Overcoming Triple Quadrupole Selectivity Challenges by Use of High-Resolution Accurate Mass Spectrometry to Enable Sensitive Quantitation of
a Small Molecule Therapeutic in Rabbit Plasma
Daniel Mulvana1, Robert Sturm1, Chet Bowen2, John Buckholz1, Raymond Biondolillo1, Kathlyn McCardle1, Chris Evans2, Barry Jones1
1Q2 Solutions, 19 Brown Road, Ithaca, NY, USA. 2GlaxoSmithKline, King of Prussia, PA, USA.
40
40
0
0
Stable-isotope
Labeled Internal
Standard (SIL IS)
NL: 5.58 × 104
Retention Time (RT): 3.07
Area: 92833
0.5
1.0
1.5
2.0
2.5
Time (min)
3.0
3.5
0
0
4.0
0.5
1.0
1.5
2.0
2.5
Time (min)
3.0
3.5
4.0
• High baseline noise from an isobaric interferent was observed in the analyte extractedion chromatogram (XIC) channel even when a resolving power of 140,000 was used.
• Negligible baseline noise was observed in the analyte channel when HRAMS selectivity
was applied to HCD generated product ion spectra at 17,500 resolving power.
• Note: RT differed from the final method because an Acquity UPLC HSS T3 1.8 µm,
2.1 x 50 mm column with an analytical flow rate of 500 µL/min was used.
Method Overview
20
0
2.0
2.2
2.4
2.6
2.8
3.0
3.2
Time (min)
3.4
3.6
3.8
4.0
• HRAMS using a Q Exactive was investigated as an alternative technique.
Initial efforts using MS1 multiplexed targeted selected ion monitoring (MSX)
were unsuccessful due to the isobaric interference, even at 140,000 resolving
power.
• Improved selectivity, and thereby sensitivity, was achieved using HCD to
enable MS/MS detection.
• Using HCD and 80 µL of rabbit plasma, a 10 pg/mL LLOQ was achieved.
Additional method refinement enabled a LLOQ of 2 pg/mL using 200 µL of
rabbit plasma.
Exact Mass
[M+H]+
GSKA
390.065 Da
m/z 391.073
SIL IS
397.088 Da
m/z 398.096
40
Chromatographic Conditions
LC System:
Dionex/UltiMate 3000 RSLC (Thermo Scientific)
Column:
XBridge BEH C18 (2.1 x 50 mm; 2.5 µm particle size;
Waters)
Column Temperature: 55 °C
Injection Volume: 30 µL
Mobile Phases*:
A: 1000:5 Water (MilliQ)/Ammonium Hydroxide
B: Methanol (NOWPak)
Flow Rate:
250 µL/min
Retention Time: 2.80 min
*Teflon mobile phase bottles were used in place of glass mobile phase bottles.
Time (min) Flow Rate (μL/min) Mobile Phase B (%)
Gradient:
• The triple quadrupole method was originally validated in rat plasma using a hydrophiliclipophilic-balanced (HLB) solid-phase extraction (LLOQ = 0.25 ng/mL). Background
interferences prevented reduction of the LLOQ to levels needed for rabbit plasma.
The method was modified to use double liquid-liquid extraction with MTBE to improve
recovery.
0.00
250
45
3.00
250
95
4.00
250
95
5.00
250
45
6.50
250
45
Mass Spectrometry Conditions
Scan Rate vs Chromatography
• Orbitrap transient times as a function of required resolving power are displayed below.
• The validated UHPLC triple quadrupole method exhibited a baseline peak width of ~3 sec.
Resolving
Power
at m/z 200
Approximate
Scan Speed
(Hz)
Approximate
Scan Time
(ms)
Transient
Length
(ms)
Suggested
Maximum Injection
Time (ms)
17,500
13
77
64
50
35,000
7
145
128
110
70,000
3
290
256
240
140,000
1.5
580
512
500
• The maximum injection time was matched to the orbitrap transient length to maximize
sensitivity and minimize duty cycle.
