Bioanalytical Applications of Liquid
Chromatography Mass Spectrometry
液相層析質譜術於生物分析之應用
Jenn-Feng Sheen
National Formosa University
Department of Biotechnology
May, 10, 2010, 雲科大
Bioanalytical Applications
 Drug Development
 Determination of drugs and metabolites in plasma or
other biofluids.
 Food Safety
 Melamine dosing, Pesticides residue, myotoxins,
additives.
 Life Science
Proteomics, metabolomics, polysaccharides
 Clinical Chemistry


Neonatal Screening, Therapeutic Drug
Monitoring, Occupational Biomonitoring
 Forensic Science
 Drug Abuse
Liquid Chromatography Mass
Spectrometry
 Characterization of organic compounds
(bimolecular or not) in complicate or relatively
simple matrices (samples, specimens).
 Qualitative and quantitative information are
both obtainable.
 It could be considered as a ultra sensitive and
specific probe for the nature.
Brief Introduction of LC-MS/MS
 A hyphened analytical system.
 LC separation + MS/MS identification.
 Suitable for wild range of compound-matrix
combinations analysis.
 Easy-to-use.
 General high sensitivity.
Liquid chromatography tandem mass spectrometry (LC–MS/MS), has led to major
breakthroughs in the field of quantitative bioanalysis since the1990s due to its inherent
specificity, sensitivity, and speed. It is now generally accepted as the preferred
technique for quantitating small molecule drugs, metabolites, and other xenobiotic
biomolecules in biological matrices (plasma, blood, serum, urine, and tissue).
API-MS Interface
 Electrospray Ionization, ESI
Preformed ion, charge residue
 Atmospheric Pressure Chemical Ionization, APCI
Heated pneumatic nebulizer
LC/MS interface
Heat
N2
N2
760 torr
gas
+
vaper
MH+
M
H3O+
H2O
H2O
H2O
Heat
Corona discharge needle
2-6 kV
Gas phase ion-molecular reaction , IMR
MS
Limitations of LC-MS/MS
 Major in the compatibility between LC and MS.
 Limited acceptable LC flow rate, ESI(< 200 uL/min),
APCI(<1 ml/min).
 Not allowed for nonvolatile Salts, e.g. phosphate,
borate.
 TFA suppresses the ES- mode.
 Ion competition in ESI (matrix effect).
 Limited buffer concentration, %Org/water, ion-pairing
or ion-exchange agents (ESI).
 Poor sensitivity for neutral compounds.
Mass Spectrometry Reviews, 2003, 22, 195– 214
Sample Preparation
Adequate sample preparation is a key aspect of quantitative
bioanalysis and can often be the bottlenecks during highthroughput analysis.
 Fail sample preparation can cause:
Interference
 Extraction efficiency variation
 Ionization suppression/enhancement

Dilute (DL) & Shoot
 For samples does not contain protein (e.g.
urine or bile).
 Sample firstly diluted with water or initial
mobile phase and then injected onto LC
column. Quick, but dirty.
 Poor robustness could be concerned.
 Variations in column performance and
ionization.
 Suitable for high concentration applications
which a extensive dilution can be applied.
Protein Precipitation (PPT)
 Samples contains proteins (e.g. plasma or
serum) are mixed with two times (or more)
volume of organic solvents (e.g. methanol or
acetonitrile).
 Vortex and centrifuge are needed.
 The supernatant is transferred for injection.
 Note that analyte may be lost due to poor
solubility.
 Be careful to matrix effect and system stability.
Liquid-Liquid Extraction (LLE)
 Applicable for samples with or without
proteins.
 Usually, large phase ratio between organic
solvent and sample is used to ensure a good
extraction efficiency.
 Nitrogen Drying is often applied.
 More polar solvents (e.g. ethyl acetate,
chloroform) give less clean extracts.
 Cost-effective but not environment-friendly.
Solid Phase Extraction (SPE)
 Applicable for samples with or without
proteins.
 Base on serious procedures including:

