/N: 7 x
N
N
N
3
2.8
Agilent 1290 Infinity LC
method 1: 1.0 mL/min
Cl
N
2
1.8
1.6
1.4
1.2
Conventional ESI
Area RSD [%] = 13.8
1
0.8
0.6
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95
Counts vs. acquisition time (min)
A1) Overlaid MRM chromatograms obtained using
AJS in comparison to conventional orthogonal ESI
for 1.5 ng/mL alprazolam spiked in human plasma.
B1) MRM chromatogram at the LLOQ for alprazolam
spiked in human plasma using AJS and flow rate =
1 mL/min (LLQQ = 0.06 ng/mL, 19.3 fg on column).
1
x10 1
8
7.8
7.6
7.4
7.2
7
6.8
6.6
6.4
6.2
6
5.8
5.6
5.4
5.2
5
4.8
4.6
4.4
4.2
1
1
B1) Agilent Jet Stream, 1.0 mL/min,
max pressure = 570 bar
Analysis time < 0.6 min
x10 1
7.4
7.2
7
6.8
LLOQ in human plasma = 0.06 ng/mL
(19.3 fg on column)
Area RSD [%] = 8.5
Accuracy [%] = 88.4
6.6
6.4
6.2
1
C1) Agilent Jet Stream, 1.2 mL/min,
max pressure = 690 bar
1
Analysis time < 0.5 min
LLOQ in human plasma = 0.06 ng/mL
(19.3 fg on column)
Area RSD [%] = 15.3
Accuracy [%] = 101.8
6
5.8
5.6
5.4
5.2
5
4.8
4.6
4.4
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95
Counts vs. acquisition time (min)
1
0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32 0.36 0.4 0.44 0.48 0.52 0.56 0.6 0.64 0.68 0.72 0.76 0.8 0.84
Counts vs. acquisition time (min)
B2) Agilent Jet Stream, 1.0 mL/min,
max pressure = 570 bar
C2) Agilent Jet Stream, 1.2 mL/min,
max pressure = 690 bar
Human Plasma
Human Plasma
Y = 28.1397 x + 4.6217
Y = 34.9686 x + 7.5606
Concentration range: 0.2 nM – 5000 nM
(0.06 ng/mL – 1544 ng/mL)
r2 = 0.9987, weight = 1/x
RSD [%] range: 0.7 - 15.3
Mean Accuracy [%] range: 80.0 – 115.2
Concentration range: 0.2 nM – 5000 nM
(0.06 ng/mL – 1544 ng/mL)
r2 = 0.9995, weight = 1/x
RSD [%] range: 0.3 – 13.6
Mean Accuracy [%] range: 88.4 – 113.6
B2) Calibration curve obtained using AJS. 4.5 orders
of linear dynamic range in human plasma was
demonstrated.
B3) Calibration curve obtained at flow rate
1.0 mL/min for alprazolam spiked pure solvent. The
amount on column for these samples corresponds to
the amount on column obtained for the spiked and
treated human plasma samples. The comparison
between the slopes of the calibration curves
obtained in spiked human plasma (slope = 34.97)
and in pure solutions (slope = 34.25) shows that the
slopes are practically the same, suggesting the
absence of any significant matrix effect on
quantification.
Agilent 1290 Infinity LC
method 2: 1.2 mL/min
-
–
C1) MRM chromatogram at the LLOQ for alprazolam
spiked in human plasma using AJS and flow rate =
1.2 mL/min (LLQQ = 0.06 ng/mL, 19.3 fg on column).
Increasing the flow rate resulted in a run time < 0.5 min
without sacrificing quantitative data quality.
B3) Agilent Jet Stream, 1.0 mL/min,
max pressure = 570 bar
Pure solvents
Y = 34.2455 x + 3.9033
[nM]
[nM]
Concentration range: 0.2 nM – 5000 nM
(0.015 ng/mL – 1544 ng/mL)
r2 = 0.9999, weight = 1/x
RSD [%] range: 0.3 – 19.4
Mean Accuracy [%] range: 98.1 – 108.3
[nM]
C2) Calibration curve obtained using AJS at flow rate
= 1.2 mL/min. 4.5 orders of linear dynamic range,
good accuracy and precision were demonstrated in
human plasma at 1.2 mL/min.
Figure 1.
A1) Overlaid MRM chromatograms obtained using AJS in comparison to conventional orthogonal ESI. B1) and C1) MRM chromatograms at the LLOQ
(0.06 ng/mL, 19.3 fg on column) using AJS and flow rates = 1.0 and 1.2 mL/min, respectively. B2) and C2): Calibration curves obtained using AJS at
1.0 mL/min and 1.2 mL/min, respectively. B3) Calibration curve of alprazolam in pure solvents obtained using AJS at 1.0 mL/min shows practically the
same slope in comparison to human plasma indicating the absence of any significant matrix effect.
5990-4933EN
84
Fast analysis of polyaromatic
hydrocarbons using the Agilent 1290
Infinity LC and Eclipse PAH columns
Application Note
Environmental
Author
mAU
2 mL/min, Pmax=350 bar
80
6
Gerd Vanhoenacker
60
5
40
Research Institute for Chromatography
10
Aceton
20
1
Kennedypark 26, B-8500 Kortrijk,
9
4
2
7 8
3
11
12 13
14
15
16
0
0
2
4
6
8
10
Time (min)
Belgium
mAU
4 mL/min, Pmax=700 bar
80
1
2
3
4
5
6
7
8
60
40
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
9
10
11
12
13
14
15
16
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Dibenzo(a,h)anthracene
Benzo(g,h,i)perylene
Indeno(1,2,3-cd)pyrene
4
5
20
0
0
1
2
3
Time (min)
Abstract
The Agilent 1290 Infinity LC System has a broader power range (the combination of
pressure and flow capabilities) than any other commercially available system. This
is extremely useful for method transfer from one (U)HPLC to the Agilent 1290
Infinity LC System and allows the analyst to develop methods that are impossible to
run on these other systems.
The flow and pressure capabilities are illustrated by a separation of 16 polyaromatic
hydrocarbons (PAHs) at high pressure and flow rate. At 2 mL/min, the analysis time
is approximately 11 min. Doubling the flow rate and gradient speed allows the sample to be analyzed in 5.5 min with a maximum pressure of 700 bar. The combination
of high flow (4 mL/min) and pressure is useful in this case to increase the sample
throughput. The separation of the PAHs is shown in Figure 1.
5990-4934EN
85
mAU
2 mL/min, Pmax=350 bar
80
6
60
5
40
10
Aceton
20
9
4
1
2
11
7 8
3
12 13
14
15
16
0
0
2
4
6
8
10
Time (min)
mAU
4 mL/min, Pmax=700 bar
80
1
2
3
4
5
6
7
8
60
40
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
9
10
11
12
13
14
15
16
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Dibenzo(a,h)anthracene
Benzo(g,h,i)perylene
Indeno(1,2,3-cd)pyrene
4
5
20
0
0
1
2
3
Time (min)
Figure 2.
Analysis of 16 PAHs on the 1290 Infinity LC. Sample: standard solution of 16 PAHs, 50 µg/mL each.
Configuration
• G4220A 1290 Infinity Binary Pump
with Integrated Vacuum
Degasser
• G4226A 1290 Infinity Autosampler
• G1316C 1290 Infinity Thermostatted
Column Compartment
Conditions
Column:
Mobile phase:
Flow rate and gradient:
ZORBAX Eclipse PAH 4.6 mm × 50 mm, 1.8 µm
A = water, B = acetonitrile
2 mL/min 0–0.33 min
40% B
0.33–10 min
40–100% B
4 mL/min 0–0.17 min
40% B
0.17–5 min
40–100% B
0.2 µL
Sig = 220/4 nm or 254/10 nm, Ref = off, 40 Hz
25 °C
Injection volume:
Detector:
Temperature:
• G4212A 1290 Infinity Diode Array
Detector
5990-4934EN
86
Fast analysis of fat soluble vitamins
using the Agilent 1290 Infinity LC
System and ZORBAX RRHT and
RRHD 1.8 µm columns
Application Note
Food Analysis
Author
Michael Woodman
Agilent Technologies, Inc.
Chemical Analysis Solutions
2850 Centerville Road
Wilmington, DE 19808
USA
Abstract
The Agilent 1290 Infinity LC has significant capabilities for a wide range of HPLC
and UHPLC applications. With a broader power range (that is, the combination of
pressure and flow capabilities) than any other commercially available system, and
the flexibility to operate a wide range of column dimensions and particle sizes, it is
extremely useful for method transfer from any HPLC or UHPLC to the 1290 Infinity
system. It allows the user to access capabilities not otherwise available.
Introduction
The speed and high resolution are demonstrated by a separation of fat-soluble vitamin isomers and esters, at a high pressure and flow rate. At 2 mL/min, utilizing a
simple 1-min gradient and a 3.0 x 50 mm, 1.8 µm column, the analysis time is only
3 min including the late eluting retinyl palmitate component. The separation of the
main components is shown in Figure 1.