40
6. Repeat Steps 3 and 4 and transfer to same evaporation plate.
40
20
• The approximate scan time was matched with the best resolution for analysis based on
obtaining at least 10 scans across the chromatographic peak.
60
60
20
0
100
Q Exactive Transient Times for Various Resolution Settings
0
60
7. After evaporation, reconstitute in water/methanol.
• To facilitate adequate peak sampling on the Q Exactive, chromatography was modified to
generate a baseline peak width of ~6 sec for mid/high analyte concentrations by reducing
flow rate and changing column particle size from 1.7 to 2.5 µm.
20
5. Transfer 600 µL of the organic layer to a clean collection plate and evaporate.
Acquisition Mode
Targeted MSX (MS1)
Quadrupole Isolation Width
±0.5 m/z units
Automatic Gain Control Target
5 × 105
Maximum Injection Time
240 ms
Normalized Collision Energy
‒
Orbitrap Resolution
140,000
Accurate Mass Processing Analyte: m/z 391.073
(±3 mmu mass tolerance)
SIL IS: m/z 398.096
60
90
Method Development Challenges
80
Mobile Phase Considerations
70
• The validated rat plasma method used a basic mobile phase (0.1% ammonium
hydroxide/acetonitrile). Improved sensitivity was achieved using 0.5% ammonium
hydroxide/methanol.
• Sodium adducts were observed to contribute to reduced analyte response.
• Previous in-house data showed methanol solvent in glass reservoirs contains high
levels of sodium. We now routinely use mobile phase solvents shipped in NOWPak
(HDPE/PTFE lined).
• Additionally, use of Teflon mobile phase reservoirs reduces sodium adduction. These
are used for some sensitive methods in-house.
Mass Spectrometric Detection
• The Q Exactive enabled very high resolution mass detection, up to 140,000 resolving
power. This is usually sufficient to resolve most isobaric interferences seen under unit
mass resolution conditions.
• Targeted MSX was evaluated but was not found to sufficiently reduce background
interference using 3 millimass unit (mmu) processing.
• HCD (MS2) fragmentation provided added selectivity, enabling low detection limits.
LLOQ HCD Mass Spectrum
(Zoomed in)
NL: 1.37 × 104
159.117
SRM Transition
Interferent
158.976
20
0.5
20 psi
50%
370 °C
400 °C
60
Analyte
80
Analyte
NL: 1.59 × 105
RT: 4.08
Area: 371191
80
60
40
40
20
0
20
100
80
RT: 4.09
Area: 88801
SIL IS
NL: 4.13 × 104
10
80
60
60
40
40
20
20
3.6
3.7
3.9
3.8
4.0
4.1
Time (min)
4.2
4.3
4.4
Analyte channel
XIC: Interferent m/z 159.117 ± 0.003
NL: 4.71 × 104
IS channel
XIC: Interferent m/z 159.117 ± 0.003
NL: 2.73 × 105
60
398.23
[M+H]+
159.5
160.0
2.4
2.6
SIL IS
NL: 5.45 × 104
2.8
3.0
Time (min)
3.2
16
2.7
2.8
Time (min)
2.9
0
2.5
3.0
100
RT: 2.73
Area: 8858
2.6
2.7
2.8
Time (min)
2.9
3.0
60
60
40
40
422.18
30
3.6
3.7
3.9
3.8
4.0
4.1
Time (min)
4.3
4.2
4.5
4.4
398.24
[M+H]+
20
20
2.6
Analyte
NL: 6.07 × 106
2.7
2.8
Time (min)
2.9
3.0
SIL IS
NL: 3.88 × 105
392.20
100
RT: 2.73
Area: 10948947
80
60
60
40
40
20
20
100
400.20
60
50
40
[M+Na]+
390
395
408.38
406.42
393.46
400
405
409.44
413.43 415.32
410
m/z
415
427.44
420
420.24
402.30
424.43
425
4
0
200
10
392.43
390.39
0
390
408.40
419.95
406.37
412.46 415.33
397.33
395
422.34
400
405
m/z
410
415
420
2.6
2.7
2.8
Time (min)
2.9
3.0
• HRAMS enabled the analyte and SIL IS to be separated from the interferent ion
to improve selectivity and overall sensitivity.