condition of the sorbent cartridge, loading of the sample
(preconditioned), wash with weak solution (low elution strength) and
elution of the analyte with strong solution.
 More clean sample solution is generally
resulted.
 Less matrix effect and system instability
problem.
 High cost and labor intensive.
On-Line SPE- direct sample (plasma) analysis
without sample manipulation and preparation.
R.N. Xu et al. / Journal of Pharmaceutical and Biomedical Analysis 44 (2007) 342–355
氟離子加成電灑法於中性氫氧基藥物之分析
• Neutrals exhibit unsatisfied response in ESI-MS.
• Chemical derivatization complicate the analytical process.
Anionic attachment-ESI
Neural hydroxyl drugs
Anionic Adduct Ions
NH4Br NH4Cl NH4F
5 ul/min 3 ppm 0.2 mM NH4F
MEPH-F-2-1 1 (0.022) Sm (Mn, 2x0.50)
氟離子加成之ESI-MS質譜
Scan ES3.13e7
201
100
[MF][M+FHF]-
%
221
202
0
150
mephenesin
m/z
160
170
180
190
200
210
220
230
240
250
260
270
5 ul/min 3 ppm 0.2 mM NH4F
GUAI-F-2-1 1 (0.022) Sm (Mn, 2x0.50)
100
[MF]-
Scan ES2.48e7
217
[M+FHF]-
%
guaifenesin
Mephenesin, MW=182.22
237
218
0
150
Simvastatin, MW=418.57
160
170
180
190
200
210
255
220
230
240
250
260
270
280
m/z
300
290
5 ul/min 3 ppm 0.2 mM NH4F
SV-F-2-1 1 (0.022) Sm (Mn, 2x0.50)
457
437
100
[MF]Guaifenesin, MW=198.22
Scan ES6.44e6
[M+FHF]-
simvastatin
%
458
438
399
493
463 469
0
350
360
370
380
390
400
410
420
430
440
450
460
470
480
490
500
510
520
530
540
m/z
550
5 ul/min 3 ppm 0.2 mM NH4F
PODO-F-2-1 1 (0.022) Sm (Mn, 2x0.50)
100
Inositol, MW=180.16
Podophyllotoxin,[M-H]
MW=414.41
[MF]-
[M+FHF]-
433
Scan ES8.78e6
[M+FHF][M+FHF]453
%
434 445
255
本研究所選擇中性氫氧基藥物之化學結構。
459
0
260
280
300
320
340
360
380
400
420
440
460
480
500
m/z
520
5 ul/min 3 ppm 0.2 mM NH4F
INOSITOL-F-2-1 1 (0.022) Sm (Mn, 2x0.50)
[MF]-
100
Scan ES1.41e7
199
[M+FHF]-
%
179
219
221
0
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
m/z
280
氟離子加成離子之子代離子質譜圖
5 ul/min 3 ppm 0.2 mM NH4F
MEPH-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50)
陰離子加成離子之氣相穩定性
Daughters of 201ES8.87e6
107
107
100
%
0
50
60
70
80
90
100
110
120
130
140
150
[M-H]-
181
mephenesin
160
170
180
190
200
m/z
220
210
5 ul/min 3 ppm 0.2 mM NH4F
GUAI-F-2--D-1 1 (0.022) Sm (Mn, 2x0.50)
[M-H]-
H+
X-
(proton bonded mixed dimmers of anions)
123
100
%
Daughters of 217ES9.20e6
123
guaifenesin
0
50
60
70
80
90
100
110
120
130
140
150
160
170
[M-H]-
197
180
190
200
210
220
230
240
m/z
250
5 ul/min 3 ppm 0.2 mM NH4F
SV-F-2-1-D-1 1 (0.022) Sm (Mn, 2x0.50)
Cai, Y.; Cole, R. B. Anal. Chem. 2002, 74, 985-991
%
115
399
[M-H]-
simvastatin
283
115
0
100
Daughters of 437ES1.10e6
399
100
417
m/z
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
5 ul/min 3 ppm 0.2 mM NH4F
PODO-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50)
383
100
413 [M-H]-
podophyllotoxin
%
0
250
Daughters of 433ES1.31e6
383
413
m/z
260
270
280
290
300
310
320
330
340
350
360
370
380
390
400
410
420
430