5990-4882EN
87
The speed, resolution and flexibility of
the system are further demonstrated by
a separation of vitamins D2 and D3. At
2 mL/min, utilizing a simple isocratic
condition and a 3.0 mm × 150 mm,
1.8 µm column, the analysis time is only
3 min. The separation of the main components, at three column temperatures
including sub-ambient, is shown in
Figure 2. Sub-ambient column temperature control, a standard feature of the
Agilent Thermostatted Column
Compartment, has significant advantages for many difficult isomer separations, including enantiomeric separations, and for shape-selective separations such as polycyclic aromatic
hydrocarbons.
Configuration
4
1
x
3
4
5
6
7
8
5
1
Retinol (A-alcohol)
A-acetate
delta tocopherol
beta/gamma tocopherol
alpha-tocopherol
alpha-E-acetate
a tocotrienol
A-palmitate
3
x
6
7
Figure 1
Analysis of important vitamins A and E components on the 1290 Infinity LC. Sample: solution of alcohols and esters of retinol and tocopherol. Conditions: 2.0 mL/min, 90% to 100% ACN at 1 min, hold to 3,
run 4 min, ZORBAX RRHT StableBond C18, 3 mm × 50 mm, 1.8 µm, 45 °C.
• G4220A 1290 Infinity Binary Pump
with Integrated Vacuum
Degasser
• G4226A 1290 Infinity Autosampler
45 °C
• G1316C 1290 Infinity Thermostatted
Column Compartment
• G4212A 1290 Infinity Diode Array
Detector
30 °C
Conclusion
Taking advantage of the combined high
flow and high pressure capability of the
system allows one to use high efficiency 3-mm id columns (having up to 40%
higher efficiency than comparable
2.1-mm id columns) to produce rapid
separations with remarkable resolution
while conserving solvent over the use
of 4.6-mm id columns.
15 °C
Figure 2
Analysis of vitamins D2 and D3 (order of elution) on the 1290 Infinity LC. Sample: standard mix
(Sigma-Aldrich). Conditions: 2.0 mL/min, 75/25 ACN/MeOH isocratic, 280 nm UV ZORBAX RRHD
StableBond C18, 3 mm × 150 mm, 1.8 µm, 45 °C, 30 °C and 15 °C.
5990-4882EN
88
High resolution of complex lipids
(triglycerides) using the Agilent
1290 Infinity LC System and ZORBAX
RRHT and RRHD 1.8 µm columns
Application Note
Lipid Analysis
Author
Michael Woodman
Agilent Technologies, Inc.
Chemical Analysis Solutions
2850 Centerville Road
Wilmington, DE 19808
USA
Abstract
The Agilent 1290 Infinity LC has significant capabilities for a wide range of HPLC
and UHPLC applications. With a broader power range (that is, the combination of
pressure and flow capabilities) than any other commercially available system, and
the flexibility to operate a wide range of column dimensions and particle sizes, it is
extremely useful for method transfer from any HPLC or UHPLC to the 1290 Infinity
system. It allows the user to access capabilities not otherwise available.
Introduction
The typical HPLC resolution is shown by a separation of complex triglycerides in
vegetable oil. Using a 24-min gradient and a 3.0 mm × 150 mm, 1.8 µm column, the
analysis time of 35 min is typical; however, resolution is insufficient for good compositional investigation of the mixture. The separation of the main components is
shown in Figure 1.
5990-4881EN
89
The high resolution of the system is
further demonstrated by separation on
a much longer column, using more of
the power range of the system. At
0.29 mL/min, incorporating a shallow
gradient condition and an RRHD,
2.1 mm × 400 mm, 1.8 µm column, the
separation is dramatically improved.
The separation of the main components
is shown in Figure 2. Subambient column temperature control, a standard
feature of the Agilent Thermostatted
Column Compartment, has significant
advantages for many difficult isomer
separations, including enantiomeric
separations, and for shape-selective
separations such as polycyclic aromatic
hydrocarbons.
Configuration
• G4220A 1290 Infinity Binary Pump
with Integrated Vacuum
Degasser
Figure 1
Analysis of vegetable oil components on the 1290 Infinity LC. Sample: soybean oil, 10 mg/mL, 10 µg on
column. Conditions: 0.6 mL/min, 20% to 60% IPA vs. ACN at 24 min, hold to 30, run 35 min, ZORBAX
RRHT StableBond C18, 3 mm × 150 mm 1.8, µm, 30 °C. Maximum operating pressure, 370 bar.
• G4226A 1290 Infinity Autosampler
• G1316C 1290 Infinity Thermostatted
Column Compartment
• G4212A 1290 Infinity Diode Array
Detector
Conclusion
The high resolution and pressure capability of the system allows one to use
high efficiency 2.1-mm id columns, generating approximately 97,000 theoretical plates and having approximately
400% lower solvent consumption compared to 4.6-mm id columns. With nearly 3 times higher efficiency, run time
was increased by only about 80%. The
low flow rate and high resolution facilitate the interfacing of the separation to
high resolution TOF and QTOF mass
spectrometers to produce high confidence peak identification and
compositional information.
Figure 2
Analysis of soybean triglycerides on the 1290 Infinity LC. Sample: soybean oil, 10 mg/mL, 30 µg on
column. Conditions: 0.29 mL/min, 10% to 40% MTBE vs. ACN at 42 minutes, hold to 55 minutes, run
60 minutes, 210 nm UV. ZORBAX RRHD StableBond C18, 2.1 mm × 400 mm (2–150 and 1–100 mm
length in series), 1.8 µm, 20 °C. Operating pressure 730 bar.
5990-4881EN
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Analysis of impurities in fine chemical
octyl-dimethyl-4-aminobenzoate using
the Agilent 1290 Infinity LC System and
ZORBAX RRHT and RRHD 1.8 µm
columns
Application Note
Fine Chemicals
Author
Michael Woodman
Agilent Technologies, Inc.
Chemical Analysis Solutions
2850 Centerville Road
Wilmington, DE 19808
USA
Abstract
The Agilent 1290 Infinity LC has significant capabilities for a wide range of HPLC
and UHPLC applications. It exhibits a broader power range (that is, the combination
of pressure and flow capabilities) than any other commercially available system and
the flexibility to operate a wide range of column dimensions and particle sizes.
Additionally, advanced optical design in the diode array detector allows a wide
dynamic range and high sensitivity, both of which are critical in the monitoring of
small impurities in fine chemicals.
Introduction
The combined benefits are demonstrated by a separation of impurities found in a
sample of octyl-dimethyl-4-aminobenzoate (Figure 1). The high pressure capability
of the system allows the use of methanol, as well as acetonitrile, to explore the
selectivity of the two solvents. At 1.5 mL/min, using a simple 2-min gradient and a
3.0 mm × 50 mm 1.8 µm column, the analysis time is only 3 min. The separation of
the main components is shown in Figure 2.
5990-4880EN
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Figure 1
Structure of the cited compound.
The speed, resolution and flexibility of
the system are further demonstrated by
a separation of the sample using
methanol or acetonitrile with low solvent consumption 2.1-mm id, 1.8 µm
columns. The flow rate and gradient
conditions are optimized for each solvent, to produce a gradient separation
with maximum pressure of approximately 850 bar, a conservative setting
for the 1200-bar capability of the
Agilent 1290 Infinity LC. The separation
of the main components, with the two
organic solvents, is shown in Figure 3a
(acetonitrile, top panel) and 3b
(methanol, lower panel), where the
chromatograms are zoomed to the
region of peaks shown from approximately 1.2-2.5 min in Figure 2.
Figure 2
Initial separation conditions showing a need for greater resolution and selectivity. Sample: Octyl
dimethyl para-aminobenzoate, 1 mg/mL. Gradient: 1.5 mL/min, 40% to 90% ACN/water over 2 minutes.
Up to 460 bar on ZORBAX StableBond RRHT C18, 3 mm × 50 mm, 1.8 µm, 40 °C. 0.75 minute retention:
4-amino-benzoic acid; 2.1 minute retention: Octyl dimethyl para-aminobenzoate.
Configuration
• G4220A 1290 Infinity Binary Pump
with Integrated Vacuum
Degasser
• G4226A 1290 Infinity Autosampler
• G1316C 1290 Infinity Thermostatted
Column Compartment
• G4212A 1290 Infinity Diode Array
Detector
Conclusion
The combined high flow and high pressure capability of the system allows
one to use high efficiency columns,
producing rapid separations with
remarkable resolution while conserving
solvent over the use of 4.6-mm id
columns. Impurity detection, due to
high detector sensitivity and stability, is
estimated to be < 0.01%.