• Sodium adduction was decreased by use of Teflon mobile phase reservoirs for
mobile phase solutions.
• The difference between the HCD product ion and interferent ion was only 0.141 Da.
• A 2-fold gain in sensitivity was observed in UHPLC runs on the Q Exactive.
• Unit resolution mass spectrometers were unable to resolve the interferent from
the analyte and SIL IS ions.
• Note: These data were collected on a TSQ Vantage MS equipped with the same
HESI-II source in Q1 only mode.
800
1000 1200 1400
Nominal Concentration (pg/mL)
1600
1800
2000
QC1
6.00
QC2
60.0
QC3
800
QC4
1600
Mean
2.29
6.41
62.8
799
1570
SD
0.178
0.325
1.88
26.3
61.5
CV (%)
7.8
5.1
3.0
3.3
3.9
RE (%)
14.5
6.8
4.7
-0.1
-1.9
18
18
18
18
18
• Quality control (QC) sample statistics indicate good accuracy and precision
from three method validation runs across the linear dynamic range of 2.00 to
1600 pg/mL.
2.6
SIL IS
NL: 3.62 × 105
0
2.5
100
Analyte
NL: 0
600
LLOQ QC
2.00
2.7
2.8
Time (min)
2.9
3.0
• The LLOQ in rabbit plasma was established at 2 pg/mL because adequate
precision was not demonstrated at 1 pg/mL (data not shown).
Inter-Lot Accuracy and Precision
GSKA Concentration (pg/mL)
2.6
RT: 2.74
Area: 651550
2.7
2.8
Time (min)
2.9
3.0
SIL IS
NL: 3.42 × 105
Plasma Lot
LLOQ
Replicate 1
2.00 pg/mL Replicate 2
Replicate 3
Mean
SD
CV (%)
RE (%)
n
Lot 1
2.01
2.06
1.79
1.95
0.144
7.4
-2.5
3
Lot 2
1.80
1.66
2.33
1.93
0.353
18.3
-3.5
3
Lot 3
2.11
1.86
2.22
2.06
0.184
8.9
3.0
3
Lot 4
1.94
1.46
2.13
1.84
0.345
18.8
-8.0
3
Lot 5
2.55
2.12
2.00
2.22
0.289
13.0
11.0
3
Lot 6a
1.65
1.69
1.57
1.64
0.0611
3.7
-18.0
3
ULOQ
Replicate 1
2000 pg/mL Replicate 2
Replicate 3
Mean
SD
CV (%)
RE (%)
n
2290
2290
2370
2320
46.2
2.0
16.0
3
1960
1930
1960
1950
17.3
0.9
-2.5
3
1930
1880
1900
1900
25.2
1.3
-5.0
3
1900
1970
1990
1950
47.3
2.4
-2.5
3
1930
1890
1900
1910
20.8
1.1
-4.5
3
1980
1970
1920
1960
32.1
1.6
-2.0
3
aPlasma
Inter-lot
1.94
0.283
14.6
-3.0
18
2000
151
7.6
0.0
18
lot with 2% hemolysis.
• Adequate inter-lot accuracy and precision and inter-lot selectivity were
observed when processing the HCD data with a ±0.003 Da XIC mass tolerance.
• QC sample statistics indicate good accuracy and precision from six individual
lots of rabbit plasma, including one lot with 2% hemolysis.
RT: 2.73
Area: 612583
• In addition, there were no chromatographic interferences observed among the
same six individual lots of rabbit plasma, including one lot with 2% hemolysis.
80
80
60
60
Conclusions
40
40
• Sensitivity was a challenge in the development of the GSKA method in
rabbit plasma due to isobaric interference and sodium adduction.
20
20
• Isobaric interference was not resolvable by MS1 high-resolution mass
spectrometry alone. HCD (MS2) fragmentation was required for selectivity
and sensitivity.