440
450
5 ul/min 3 ppm 0.2 mM NH4F
INOSITOL-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50)
100
179
0
50
[M-H]-
inositol
%
Daughters of 199ES5.03e6
179
199
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
m/z
230
氟離子加成法分析血漿中(a) mephenesin及
(b) guaifenesin 之質譜層析圖。
(a)
100
201>107 m/z
Blank plasma
0.5 ml plasma, liq-liq, postinfusion of 0.2 mM NH4F
%
79
100
%
201>107 m/z
0.05 ng/ml
48
100
%
201>107 m/z
5 ng/ml
0
1.00
2.00
3.00
4.00
Time
5.00
(b)
100
217>123 m/z
Blank plasma
%
72
100
217>123 m/z
0.05 ng/ml
%
65
100
%
217>123 m/z
5 ng/ml
0
Time
1.00
2.00
3.00
4.00
Hydrophilic Interaction Liquid
Chromatography (HILIC)
- It was introduced by Alpert (1990) and later used by Strege in tandem with
MS in drug research (1998).
-HILIC is similar to NPLC in that elution is promoted by the use of polar mobile phases, but is unique in
that the presence of water in the mobile phase is crucial for the establishment of a stagnant enriched
aqueous layer on the surface of the stationary phase into which analytes may selectively partition, as
described by Alpert.
HILIC-retention of small polar
compounds
1. Uracil
2. 5-fluorocytosine
3. cytosine
Monolithic Chromatography
-Bimodal Pore Structure
Onyx™ is a silica-based monolithic HPLC column. This technology creates
highly porous rods of silica with a revolutionary bimodal pore structure.
Mesoporous Structure
Creates large surface area
The mesopores form the fine
porous structure (130Å) of the
column interior and create a
very large surface area on
which adsorption of the target
compounds can occur. The
unique
combination
of
macropores and mesopores
enables
Onyx™
monolithic
HPLC columns to provide
excellent separations in a
fraction of the time compared to
a standard particulate column.
Macroporous Structure
Allows rapid flow (up to 9mL/min)
at low pressures
Each macropore is on average 2 μm
in diameter and together form a
dense network of pores through
which the mobile phase can rapidly
flow at low pressure dramatically
reducing separation time.
Excellent performance with minimal HPLC system stress
Turbulent Flow Chromatography
 Allows direct injection of biological samples
into an MS/MS system.
The large interstitial spaces between
the column particles and the high
linear mobile phase velocity creates
turbulence within the TurboFlow
column.
The turbulent flow of the mobile
phase quickly flushes the large
sample compounds through the
column to waste before they have
an opportunity to diffuse into the
particle pores.
http://www.cohesivetech.com/technologies/turboflow/index.asp
Mass Spectrometry Detection
 Which ion mode is good ?
ES+, ES-, AP+ and AP=>Base on your target structure
 Basic compounds => positive mode
 Acidic compounds => negative mode
 Neutral compounds => poor sensitivity
 High polar (ionic) => poor sensitivity
 Perfect structure =>surfactant-like