Figure 3
Results using ACN vs. MeOH with the same gradient slope on the 1290 Infinity LC. Sample: ODPABA
working standard, 1 mg/mL. Conditions: ACN gradient 0.6 mL/min, 40% to 90% ACN/water over
7.4 minutes. Up to 850 bar on ZORBAX StableBond RRHD C18 2.1 mm × 150 mm, 1.8 µm, 40 °C. Methanol
gradient 0.52 mL/min, 50% to 100% MeOH/water over 5.7 minutes. Up to 850 bar on ZORBAX
StableBond RRHD C18 2.1 mm × 100 mm 1.8 µm, 40 °C. The increased selectivity of methanol allowed a
shorter column to be used, decreasing run time and avoiding the use of more expensive acetonitrile
mobile phase.
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New dynamic MRM mode improves
data quality and triple quad quantification in complex analyses
Technical Overview
Authors
Abstract
Peter Stone, Thomas Glauner,
Multiple Reaction Monitoring (MRM) mode has become the preferred method for the
Frank Kuhlmann, Tim Schlabach and
quantitative analysis of known or target compounds using triple quadrupole mass
Ken Miller
spectrometry. The current solution for MRM analysis uses time segmentation, where
Agilent Technologies,
a method is divided into a series of time segments and predefined sets of MRM tran-
Santa Clara, CA, USA
sitions are monitored for each segment. As sample complexity increases (e.g. quantifying very low levels of hundreds of pesticide residues in a wide variety of food matrices), very real practical limitations in the time-segmentation methodology become
apparent. A better solution is required.
New dynamic MRM methods on the Agilent 6400 Series triple quad instruments create new capability to tackle large multi-analyte assays and to accurately quantify
exceedingly narrow peaks from fast Agilent 1200 Series RRLC and 1290 Infinity
UHPLC separations. Examples of pesticide analysis and rapid screening of drugs of
abuse are highlighted. Dynamic MRM methods yield equivalent, or better, quality data
and results as compared to traditional time segment based methods – plus easier
method development and modification.
5990-3595EN
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Introduction
Utilizing multiple reaction monitoring (MRM) with a triple
quadrupole tandem mass spectrometer enables extraordinary
sensitivity for multi-analyte quantitative assays. The first
quadrupole (Q1) selects and transmits a precursor ion with a
specific m/z. This ion is then fragmented in the second
quadrupole (Q2 collision cell), and a specific product ion with
a defined m/z is selected and transmitted in the third
quadrupole (Q3). See Figure 1. The combination of a specific
precursor mass and a unique product ion is generally an
unambiguous and sensitive method to selectively monitor and
quantify a compound of interest. Since two stages of mass
selection are utilized, MRM assays are particularly useful for
the specific analysis of target compounds in complex mixtures
and matrices. MRM mode has become the preferred method
for the quantitative analysis of known or target compounds.
Figure 1
A schematic diagram of MRM mode on a triple quadrupole instrument. The
precursor ion is selected in Q1, fragmentation occurs in Q2, and the product
ion is selected by Q3. Since two stages of mass selectivity are utilized, there
is very little interference from background matrix resulting in excellent
sensitivity.
narrow LC peak to allow for reliable quantitation. Both of
these factors can lead to compromises in data quality.
There is an additional challenge using time segments. In order
not to compromise any data, the change from one segment to
the next must occur during a time when no peaks are eluting
from the LC column. In complex analyses such as pesticide
analysis, where many co-eluting peaks are monitored at
almost every time point during the chromatogram, this can be
a formidable challenge as is highlighted in Figure 2.
Furthermore, there is always the risk that adding analytes to a
method may require complete redevelopment of a method to
introduce these chromatographically quiet zones where
segment changes can occur.
The Limitations of Time Segment Methods
The current solution to complex sample analysis is time segmentation. A method is developed with multiple predefined
time segments and the triple quad MS is programmed to perform MRM assays for only those analytes that elute during
each segment. Figure 2 shows an example of a method with
four time segments. One set of MRM transitions is analyzed
during segment 1, another set during segment 2, etc. The benefit of such a method is that, rather than performing MRM
scans for all analytes during the entire method, during any
given segment the triple quad only monitors MRM transitions
for the analytes that elute in that segment. The result is that
there are fewer MRM transitions during each MS scan, allowing the mass spec method to use a longer dwell time and/or
to reduce the overall cycle time for each MRM scan so that
there are more data points per peak.
However, there are some limits to what can be accomplished
with time segment methods. As the number of analytes in a
method increases, so too will the number of concurrent MRM
transitions in each segment. It will be necessary to either
reduce the dwell times for these transitions or to increase the
cycle time for each MS scan. Reducing dwell times (the
amount of time required for the triple quad to analyze a single
MRM transition) can compromise MS data integrity by introducing collision cell cross-talk (insufficient clearing of the collision cell between individual MRM experiments such that
some product ions from a previous MRM may be detected in
the subsequent MRM). Maintaining the same dwell time but
increasing the overall MS cycle time may mean that not
enough data points are collected during the elution of a very
Figure 2
Dividing the chromatogram into time segments. Detection of a complex
pesticide mixture demonstrates the advantages and some of the limitations
of time segment based MRM quantitation.
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Introducing Dynamic MRM Mode
This approach addresses the limitations of the time segment
methods for a large batch of compounds by replacing the
group segmentation with individual time windows for every
analyte transition and by
dramatically reducing, on average, the number of individual
MRM transitions that are monitored during each MS scan.
This approach is demonstrated in Figures 3-5.
Agilent’s new and unique analytical method approach is now
available on all 6400 Series Triple Quadrupole LC/MS systems. MassHunter acquisiton software allows the user to
choose conventional MRM or dynamic MRM mode. Ion transitions and a retention time window for each analyte are
stored in a method. MRM transition lists are then built
dynamically throughout an LC/MS run, based on the retention time window for each analyte. In this way, analytes are
only monitored while they are eluting from the LC and valuable MS duty cycle is not wasted by monitoring them when
they are not expected. An added benefit of this approach is
that MassHunter MS Optimizer software can readily determine and store optimal transition ions for each target analyte,
greatly simplifying dynamic MRM method set up.
Figure 3
Dynamic MRM method does not require time segments. Extracted ion
chromatogram of a 250 pesticide mix spiked into tap water (500 total
transitions, 2.5 pg on-column) using a dynamic MRM method run on a 1290
Infinity LC and a 6460 Triple Quadrupole LC/MS system with Agilent Jet
Stream technology.
Dynamic MRM removes the requirement to resolve compounds to baseline and to create well-defined segments in
the chromatogram where no compounds elute. This reduces
the potential method impact of adding analytes and of retention time shifts. The inevitability of multiple co-eluting peaks
is of lesser concern with dynamic MRM as long as the individual ion transitions are unique. Figure 5 shows an expanded
region of the analysis in Figure 3, with 22 compounds eluting
between 5.58 and 6.51 min with substantially overlapped
peaks. All analytes can be accurately quantified because their
ion transitions are mutually exclusive, allowing total exclusion
of background and interferences.
Figure 4
Dynamic MRM methods are based on individual retention time windows for
each MRM transition. 24 pesticide transitions from the analysis in Figure 3
are highlighted and their retention time windows are shown.
Note that, on average, the number of transitions which are monitored at
any point in the chromatogram is dramatically reduced relative to time
segment methods, allowing much faster MS scan cycle times. Also note
that this MS cycle time is held constant (60 ms in this case) in order to
assure the highest possible data quality and quantitative result.
5990-3595EN
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peak. In Figure 5 an example of an
ultrafast application is shown with a
peak width for the first peak of
184 msec. In this example, it is demonstrated that a data rate of 160 Hz is
needed to get the most out of a chromatogram in terms of resolution, peak
width and peak height. In Table 1, the
results are combined.
mAU
2.5 Hz
0
0
0.1
0.2
0.3
0.4
mAU
0.5
min
5 Hz
0
0
0.1
0.2
0.3
0.4
mAU
0
0.1
0.2
0.3
0.4
0.5
0
0.1
0.2
0.3
0.4
0.5
0
0.1
0.2
0.3
0.4
0.5
mAU
mAU
min
40 Hz
0
mAU
min
80 Hz
0
0
The limit of detection for Anthracene
was evaluated using the DAD at 2.5 Hz
and both available detector cells. The
injected concentration was as low as
10.5 pg/µL. In Figure 6, the overlaid
chromatograms of both cells are
shown. The results are combined in
Table 2. The limit of detection for a
signal-to-noise ratio of 2 is 87 fg for the
60-mm path length detector cell.
min
20 Hz
0
Limit of detection for
Anthracene
min
10 Hz
0
The chromatographic conditions combine an ultrafast gradient with high flow
rate and automatic delay volume reduction (ADVR) for the auto sampler.