424.37
425
400
GSKA Concentration (pg/mL)
RT: 2.73
Area: 673149
Primary Carryover Blank
20
402.22
6
CV (%) = (SD/Mean) × 100
RE (%) = [(Mean − Nominal)/Nominal] × 100
0
2.5
80
0
2.5
NL: 3.52 × 106
30
20
8
Inter-Day Method Accuracy and Precision
Upper Limit of Quantitation (ULOQ; 2000 pg/mL)
70
40
10
n
100
100
400.26
[M+Na]+
12
Excellent linear dynamic range was observed with this method on the Q Exactive
with HCD.
80
80
420.22
14
2
90
50
0
Analyte
NL: 6.34 × 103
RT: 4.08
Area: 122596
Teflon Mobile Phase Bottle
NL: 2.63 × 106
18
LLOQ (2 pg/mL)
0
2.5
0
3.5
4.5
60
10
20
159.0
m/z
RT: 4.07
Area: 502609
• Note: RT was different than the final assay because a Waters XBridge BEH C18
2.5 µm, 2.1 x 50 mm column with an analytical flow rate of 100 µL/min was used.
392.41
40
158.5
Analyte
NL: 2.24 × 105
2.6
4.0
• A 1.5-fold gain in sensitivity was observed by increasing the ammonium
hydroxide concentration in Mobile Phase A.
70
0
0
3.5
RT: 2.73
Area: 634939
Regression Method = Linear; Weighting Factor = 1/x2
Response = Slope x Concentration + Intercept
Slope = 0.009153; Intercept = -0.006931; r-Squared = 0.9982
0
100
80
20
80
3.0
20
90
40
100
2.5
Time (min)
0.5% Ammonium Hydroxide in Water
100
60
0
3.5
RT: 2.82
Area: 705437
60
30
2.0
Standard Curve
0
Glass Bottles vs Teflon Bottles as Mobile Phase Reservoirs
0
20
80
Glass Mobile Phase Bottle
100
20
• Note: RT was different than the final assay because an Acquity UPLC HSS T3
1.8 µm, 2.1 x 50 mm column with an analytical flow rate of 500 µL/min was used.
100
SIL IS channel
XIC: m/z 158.976
± 0.003
NL: 3.88 × 105
1.5
RT: 2.81
Area: 7147
0
40
0
2.5
0
1.0
4.0
0.1% Ammonium Hydroxide in Water
20
80
3.5
3.0
40
100
40
100
2.5
2.0
Time (min)
1.5
1.0
SIL IS
NL: 6.40 × 104
60
• A 2-fold gain in sensitivity was observed by changing Mobile Phase B to 100%
methanol.
20
40
0
158.0
40
20
40
60
50
40
100
HCD (MS2)
±2.0 m/z units
1 × 106
50 ms
40%
17,500
Analyte: m/z 158.976
SIL IS: m/z 158.976
Analyte channel
XIC: m/z 158.976
± 0.003
NL: 6.08 × 103
80
100
60
0
0
RT: 1.75
Area: 160126
80
Mobile Phase: Increasing Percentage of Ammonium Hydroxide
in Mobile Phase A
Is the SRM Interference Observed and Resolved in the
Q Exactive HCD Data?
100
0
100
60
80
Instrument:
Q Exactive
Auxiliary Gas Pressure:
Source:
HESI-II
S-Lens Radio Frequency Level:
Polarity:Positive
Capillary Temperature:
Spray Voltage:
3.8 kV
Heater Temperature:
Sweep Cone Pressure: 0 psi
SIL IS
NL: 2.70 × 104
RT: 1.02
Area: 64550
80
20
60
SIL IS
NL: 3.70 × 105
Instrument Response
(Analyte/SIL IS Peak Area Ratio)
60
SIL IS
NL: 1.83 × 104
Analyte
NL: 2.69 × 104
RT: 1.75
Area: 63696
80
Relative Abundance (%)
80
60
20
RT: 1.01
Area: 45434
80
100
Analyte
NL: 0
80
Relative Abundance (%)