ESI concerns compound’s solution acidity/basicity (pKa)
APCI concerns it’s gas phase proton affinity (PA)


ESI usually is more sensitive than APCI
Compounds with electronegative aromaticity
and nitroaromaticity can perform radical ion
formation in AP- mode. (poor stability)
 MRM is always used in TSQ.
 Note that the molecular ion species may be
different in different mobile phase.
 Remind that flow rate, water content, buffer
concentration all have limits.
 The most important is matrix effect problem.
大氣壓下電子捕捉化學離子化法於酸性藥物PFB-衍生物之分析
Matrix Effect: APCI < ESI
Sensitivity: APCI- < ESI-
Deprotonation
R-COO-
R-COOH
[M - H ]-
Negative APCI
Negative ESI
Electron Capture
R-COO-PFB
Negative APCI
R-COO-
[M – PFB]-
Thioctic acid
MW = 206.23
Flufenamic acid
MW = 281.23
Estradiol
MW = 272.39
Quattri Ultima
三種具有酸性質子之西藥結構。
205
100
未衍生藥物及其PFB-衍生物
負離子APCI質譜圖
Thioctic acid-PFB
[M-181]-
(a)
MeOH/CAN/Water= 60/20/20, 0.5 ml/min
%
0
180
190
200
210
205
100
220
230
240
250
260
270
280
290
m/z
310
300
[M-H]-
(b)
[M-H+32]-
237
Thioctic acid-STD
%
207
0
180
190
200
210
220
230
240
250
260
270
280
290
m/z
310
300
280
100
(c)
Flufenamic acid-PFB
[M-181]-
%
80%MeOH, 0.5 ml/min
281
0
200
m/z
210
220
230
240
250
260
270
100
%
280
290
300
310
320
330
340
350
280
[M-H]-
(d)
Flufenamic acid-STD
281
0
200
m/z
210
220
230
240
250
260
100
270
280
303
(e)
300
330
340
350
Estradiol-PFB
90%MeOH, 0.3 ml/min
m/z
220
240
260
280
300
320
340
360
380
400
303
100
APCI parameters: Corona: 15 A, Cone Voltage: 30 V,
Sourec temp: 90 oC, Desolvation temp: 600 oC,
Nabulizer gas: Max, Desolvation Gas: 400 l/hr.
320
271
304
0
200
310
[M-181+32]-
[M-181]-
%
290
(f)
[M-H+32]-
[M-H]-
%
271
STD
Estradiol-STD
0
m/z
220
240
260
280
300
320
340
360
380
400
Thioctic Acid-PFB
100
AP-
未衍生藥物及其PFB-衍生物在
負離子APCI下之靈敏度比較
3.18
4.36
3.75
45.2 ppb
SIM (m/z 205)
%
Thioctic Acid-STD
0
Time
1.00
10-25 fold enhancement
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
Flufenamic acid-PFB
3.15
100
2.63
2.13
2.65 ppb
SIM (m/z 280)
%
Flufenamic Acid-STD
1.28
0
Time
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Estradiol-PFB
100
2.72
Mobile Phase: 80%CH3OH(aq), 1 ml/min.
Corona: 20 A, Probe Temp: 600 oC
5.9 ppb,
SIM (m/z 303)
2.16
1.68
%
Estradiol-STD
0
Time
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
Flunitrazepam在標準介面下之全掃瞄質譜圖
(a) ESI正離子全掃瞄質譜圖(4 kV, 400 oC)、
(b) ESI負離子全掃瞄質譜圖(-2.5 kV, 400 oC)、
(c) APCI正離子全掃瞄質譜圖(15 A, 500 oC)、
(d) APCI負離子全掃瞄質譜圖(15 µA, 500 oC)。
314.1
100
(a)
ES+
1.23e8
%
315.2
0
m/z
295
分析物 Flunitrazepam (5 g/ml)溶於
80%ACN(aq) 、注入流速為 40 µl/min。
[MH]+
300
305
310
315
320
325
330
335
100
ES3.86e7
(b)
%
0
290
m/z
295
300
305
100
310
[MH]+
(c)
315
320
325
330
314.1
335
340
AP+
1.27e8
%
315.2
Flunitrazepam
MW = 313.29
0
m/z
295
300
305
310
[M]
100
(d)
315
320
325
330
313.1
335
AP2.57e8
%
314.1
0
m/z
295
300
305
310
315
320
325
330
335
Matrix Effect
 Matrix effect is a phenomenon observed
when the signal of analyte can be either
suppressed or enhanced due to the coeluting components that originated from the
sample matrix.
 When a rather long isocratic or gradient
chromatographic program is used in the
quantitative assay, matrix effect may be not
present at the retention time for an analyte.
R.N. Xu et al. / Journal of Pharmaceutical and Biomedical Analysis 44 (2007) 342–355
Matrix Effect
 The difference in response between a neat
solution sample and the post-extraction
spiked sample is called the absolute matrix
effect.
 The difference in response between various
lots of post-extraction spiked samples is
called the relative matrix effect.
Matuszewski et al. [Anal. Chem. 2003, 75, 3019]
Matrix effect can be resulted from:
 Ionization reason





Endogenous compounds, e.g. lipids
Exogenous compounds, e.g. vial polymers
Anticoagulants, e.g. Li-heparin
Source design, e. g. Sciex, Waters, Thermal…
Ionization mode, e.g. ES vs AP
 Extraction efficiency reason

Sample lots, e.g. differ plasma bags, volunteers
Matrix Effect Probing
 For ion suppression/enhancement effect,


Compare ion signals of the analytes postspiked at mobile phase and sample extracts
solution.
Use post-column infusion method,