0.5
0.1
0.2
0.3
0.4
mAU
0.5
min
160 Hz
0
0
0.1
0.2
0.3
0.4
0.5
min
Chromatographic method
Column:
Agilent ZORBAX RRHD Eclipse Plus C18, 50 mm × 2.1 mm, 1.8 µm
Sample:
Set of 9 compounds, 100 ng/uL each, dissolved in water/ACN
(65/35)
1. Acetanilide, 2. Acetophenone, 3. Propiophenone, 4. Butyrophenone
(200 ng/mL), 5. Benzophenone, 6. Valerophenone, 7. Hexanophenone,
8. Heptanophenone, 9. Octanophenone (p/n 5188-6529)
Injection volume:
1µl with Automatic Delay Volume Reduction (ADVR)
Column temperature: 60 °C
Mobile phases:
water(A) and Acetonitrile (B)
Gradient:
at 0 min 35% B, at 0.3 min 95% B
Flow:
1.5 mL/min
Stop time:
0.6 min
DAD:
2.5 up to 160 Hz, 245/10 nm, Ref 360/80
Figure 5
Influence of data rate on resolution and peak width.
Data rate
(Hz)
Resolution
peak 5
Peak width
last peak
(min)
Peak height
(mAU) of
peak 3
160
1.89
0.00307
1171.2
80
1.83
0.00323
1131.1
40
1.57
0.00381
1006.4
20
1.06
0.00565
738.6
10
0.56
0.0102
431.2
5
–
0.0203
217.1
2.5
–
–
–
Table 1
Influence of data rate on resolution, peak width and peak height.
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The Importance of Constant MS Scan
Cycle Time in Dynamic MRM Methods
Agilent’s dynamic MRM approach uses a constant sampling
time across chromatographic peaks. Even data point spacing
with adequate sampling across the peak provides the best
and most precise representation of the peak. To maintain a
constant cycle time, the individual MRM dwell time is also
adjusted to keep a constant sampling rate across all peaks,
even though the number of ion transitions being monitored
will change dynamically and may vary cycle to cycle, dependent on elution time and the number of concurrent analytes.
Because dynamic MRM yields generally fewer concurrent ion
transitions per unit time than traditional time segments, MS
cycle times can be reduced and individual transition dwell
times are typically longer than traditional time segmented
methods. While the Agilent 6460 and 6430 Triple Quadrupole
LC/MS systems are capable of 1 ms dwell times, this is typically only required in the most extreme assays. Note: with the
proprietary axial acceleration technology present on all
Agilent triple quadrupole and Accurate Mass Q-TOF collision
cells, all product ions are cleared from the collision cell in less
than 600 µs so that there is no MRM cross-talk with the
shortest dwell times (1).
Figure 5
Extracted ion chromatogram of 11 pesticides and 11 qualifier ions. In spite
of significant co-elution, well-chosen MRM transitions allow for accurate
quantification of all sample components.
Furthermore, by maintaining a constant dynamic MRM cycle
time, MS methods can be matched to analyte peak widths to
ensure that a statistically adequate number of data points is
acquired for each analyte to yield excellent analytical accuracy and precision. This approach yields uniform data points
across any given analyte peak and results in good peak symmetry — a distinct improvement over constant dwell time
approaches.
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Dynamic MRM Easily Accommodates Fast
UHPLC
HPLC or UHPLC separations with the Agilent 1200 RRLC or
1290 Infinity LC systems can reduce method times dramatically without sacrificing peak capacity or chromatographic resolution. Individual peak widths may be reduced to just a few
seconds. In the extreme, peak widths may be less than one
second wide. Dynamic MRM methods require on average,
fewer ion transitions to be monitored concurrently in a chromatogram. MS cycle times are much faster than with time
segment methods and allow collection of many data points
across narrow peaks, as is shown in Figure 6, for excellent
quantitative results.
Figure 6
Dynamic MRM allows accurate quantification of narrow LC peaks. A
pesticide analysis gave this 6-sec wide peak for atrazine (5pg on-column). A
dynamic MRM method allowed for collection of sufficient data points to
assure an excellent quantitative result. The MS scan cycle time was 350 ms
and remained constant across the peak. Quantitative precision showed a
peak area %RSD < 3.5 for this compound.
Linearity with dynamic MRM methods is at least as good as
traditional time segment approaches. Typically, linear correlation coefficients are excellent and assay linearity exceeds
three orders of dynamic range. Figure 7 shows a calibration
curve for the pesticide compound oxamyl, with excellent sensitivity, linearity, and dynamic range. Triplicate injections of a
25 pg sample on-column yielded a peak area %RSD of only
1.08.
Figure 7
Dynamic MRM methods provide excellent quantitative data. Linearity of
oxamyl from 0.1 pg to 100 pg on-column, R2 = 0.9992.
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Application of dynamic MRM
Rapid Screening for Drugs of Abuse
Pesticide Screening
A further example of dynamic MRM capability is the fast
screening for 100 drugs of abuse in oral fluids over a 5 minute
gradient — a typical work-place drug test.
A challenging real world application for dynamic MRM is the
quantitative analysis of a very complex sample run at ultra
high pressure using a high resolution column and a fast gradient. 300 pesticides with internal standards were run on a subtwo micron column with a 15 min gradient at pressures
exceeding 800 bar, or 11600 psi. A dynamic MRM method
with 600 transitions was created using
retention time windows of only 0.5 min. Results of this analysis are shown in Figure 8.
This is a particularly challenging analysis given the timescale
of the assay relative to the number of analytes in the target
screen. Further, since qualifier ions and quantifier ions were
necessary for confirmatory purposes, a total of 200 dynamic
MRM transitions were employed during the analysis, covering
classes of analytes such as opiates, amphetamines, cannabinoids and benzodiazepines, among others.
In this example, peak widths were approximately 2 sec. A
dynamic MRM method using retention time windows of only
12 sec was used. The maximum number of concurrent dynamic MRM transitions was never more than 52. The assay
showed excellent sensitivity, (LOD = 23 fg on-column) and linearity. External calibration linearity: (R2 = 0.9987) for one of
the spiked analytes (Prazepam) is described in Figures 11 and
12, respectively.
Comparison of dynamic MRM with conventional time segment methods reveals an excellent correlation. Eight pesticides were injected in 20 replicates at the 10 pg level and
both average area and relative standard deviation were calculated. As shown in Figure 9, the correlation in peak areas
derived with both the dynamic and time-based MRM methods
was outstanding with R2 = 0.99992. The peak area relative
standard deviations for the time segment based method was
less than 6% and less than 4% for the dynamic MRM method.
Figure 9
Comparing pesticide peak areas with dynamic MRM and time segment
based methods.
Figure 8
Dynamic MRM analysis allows quantification of 300 pesticides using
internal standards in a 15 min method. Data was generated with an Agilent
1200 Infinity LC and 6460 Triple Quadrupole LC/MS system with Agilent Jet
Stream technology.
5990-3595EN
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Figure 12
Linearity of Prazepam from 23 fg to 100 pg on-column, R2 = 0.9987.
Figure 10
Detection of 10 spiked benzodiazepine drugs in an oral fluid extract using a
dynamic MRM method with >200 MRM transitions and 12 sec retention time
windows. This study was performed on an Agilent 1290 Infinity LC and 6460
Triple Quadrupole LC/MS system with Agilent Jet Stream technology.
Compound
Clobazam
Clonazepam
Flunitrazepam
Flurazepam
Lorazepam
Lormetazepam
Midazolam
Oxazepam
Oxazolam
Prazepam
Temazepam
LOD (fg on-column)
126
91.5
47.5
43.0
186
45.9
1.2 pg
145
235
23.7
156
Figure 11
Limit of detection (LOD) of Prazepam
is 23.7 fg with a S/N of 17.3.
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Summary
References
New dynamic MRM methods on the Agilent 6400 Series triple
quad instruments create new capability to tackle large multianalyte assays and to accurately quantify exceedingly narrow
peaks from fast Agilent 1200 Series RRLC and 1290 Infinity
UHPLC separations. The number of MRM transitions is adjusted
dynamically throughout the LC run, selecting only transitions
with relevant retention time windows. This means that, on average, many fewer MRM transitions are monitored during a typical MS scan than would be the case with a time segment based
method – with the added benefit that dynamic MRM methods
are less demanding to develop and adapt.
(1) Agilent publication 5989-7408EN: Ion optics innovations
for increased sensitivity in hybrid MS systems
Fewer transitions allow methods with shorter MS scan cycle
times (more scans/second) and the ability to provide excellent
quantification of very narrow (even sub-second) RRLC and
UHPLC peaks. Importantly, this dramatically shortened MS scan
cycle time is kept constant so optimized sampling and consistent accurate quantitation is ensured (the same cannot be said
for methods that vary MS scan cycle time).
Practically, dynamic MRM methods can be used to accurately
quantify hundreds of individual analytes, plus their internal standards and qualifier ions, in a relatively short LC run. Compared
to benchmark time segment methods, dynamic MRM methods
achieve similar sensitivity, linear dynamic range, and
quantitative accuracy, with better precision.