100
Analyte
NL: 1.28 × 104
RT: 1.02
Area: 34030
4. Mix on hematology mixer for 10 min.
Matrix Blank with Internal Standard
80
Relative Abundance (%)
0
100% Methanol
100
Relative Abundance (%)
80
20
90:10 Acetonitrile/Methanol
100
Relative Abundance (%)
SIL IS
NL: 8.60 × 103
3. Add 800 µL of MTBE to each well using the Microlab STAR (Hamilton Company).
Relative Abundance (%)
RT: 1.00
Area: 21675
40
Relative Abundance (%)
Relative Abundance (%)
100
2. Add plasma sample (200 µL) plus SIL IS to the 96-well plate.
Relative Abundance (%)
Analyte
Normalized Target
Level (NL): 1.27 × 104
40
Relative Abundance (%)
0
60
• At low analyte concentrations on the Q Exactive, the baseline peak width was
decreased due to limits of detection at the shoulders of the peak elution profile.
60
80
20
100
Mobile Phase: Changing Mobile Phase B to 100% Methanol
Relative Abundance (%)
Triple Quadrupole Selected Reaction Monitoring (SRM)
Transition Noise (1 pg/mL)
100
40
Method Development Changes to Improve Sensitivity
1. Wash a 2-mL round 96-well plate by adding 500 µL of MTBE to remove
possible contaminants. Dry plate.
Relative Abundance (%)
• Triple quadrupole rat and human plasma methods for the small molecule
GSKA were challenged by high chemical noise due to isobaric interference.
Efforts to generate cleaner extracts included double liquid-liquid extraction
with methyl tert-butyl ether (MTBE) and pre-rinsing of the 96-well plate with
extraction solvent.
80
High Baseline Noise
Sample Extraction
Introduction
100
80
Analyte
NL: 1.49 × 105
Relative Abundance (%)
Results:
• An LLOQ of 2 pg/mL using 200 µL of rabbit plasma was achieved.
• The method was validated to full regulatory compliance.
60
RT: 1.01
Area: 416902
Relative Abundance (%)
Method:
• GSKA was extracted from rabbit plasma using a double liquid-liquid extraction.
• Chromatographic separation was performed by ultra-high-performance liquid
chromatography (UHPLC).
• Heated electrospray ionization (HESI) was used for detection on a
Q Exactive (Thermo Scientific) mass spectrometer. Higher energy collisioninduced dissociation (HCD) was used to enable tandem mass spectrometric
(MS/MS) detection.
80
100
Analyte
NL: 6.33 × 104
RT: 1.00
Area: 153826
Sample Preparation
HCD (MS2) at 17,500 Resolving Power
MSX (MS1) at 140,000 Resolving Power
100
Relative Abundance (%)
Purpose:
• Validated triple quadrupole rat and human plasma methods were used as
starting points for the development of a rabbit plasma method for the small
molecule GSKA. The lower limit of quantitation (LLOQ) for the rat plasma
method was 250 pg/mL using 25 µL of plasma and the LLOQ for the human
plasma method was 10 pg/mL using 200 µL of plasma.
• A lower LLOQ (1 pg/mL) was desired for the rabbit plasma method;
however, interfering chemical noise due to isobaric interference hindered
this effort.
• High-resolution accurate mass spectrometry (HRAMS) was investigated
as an alternative technique to enable higher sensitivity by increased
selectivity.
Results
Method
Relative Abundance (%)
Targeted Multiplexed MSX (MS1) vs HCD (MS2) on the Q Exactive
(2.5 ng/mL)
Overview
0
2.5
2.6
2.7
2.8
Time (min)
2.9
3.0
0
2.5
2.6
2.7
2.8
Time (min)
2.9
3.0
Excellent selectivity and negligible carryover were observed with this method
on the Q Exactive with HCD.
• Sodium adduction was minimized by use of non-glass mobile phase
solvent reservoirs.
• The method for GSKA in rabbit plasma was successfully developed,
validated, and used in regulated preclinical sample analysis.
Acknowledgment: Suzanne Spencer for poster creation