Key Points
• Key enabling technology for fast, accurate LC/MS quantitation
of complex samples
• Matches performance of Agilent 6400 series triple quad with
separation power of 1200 Series RRLC and UHPLC with 1290
Infinity LC
• Many data points collected across very narrow peaks for
accurate LC/MS quantitation
• Constant MS scan cycle time ensures accurate quantitation
• Equivalent and better quality data and results than traditional
time segment based methods – plus easier method development and modification with MassHunter Optimizer software
• Up to 4,000 ion transitions per LC run
• Diverse applications: pesticide analysis, drug screening,
targeted protein quantitation
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102
Performance characteristics of the
Agilent 1290 Infinity Binary Pump
More resolution and speed for conventional,
superficially porous and sub-2-micron column
packing material
Technical Note
Introduction
The Agilent 1290 Infinity Binary Pump uses new technology to overcome the challenges of pumping LC solvents at ultrahigh pressure and high flow rates. This
includes heavy duty drive motors on the pistons; new material for the pistons themselves to withstand the workload and to actively transfer heat from the seals; micro
fluidic heat exchangers; and the Jet Weaver, a micro fluidic mixing device. The
pump can deliver flow in the range of 0.05 – 5 mL/min at pressures up to 1200 bar.
The Agilent 1290 Infinity Binary Pump contains two identical high pressure pumps
(1200 bar) driven by two independent high performance motors each; a two-channel
high efficiency solvent degasser and 2 × 2-channel inlet solvent selection valve,
automatic purge valve and low-volume mixing device, the Jet Weaver, integrated
into a single housing.
One of the main performance criteria of any LC system is precision of retention
times, which is influenced primarily by the pumping device. It is important that the
desired flow rate is delivered precisely and that for gradient operation the mobile
phases are mixed reliably and accurately over the complete gradient range. Because
of method transferability and predictability, it is also important that the flow rate
and solvent composition in the blending (binary) operation mode are generated
accurately. In this Technical Note, the precision of retention times for gradient and
isocratic applications is evaluated. Further, based on tracer experiments, it is
demonstrated that linear and stepwise gradients are delivered with excellent accuracy and precision.
5990-4536EN
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The new design
The new pump design offers:
• “Infinite” power range (up to
2 mL/min at 1200 bar and 5 mL/min
at 800 bar) enables unprecedentedspeed and resolution, as well as compatibility with methods developed on
other platforms
• Lowest delay volumes down to
<10 µL enable ultrafast gradients for
LC/MS and LC/UV applications
Innovative Jet Weaver mixer, based on
multilayer microfluidics technology,
combines highest mixing efficiency
with lowest delay volumes to virtually
eliminate detector noise
• Dual-core microprocessor-controlled
active damping compensates for solvent properties and provides real-time
flow optimization to ensure negligible
noise and highest precision
• Silicon carbide pistons with superior
thermal behavior provide higher seal
lifetime and instrument uptime
• Integrated, high efficiency degassing
offers fast changeover of solvents for
purging and priming the pump
In Figure 1 the pump design is shown
schematically. The degasser is now
integrated into the pump housing. 2 × 2
solvents can be attached and out of
these the binary gradient can be
selected.
Each pump channel is a dual piston inseries design using novel firmware control algorithms and innovative piston
material, silicon carbide, which efficiently removes frictional heat from the
piston seal. The connection between
the primary piston to the secondary piston has an integrated heat exchanger to
remove the heat generated during solvent compression. Each pump channel
has one passive inlet valve and one
Figure 1
Design of 1290 pump.
passive outlet valve on the primary
head. Each piston is independently and
precisely driven by a motor with 65000
steps per resolution providing volume
displacement resolution as good as
300 pL/step.
The movement of the pistons is under
intelligent control with a feedback loop
to ensure that active damping results in
a ripple-free flow. The piston drive
tunes itself for the compressibility characteristics of the solvent and the
hydraulic characteristics of the system
to maintain the ripple-free state. This in
conjunction with the smooth-motion
control, which reduces pressure pulsation caused by the movement of the
piston, combines with the efficient lowvolume mixer to ensure that pump
noise on UV traces is the lowest possible. A dedicated microprocessor in the
pump takes care of the smooth-motion
control and optimization of the pistons'
motion for real-time optimization based
on the static and dynamic parameters.
In addition to superior chromatographic
performance these features make the
pump very quiet in operation.
5990-4536EN
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For running salt or buffer-containing
eluents, the active seal wash option
can be used to extend the lifetime of
the pump seals.
The solvent selection valve allows the
choice of one of two solvents per channel. Binary gradients are created by
high-pressure mixing of solvents from
channel A and channel B. The mixing
point is located within the purge valve
thus further reducing the system gradient delay volume. The purge valve
allows software controlled flow switching to waste for purging the pump
channels.
The mixer design shown in Figure 2
combines highest mixing efficiency
with lowest delay volume. Nominal
volume mixers of V 35:< 40 µL and
V100: < 75 µL (for TFA applications) are
possible, just by rotating the mixer
assembly. The mixing device, known as
the Jet Weaver, employs a multilayer
micro fluidic design to ensure optimum
suppression of residual composition
disturbances. The Jet Weaver is currently available as two nominal
volumes: V 35:< 40 µL for normal UV
detection applications and V100:
< 75 µL for demanding situations such
as the use of TFA in UV detection. For
MS detection it might be possible to
work without the Jet Weaver only making use of natural mixing within the
system flow path. Typical applications
might be high throughput methods with
fast gradients, on high resolution
2.1-mm columns with moderate baseline noise and precision demands.
However, the performance specification
is only to be considered valid for a system with a Jet Weaver installed.
• Comparison of acetonitrile and
methanol as mobile phase
Step gradients and linear gradients
to evaluate delay volume, noise and
ripple, accuracy and precision
Equipment and material
The instrument used was an Agilent
1290 Infinity LC system, equipped with
the following modules:
• Agilent 1290 Infinity Binary Pump
with vacuum degasser
• Agilent 1290 Infinity high performance
pump
• Agilent 1290 Infinity TCC
• Agilent 1290 Infinity DAD SL for
160-Hz operation
• Agilent ZORBAX SB C-18 columns
with different internal diameters and
lengths, packed with 1.8-µm particles
To evaluate pump performance tracer
experiments are frequently used to
verify the system ripple at different gradient mixtures. The delay volume, and
the accuracy and precision of gradients
are also evaluated using step gradients.
Figure 3 shows a step gradient from 0
to 100% in 10% steps using the Agilent
1290 Infinity LC. As a tracer compound,
caffeine was selected. Acetone is not
ideal for testing step gradient performance because acetone is too easily
removed in the degasser at low flow
rates. For the 1290 Infinity step gradient
performance testing we recommend the
use of non-volatile compounds.
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_101.D)
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_102.D)
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_103.D)
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_104.D)
mAU
600
Figure 2
Design of Jet Weaver (mixer).
500
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_102.D)
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_101.D)
DAD1 A, Sig=273,4 Ref=380,100 (CHINA\STEP...NT200BAR\STEPGRADIENT 2009-07-08 10-13-19\STEPGRADIENT_103.D)
400
mAU
300
371.5
372
In the following experiments, the performance of this new design is demonstrated. The pump performance was
evaluated using the following:
• Step gradients and linear gradients
to evaluate delay volume, noise and
ripple, accuracy and precision
• Influence of Jet Weaver mixer
• Retention time (RT) precision at standard conditions for conventional gradient profiles
200
370.5
370
100
369.5
31
31.5
32
32.5
33
33.5
34
min
0
0
10
20
30
40
50
60 min
Chromatographic conditions:
Column substitute:
Mobile phases:
Flow rate:
Step gradients:
Restrictor temperature:
Detection:
• RT precision at ultrafast conditions
for gradient profiles
• RT precision for fast isocratic runs
60% step, overlay of 3 runs
371
Figure 3
Overlay of 3 step gradients.
5990-4536EN
105
Restriction capillaries providing a backpressure of 166 bar 1 mL/min for
the solvent used
A = water +20% isopropanol, B = water +20% isopropanol
+10 mg/l caffeine
1 mL/min
0% B to 100% B in 10 % steps, each step held for 5 min
36 °C
Signal 273 nm/4 nm bandwidth, reference 380/100 nm,
Slit 4 nm, 20 Hz
The performance results are:
• Composition Accuracy = ±0.35% or
better
• Precision from run to run = <0.1% relative standard deviation (RSD)
DAD1 A, Sig=265,10 Ref=360,100 (AE TRAINING\LINEARGRAD\LINGRAD10 2009-03-30 08-08-13\LIN_GRAD102.D)
DAD1 A, Sig=265,10 Ref=360,100 (AE TRAINING\LINEARGRAD\LINGRAD10 2009-03-30 08-08-13\LIN_GRAD103.D)
DAD1 A, Sig=265,10 Ref=360,100 (AE TRAINING\LINEARGRAD\LINGRAD10 2009-03-30 08-08-13\LIN_GRAD104.D)
mAU
800
600
• Mixing noise = 0.023% to 0.017% B
for critical steps like 10, 50 and 90%
step related to 100% step
• System delay volume = <140 µL
In Figure 4, the overlay of three consecutive linear gradients are shown.
Precision is also excellent here.
400
200
0
0
10
15
20
25
min
30
Chromatographic conditions:
Column:
Mobile phases:
Flow rate:
Linear gradients:
Temperature:
Detection:
Influence of 35-µL Jet Weaver
mixer
Using UV detection it is advisable to
use the V 35:< 40 µL mixer. If this is not
sufficient, the V100: < 75 µL mixer
should be used, especially if TFA is
used as modifier. In Figure 5, the example shows that the 35-µL mixer can mix
the mobile phases very efficiently.
5
Restriction capillaries
A = water, B = water +0.5% acetone
1 mL/min
0% B to 100% B in 30 min
36 °C
Signal 265/20 nm, reference 360/100 nm, Slit 4 nm, 20 Hz
Figure 4
Overlay of 3 linear gradients.
mAU
1
0.9
No mixer
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.02
Column:
Sample:
Mobile phase:
Flow rate:
Gradient ultrafast:
Oven temperature:
DAD:
Injection volume:
0.04
0.06
0.08
Figure 5
Comparison with and without mixer at 35/65% ACN+0.08%TFA/Water+0.1%TFA.
5990-4536EN
106
0.1
min
Agilent ZORBAX RRHD SB C18, 2.1 mm × 50 mm, 1.8 µm,
Set of 9 compounds, 100 ng/µL each, dissolved in water/ACN
(65/35)
1. acetanilide, 2. acetophenone, 3. propiophenone,
4. butyrophenone (200 ng/mL), 5. benzophenone,
6. valerophenone, 7. hexanophenone, 8. heptanophenone,
9. octanophenone (p/n 5188-6529)
water +0.1% TFA /acetonitrile +0.08% TFA with mixer and
without mixer
1 mL/min
35% to 95% B in 0.5 min
60 °C
254/5 nm, REF 400/80 nm, 80 Hz
0.5 µL, exterior needle wash 6 s
RT precision at standard conditions for conventional gradient
profiles
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4601.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4602.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4603.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4604.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4605.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4606.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4607.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4608.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4609.D)
DAD1 A, Sig=254,100 Ref=360,100 (AE TRAININ...C185UM\150X46SBC185UM 2009-04-01 08-03-33\SBC18_150X4610.D)
50
40
11.073
9.599
8.489
7.382
10
6.225
5.779
0.697
20
5.032
30
6.697
2.050
mAU
60
3.671
Retention time precision was tested
with gradient and isocratic conditions,
using standard bore and narrow bore
columns. The relative standard deviation for retention times for conventional
chromatography was evaluated using a
150 mm × 4.6 mm standard bore column. A "phenone mix" was analyzed
using gradient conditions from 30% to
75% in 8 min and a flow rate of 1.5
mL/min was applied. Figure 6 shows 10
overlaid runs, demonstrating the retention time precision obtained for this
application. The run time was 13 min.
The evaluation of the retention time
precision was found to be less than
0.04 % RSD.
0
0
2
4
6
8
10
12
Chromatographic conditions:
In Table 1 the precision data for conventional runs on 4.6-mm id columns are
combined.
Sample:
Set of 9 compounds, 100 ng/µL each, dissolved in
water/ACN (65/35)
1. acetanilide, 2. acetophenone, 3. propiophenone,
4. butyrophenone (200 ng/mL), 5. benzophenone,
6. valerophenone, 7. hexanophenone, 8. heptanophenone,
9. octanophenone
Column:
Agilent ZORBAX SB C18, 150 mm × 4.6 mm, 5 µm
Flow rate:
1.5 mL/min providing a backpressure of 159 bar
Mobile phase:
water/acetonitrile with mixer
Gradient conventional:
30% to 75% B in 8 min
Oven temperature:
30 °C
DAD:
254/10 nm, REF 360/100 nm, 20 Hz
Injection volume:
1 µL, exterior needle wash 6 s
Figure 6
Overlay of 10 chromatograms acquired on 4.6 mm id column.
Peak
RSD RT (%)
1
0.026
2
0.034
3
0.031
4
0.028
5
0.028
6
0.026
7
0.024
8
0.027
9
0.035
Table 1
Precision of retention times for conventional runs on 150 mm × 4.6 mm column.
5990-4536EN
107
min
RT precision at ultrafast conditions for gradient profiles
400
300
0.889
0.617
600
500
0.818
0.451
mAU
0.685
0.713
0.761
In Figure 7, an example of an ultrafast
separation with a gradient time of
0.5 min is presented. The flow was set
to 1.2 mL/min. All peaks elute within
1 min. Peak width at half height for the
first peak is as narrow as 0.334 sec and
for the last peak the peak width is only
0.543 sec. Even for this demanding
application, the precision of retention
times was <0.008% RSD except for the
first peak.
0.551
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST11.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST12.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST13.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST14.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST15.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST16.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST17.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST18.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST19.D)
DAD1 A, Sig=245,10 Ref=380,80 (1290DATAFI...XX\RRGRADIENTRTRSD\RRRSDRT2 2009-05-19 15-46-01\RR_UFAST20.D)
200
100
0
In Table 2, the precision data for ultrafast runs on 2.1-mm id columns are
combined.
0
0.2
Column:
Sample:
Mobile phase:
Gradient:
Flow rate:
Oven temperature:
DAD:
Injection volume:
0.4
0.6
0.8
1
Agilent ZORBAX RRHD SB C18, 2.1 mm × 50 mm, 1.8 µm,
Set of 9 compounds, 100 ng/µL each, dissolved in water/ACN
(65/35)
1. acetanilide, 2. acetophenone, 3. propiophenone,
4. butyrophenone (200 ng/mL), 5. benzophenone,
6. valerophenone, 7. hexanophenone, 8. heptanophenone,
9. octanophenone
water/acetonitrile with mixer
At 0 min 5% ACN, at 0.5 min 95% ACN
1.2 mL/min
40 °C
245/10 nm, REF 360/80 nm, 80 Hz
0.5 µL exterior needle wash 6 s
Figure 7
Overlay of 10 ultrafast runs with a gradient time of 0.5 min at 715 bar backpressure.
Peak
RSD RT over 10
consecutive runs
1
0.016
2
0.00769
3
0.00517
4
0.00575
5
0.00577
6
0.00538
7
0.00585
8
0.00571
9
0.00797
Table 2
Precision of retention times for ultra fast gradient runs.
5990-4536EN
108
min
RT precision for ultrafast isocratic runs at 670 bar backpressure
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 14-47-13\GRADIENT_PREC11.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC12.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC13.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC14.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC15.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC16.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC17.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC18.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 24-47-13\GRADIENT_PREC19.D)
DAD1 A, Sig=254,10 Ref=380,80 (ISOPRECISION\ISOGRADPREC 2009-06-08 14-47-13\GRADIENT_PREC20.D)
mAU
In Figure 8, an example for isocratic
conditions is shown. A narrow bore column was chosen and the flow rate was
set to 1 mL/min. The mobile phases
were blended in the pump, delivered by
the two pump channels and mixed in
the 35-µL mixer. The four peaks elute
within 45 sec. The standard deviation is
typically <70 msec for these peaks.
350
300
250
200
150
In Table 3, the results are combined.
100
50
0
0
0.25
Column:
Sample:
Mobile phase:
Flow rate:
Oven temperature:
DAD:
Injection volume:
0.5
0.75
min
Agilent ZORBAX RRHD SB C18, 2.1 mm × 50 mm, 1.8 µm,
1200 bar
Agilent isocratic test sample with dimethylphthalate,
diethylphthalate, biphenyl, o-terphenyl,
water/acetonitrile, dynamically blended with mixer = 20/80
1 mL/min
50 °C
254/10 nm, REF 360/80 nm, 80 Hz
0.5 µL, exterior needle wash 6 s
Figure 8
Precision of retention times on 2.1-mm column, isocratic elution, overlay of 10 runs, SD of RT less than
75 msec. 35 ?L Jet Weaver used for mixing. Backpressure was 670 bar.
Peak
MeanRT (sec)
SD (msec)
1
9.6
19
0.194
2
12.3
18
0.147
3
21.42
33
0.156
4
38.76
68
0.176
Table 3
Precision of retention times for fast isocratic runs.
5990-4536EN
109
RSD (%)
Comparison of methanol and
acetonitrile as mobile phase
Because of the recent economic situation, there has been a shortage of acetonitrile. Therefore, it is important that
the LC system can operate with
methanol instead of acetonitrile.
Methanol and its aqueous mixtures
have a higher viscosity than acetonitrile, resulting in higher back pressures
under the same chromatographic conditions. Methanol can be used with the
Agilent 1290 Infinity LC system without
exceeding the instrument pressure
limit. Figure 9 illustrates the influence
of methanol on backpressure and peak
elution.
Conclusion
The data presented in this note show
that the precision of retention time of
the Agilent 1290 Infinity LC system is
excellent for a wide range of LC applications using either narrow bore or
standard bore columns. The retention
time precision is typically less than
0.075% relative standard deviation for
conventional flow rates and ultrafast
applications with run times less than
1 min. Accuracy and precision of linear
and step gradients are excellent. Pump
ripple is typically less than 0.025%. The
total system delay volume is 105 µL
without a mixer and 140 µl with a mixer.
DAD1 B, DAD1A, DAD: Signal A, 254 nm/Bw:10 nm Ref 360 nm/Bw:100 nm (F:\ACNVSMEOH200001.D)
mAU
100
80
60
40
20
0
MeOH ( 794 bar)
0
1
2
3
4
5
6
7 min
6
7 min
DAD1 B, DAD1A, DAD: Signal A, 254 nm/Bw:10 nm Ref 360 nm/Bw:100 nm (F:\ACNVSMEOH100002.D)
mAU
100
80
60
40
20
0
ACN ( 555 bar)
0
1
Sample:
Column:
Mobile phases:
Gradient:
Flow rate:
Column temperature:
Detection:
Injection volume:
2
3
4
Gradient Evaluation mix, No.48271, sample contains: uracil,
phenol, methylparaben, ethylparaben, propylparaben,
butylparaben, heptylparaben, supplier is Sigma Aldrich
Agilent ZORBAX RRHD SB C18, 2.1 mm × 100 mm, 1.8 µm,
water/acetonitrile or methanol (B)
at 0 min 5% B, at 6 min 80% B, at 6.5 min 80% B, at 6.6 min 5% B,
at 8 min 5% B
0.5 mL/min
40 °C
254 nm, 20 Hz
1 µL
Figure 9
Comparison of acetonitrile and methanol as mobile phases.
5990-4536EN
110
5
Performance characteristics of the
Agilent 1290 Infinity Thermostatted
Column Compartment
New QuickChange valves, two heated zones
up to 100 °C, improved usability
Technical Note
Introduction
The Agilent 1290 Infinity Thermostatted Column Compartment (TCC) offers a number of performance and usability improvements:
• Valve heads can be easily changed by the user
• Operation up to 100 °C
• Two heated zones enable the operation of up to four 100-mm long columns at two
different temperatures
This Technical Note describes the design of the Agilent 1290 Infinity Thermostatted
Column Compartment.
5990-4538EN
111
The new design
The Agilent 1290 Thermostatted
Column Compartment offers a number
of significant improvements that
increase usability, flexibility and performance (Figure 1).
Usability was improved by implementing a new capillary guide to hold capillaries in position. The leak funnel was
redesigned from earlier models and
allows easier capillary installation on
optional valves
(Figure 2).
Figure 1
Optimized parts of the new Agilent 1290 Infinity TCC.
For improved data quality in routine
operations, a new door-open sensor
was introduced. This sensor prevents
analysis when the front door is not
closed properly, which would result in
non-comparable temperatures inside
the columns and variations in chromatographic results.
More flexibility is provided by longer
heat exchange carries, due to the
installation of 100-mm long columns in
one heated zone. New QuickChange
valves allow much faster and easier
maintenance operations. For example,
the change from a 1200 bar column
selection valve to a 2 position/10 port
valve can be accomplished in only a
few minutes, since valve heads are
exchanged from the front of the instrument without the need to take the
instrument stack apart, and disassemble the module. For easy installation of
capillaries attached to the valves, the
capillaries are mounted on pull-out rail
(Figure 2).
In addition, radio frequency identification (RFID) tags positioned at the rear
of the valves provide storage of valve
type, serial number, pressure range and
number of valve switches.
Different valve types can be installed:
• 2 position/10 port valve (1200 bar) for
automated column regeneration to
provide highest sample throughput
Figure 2
Ease of installation of capillaries and maintenance with new slide-out valve.
• 2 position/6 port valve (1200 bar) for
sample enrichment and sample stripping or dual column selection
• 8 position/9 port valves (400 or
1200 bar) for 8-fold column selection
used in method development, multimethod applications and walk-up LC
systems
The implementation of 8 position/9 port
valves in two clustered Agilent 1290
Infinity TCCs enables the most versatile
setup for method development and
multimethod applications. For more
details on this topic please refer to
Agilent publication number 59904095EN.
5990-4538EN
112
Equipment
The instrument used was an Agilent
1290 Infinity LC system, with the following modules:
• Agilent 1290 Infinity Binary Pump
with vacuum degasser
• Agilent 1290 Infinity Autosampler
• Agilent 1290 Infinity TCC
• Agilent 1290 Infinity DAD for 160-Hz
operation
Performance of the column
compartment
The temperature range of the Agilent
1290 Infinity TCC is 10 °C below ambient up to 100 °C. The temperature stability is ± 0.05 °C. The temperature
accuracy is ± 0.8 °C with calibration
± 0.5 °C and the heat-up/cool-down
time is 5 min from ambient to 40 °C,
and 10 min from 40 °C to 20 °C.
150
Peak 1
0.049%
Peak 2
0.029%
Peak 3
0.014%
Peak 4
0.011%
Peak 5
0.013%
Peak 6
0.015%
Table 1
Long term precision of retention times.
5.566
1.713
200
5.578
250
4.097
2.875
Norm.
3.515
2.228
Figure 3
Temperature compared between G1316C and G1312B, measured with water at different flow rates and
at 10 and 100 °C (100 °C is only specified up to 2.5 mL/min).
100
4.156
50
0
0
RSD RT over 80 runs
Temperature of solvent at column entry of G1316C and G1316B,
200 µL/min –5 mL/min of 100% water (max. thermal capacity)
0.236
In Figure 4, an example for the precision
of retention times overnight is given.
80 runs were done starting on the 24th
of March at 1:56 pm and ending with
the start of the last run on the 25th of
March at 2:38 am. It should be noted
that the air condition was switched off
at 8:00 pm. In Table 1 the results for the
relative standard deviation are combined. The cycle time, including run
time, was 9 min and 36 sec.
Conditions:
0.329
The equivalency of temperatures compared to legacy systems is important for
method transferability. Figure 3 shows
the comparison of the temperature of
pure water (highest thermal capacity)
directly at the column inlet of a new
Agilent 1290 Infinity TCC (G1316C) compared to an Agilent 1200 Series
Thermostatted Column Compartment SL
(G1316B). Setpoints are the lowest and
highest possible temperature settings
across the entire specified flow rate.
The difference of these two modules
even under these extreme conditions is
typically around or even below 0.5 °C.
1
2
3
4
5
6
7
8 min
Column:
Sample:
Agilent ZORBAX RRHD SB C18, 2.1 mm × 50 mm, 1.8 µm
Gradient Evaluation mix, No. 48271, sample contains: uracil,
phenol, methylparaben, ethylparaben, propylparaben, butylparaben,
heptylparaben, supplier is Sigma Aldrich
Mobile phase:
water (A) and acetonitrile (B)
Flow rate:
0.5 mL/min
Gradient:
At 0 min 5% B, at 5 min 60% B, at 6 min 80% B, at 7 min 5% B, at
8.5 min 5% B
Injection volume:
3 µL, draw speed 20 µL/min, exterior needle wash 6 s
Column temperature: 40 °C
DAD:
245/10 nm, Ref 360/100 nm, 20 Hz
Figure 4
First and 80th run from 1:56 pm to 2:38 am of the next day at 40 °C column temperature, blue is the first
injection, red is the 80th injection.
5990-4538EN
113
Conclusion
The Agilent 1290 Infinity Thermostatted
Column Compartment integrated into a
1290 Infinity LC System offers high performance combined with high flexibility.
Different valves can be integrated by
removing and installing different valve
heads. Columns up to 300 mm in length
can be installed. Two heated zones are
available and can be set at different
temperatures. The excellent temperature stability of the Agilent 1290 Infinity
TCCs enable highly precise retention
times during long sequences running
day and night, combined with an excellent comparability of thermal behavior
to legacy systems.
5990-4538EN
114
Performance characteristics of the
Agilent 1290 Infinity Diode Array
Detector
Low noise, low refractive index, high speed
and data security
Technical Note
Introduction
The Agilent 1290 Infinity Diode Array Detector (DAD) has a new optical design,
which uses a cartridge cell with optofluidic waveguide technology that offers high
sensitivity with low dispersion. It provides a wide linear range and a very stable
baseline for standard or ultrafast LC applications. The Agilent Max-Light cartridge
cell dramatically increases the light transmission by using the principle of total
internal reflection along a noncoated fused silica capillary, achieving a new level of
sensitivity without sacrificing resolution through cell volume dispersion effects. This
design minimizes baseline perturbations caused by refractive index variations within
the cell, either generated by gradient analysis, temperature variations or solvent
composition inhomogeneities. The stable baseline results in more reliable integration of peak areas.
In this Technical Note, the design is discussed along with the following performance data:
• ASTM drift and noise for the 10 and 60 mm path length cell
• Linearity over a wide range
• Limit of detection for anthracene for the 10 and 60 mm path length cell
• Influence of data rate on peak width, resolution and peak height
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The new design
The new design of the Agilent 1290
Infinity Diode Array Detector (Figure 1)
offers:
Lowest noise and highest linearity—
with 10 mm Agilent Max-Light cartridge
cell
• Ultra sensitivity—with unique 60 mm
Agilent Max-Light high sensitivity
cartridge cell
• More reliable and robust peak integration process due to less baseline
drift
• Multiple wavelength and full spectral
detection at a highest sampling rate
(160 Hz)—keeps pace with the analysis speed of ultra-fast LC
Figure 1
Design of the Agilent 1290 Infinity DAD.
• Programmable slit (1 to 8 nm)—provides optimum incident light conditions for rapid optimization of sensitivity, linearity and spectral resolution
• RFID tags for flow cell and lamps
ensure data traceability and usage
tracking
• Engineered for simplicity and ease of
use—cartridge design allows fast,
easy exchange of flow cell. Non-coated fused silica fibre cell optics for
robust performance and handling
The Agilent 1290 Infinity DAD incorporates electronic temperature control to
further enhance the resistance to temperature effects. Although the dispersion volume of the Agilent Max-Light
Cartridge Cell (Figure 2) is very small
(Vs = 1 µL), the path length is a standard 10 mm. However, for even higher
sensitivity the alternative Agilent MaxLight high sensitivity cell is available
with a path length of 60 mm (Vs =
4 µL). Cells are easily exchanged by
sliding them in or out of the cell holder
and they are auto-aligned in the optical
bench. The DAD light source is a deuterium lamp and the operating wavelength range covered is 190 to 640 nm.
Figure 2
Max-Light Cartridge Cell - Optofluidic waveguides.
This is detected by a diode array comprising 1024 diodes. The entrance to
the spectrograph is through a programmable optical slit which can give a
spectral resolution of 1 to 8 nm. This is
generally operated in the middle of the
range but can be closed down for optimization to 1 nm for high spectral resolution (rarely required in liquid phase
UV spectra). It can also be opened up to
8 nm for maximum light transmission
and minimum noise in the signal. The
chromatographic signals are extracted
from the diode array data within the
firmware of the module. Up to 8 individual signals can be defined, each comprising a signal wavelength (nm), a
diode bunching bandwidth, and if
required, a reference wavelength and
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bandwidth. Signals can be output up to
160 Hz (160 data points/second) for
accurate recording of the fastest (narrowest) chromatographic peaks. At the
same time the module can also output
full-range spectra to the data system at
the same rate of 160 Hz. For regulated
laboratories it is important that all the
method parameters are recorded. The
Agilent 1290 Infinity DAD not only
records the instrument setpoints but
also has radio-frequency identification
tags (RFID) incorporated into the lamp
and flowcell cartridge so that the identity and variables of these important
components are also recorded by the
system.
Equipment and material
The instrument used was an Agilent
1290 Infinity LC system, equipped with
the following modules:
• Agilent 1290 Infinity Binary Pump
with vacuum degasser
• Agilent 1290 Infininty Autosampler
• Agilent 1290 Infinity Thermostatted
Column Compartment
• Agilent 1290 Infinity Diode Array
Detector for 160-Hz operation
mAU
_0.02
_0.04
10 mm path length cell
_0.06
_0.08
_0.1
0
Baseline noise ASTM for the 10 mm
and 60 mm path length cell
The ASTM noise and drift was evaluated using a restriction capillary column,
with water as the mobile phase. The
detector was set to a 2-s response time.
The resulting ASTM noise for the
10-mm path length cell was found to be
Noise = ±2.8 µAU and the drift was
Drift = –0.04791 mAU/h. The resulting
ASTM noise for the 60-mm path length
cell was found to be Noise = ±0.6 µAU
and the drift was Drift = –0.249 mAU/h.
In Figure 3, an example chromatogram
is shown for the noise and drift behavior of both cells.
5
7.5
10
12.5
mAU
_
0.02
_0.04
15
17.5
20
22.5
min
Noise = 1.2e _3 = ±0.6 µAU
Drift = _0.249 mAU/h
_0.06
_0.08
_0.1
60 mm path length cell
0
Performance of diode array
detection
2.5
Noise = 5.6e _3 = ±2.8 µAU
Drift = _0.04791 mAU/h
2.5
5
7.5
10
12.5
15
17.5
20
22.5
min
Chromatographic Conditions for ASTM on the Agilent 1290 Infinity LC system
Column:
Restriction capillary 143 bar
Flow rate :
1 mL/min
Mobile phase:
Water
Detector:
254/4 nm, 360/100 nm, 2.5 Hz, time constant = 2 sec time, slit 4 nm
Column temperature: 36 °C
Process range:
5 to 25 min
Figure 3
Determination of ASTM noise and drift; Noise = ±2.8 µAU, Drift = –0.04791 mAU/h for the 10 mm path
length cell.
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Linearity for different caffeine
concentrations
Agilent 1290 Infinity DAD Linearity 2.58 to 2285 mAU
5.00E-02
Response factor (Amount/area)
The linearity was tested using certified
caffeine standards from 0.5 µg/mL to
500 µg/mL. For this concentration
range, very good linearity was obtained.
The coefficient of correlation was
0.99996. The response factors were all
within the 5% error range over an
absorbance range of 2.58 to 2285 mAU
(Figure 4).
5% error bars
4.80E-02
4.60E-02
4.40E-02
4.20E-02
Influence of data rate
To obtain optimum results the data rate
must be selected appropriately. If the
data rate is too low all gained resolution is lost in the detector. If data rate
is too high, the noise level may not be
appropriate for the peak width obtained
in the chromatogram and sensitivity is
lost because the signal-to-noise ratio is
decreasing. Therefore, the data rate
should be selected so that 15 to 30 data
points can be acquired over the fastest
4.00E-02
0.5
1
5
25
50
100
200
250
Amount (µg/mL)
Correlation coefficient= 0.99996
Chromatographic Method
Column:
Agilent ZORBAX RRHD SB C18, 50 mm × 2.1 mm, 1.8 µm
Sample Caffeine with 0.5,1,5,25,50,100,200,250,500 µg/mL
Injection volume:
4 µl
Column temperature: 30 °C
Mobile Phases:
water (A) and acetonitrile (B)
Flow:
0.5 mL/min, 92% water, 8% acetonitrile isocratic
Stop time:
2.5 min
DAD:
273/10 nm, Ref 380/80, 20 Hz
Figure 4
Linearity of DAD using response factors.
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500
mAU
0.175
1.339
Two cells are available for the Agilent
1290 Infinity DAD; one with the common path length of 10 mm and the second one with 60 mm path length for
highest LOD and impurity detection.
mAU
0.8
0.15
0.125
0.1
0.7
0.075
0.6
Because of the de facto standardization
on 10 mm path length in absorbance
spectroscopy regarding specific
absorption coefficients in literature data
as well as signals and spectra in
databases the absorbance values of
the 60-mm cell is also normalized on
10 mm. Hence the full length correct
unit is AU/cm, as it is for 10 mm cells.
0.05
0.5
0.025
0.4
0
_0.025
0.3
2.2 2.25 2.3 2.35 2.4 2.45 2.5 min
0.2
0.1
0
1 1.2 1.4 1.6 1.8
2 2.2 2.4 2.6 2.8 min
Chromatographic method
As a result, the output of the 60-mm
cell is divided by 6. This decreases the
noise level whereas the signal height
stays nearly the same. This is a benefit
when comparing data of different cell
lengths. The 60-mm path length cell
was found to be 4.7 times more sensitive than the 10-mm path length.
Column:
Agilent ZORBAX RRHD SB C18, 2.1 mm × 50 mm, 1.8 µm
Sample:
Anthracene 10.5 pg in 1 µL
Injection volume:
1 µL
Column temperature: 36 °C
Mobile phases:
Water and Acetonitrile
Isocratic:
Water/ACN = 35/65
Flow:
0.5 mL/min
Conclusion
Stop time:
3 min
The new design of the Agilent 1290
Infinity DAD offers lowest noise
<±3 µAU and small drift behavior
<0.5 mAU/h for the 10-mm path length
cell. The linear range typically goes up
to 2500 mAU. Data rates up to 160 Hz
enable excellent quantitation even for
peak widths <200 msec. The limit of
detection for anthracene was as low as
87 fg with a S/N ratio of 2 using the
60-mm path length cells. The new
design allows for an easy change of
detector cell. RFID tags in the lamp and
the flow cell ensure traceability of lamp
and cell usage.
DAD:
10 Hz, 251/4 nm, Ref 450/80
Figure 6
LOD of Anthracene comparing the 10 mm and the 60 mm path length cell, red is the 10 mm path length
cell and blue the 60 mm path length cell.
10 mm cell
60 mm cell
Noise PtoP (mAU)
0.01397
0.002297
Peak height (mAU)
0.714955
0.555970
S/N
51
242
LOD (FG)
412
87
Table 2
Results of LOD measurements, LOD for Anthracene = 87 fg with S/N =2 at 10 Hz
for the 60 mm path length cell and 412 fg for the 10 mm path length cell.
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www.agilent.com/chem/1290
© Agilent Technologies, Inc., 2009
October 1, 2009
Publication Number 5990-4694EN