Let your target show off at the “matrix-free region”
 Samples Lots affect both on extraction and
Ionization.
Matrix Effect Probing
(Plasma Extracts)
Sample Loop (10 µL)
Valco T
LC Pump
API-MS
Syringe Pump
(Standard Solution)
血漿萃出物基質效應之實驗裝置。
Matrix Effect Probing
100
0.40
3.03
(a)
2.59
4.63
1.79 2.04 2.27
1.38
3.81
4.19
%
ES+
314>268
4.61e5
100
1.04 1.28
0.38
4.96
4.52
1.70 2.01
2.73
2.26
3.07
4.25
3.68 3.98
4.86
AP+
314>268
5.44e4
%
(a)
0
100
0
0.21
4.87
0.54
(b)
0.89
%
2.31
2.85
100
1.67
3.64
4.55
3.94
0
0.50
1.00
1.50
2.00
2.50
3.00
1.16
0.72
4.19
3.25
1.86
2.14
1.26
0.57
3.13
3.50
4.00
4.50
ES+
286>242
1.92e5
2.05
2.31
2.57
3.64 3.86 4.09
4.52
4.30
AP+
286>242
8.76e4
Time
%
(b)
Time
5.00
0
0.50
圖5.20人類血漿萃出物對Flunitrazepam及Nifuratel
在標準ESI介面下正離子訊號之影響。
(a) Flunitrazepam [MH]+、
(b) Nifuratel [MH]+ 。
血漿萃出物溶液共注入兩次(10 µl)， 注入時間約在
0.3-0.5 min 及 3-4 min。
4.96
3.08
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
圖5.21人類血漿萃出物對Flunitrazepam及Nifuratel
在標準APCI介面下正離子訊號之影響。
(a) Flunitrazepam [MH]+、
(b) Nafuratel [MH]+ 。
血漿萃出物溶液共注入兩次(10 µl)， 注入時間約在
0.3-0.5 min 及 3-4 min。
非揮發性鹽類的離子抑制效應
2, 4-D in ESI100
SIR
m/z 221
1.11e7
%
( Infusion of test compounds )
Syringe pump
0
Waters
616
LC
pump
100
API/MS/MS
SIR
m/z 219
1.88e7
%
Valco T
Rheodyne 7010
Sample injection valve
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Time
10.00
( Injection of non-volatile buffer )
2, 4-D-PFB in ECAPCI
100
10 mM Na2HPO4 in 70% ACN(aq), 10 uL inj
SIR
m/z 221
2.37e7
%
0
100
SIR
m/z 219
3.59e7
%
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Time
9.00
Sample Lots Effect
 Compare at least five different lots
Overcome the Matrix Effect
 Normalize the biological sample, e.g. add
buffer solution.
 Change extraction solvent.
 Let targets separated from the “matrixaffected” region.
 Solid Phase Extraction (or even a complicate
protocol).
 Change Ion Mode, ES+/ES-/AP+/AP-.
 Use the gradient elution.
 Stable Isotope Internal Standard.
Determination of Unknown Leads in
Mouse Plasma by LC-MS/MS
Usually, quite limited sample volume is available for animal samples.
Two pharmaceutical compounds
were analyzed by LC-ESI-MS/MS
without the structure information.
STD 100
By using 20 µL plasma sample,
1 ng/ml sensitivity was obtained
for both compounds.
STD 1.00
DCB-02-24
MRM of 3 Channels ES+
366.1 > 132
9.18e4
100
%
IS
MRM of 3 Channels ES+
285.1 > 153.8
1.10e5
100
Unknown 1
0
DCB-02-24
100
Unknown 2
1.00
1.50
2.00
2.50
IS
0
DCB-02-22
MRM of 3 Channels ES+
285.1 > 153.8
4.00e3
100
Unknown 1
32
DCB-02-22
100
Unknown 2
%
0
0.50
100
%
MRM of 3 Channels ES+
237.1 > 193.9
2.08e5
%
MRM of 3 Channels ES+
366.1 > 132
8.96e4
%
0
DCB-02-24
%
DCB-02-22
3.00
3.50
Time
4.00
MRM of 3 Channels ES+
237.1 > 193.9
1.38e4
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Time
4.00
Subject Analysis
76 samples
R2=0.998
Determination of Specific Polypeptide in Fish and Rat Plasma
by LC-ESI-MS/MS
The determination of an unknown
polypeptide (Mw=2334.8) in animal
biological fluids was required.
In ESI-MS, the polypeptide gave
multiple charged ions (Fig. 1).
CV=31
0 9 -M a y-2 0 0 6 0 9 :3 4 :0 6
P L 1 (0 .1 7 6 ) S m (M n , 2 x 0 .5 0 )
Scan ES+
1 .1 2 e 7
779.47
100
m/z 779 = [M+3H]3+
[M+4H]4+584.91
%
Fig. 1
[M+2H]2+
468.03
381.53
In ESI-MS/MS, the parent ion at
m/z 779 ([M+3H]3+) produced the
major product ion at m/z 110 (Fig.
2). The mass transition of 779/110
was used for the SRM detection.
353.37
1167.95
674.58
784.98
143.08
899.11
260.88
0
m /z
200
400
600
800
1000
1200
1400
1600
CV=31 CE60
2000
0 9 -M a y-2 0 0 6 0 9 :4 3 :2 3
P L M S 2 -7 7 9 1 (0 .1 7 6 ) S b (1 ,4 0 .0 0 )
D a u g h te rs o f 7 7 9 E S +
3 .6 2 e 6
110.06
100
1800
m/z 110 was selected for quantization.
%
Fig. 2
120.23
86.22
177.15
84.08
176.83
285.25
195.15
223.45
286.13
343.10
0
50
m /z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
By using 50 L of fish and rat plasma sample, the LLOQ was established at 62.5 ng/mL
(26.7 x10-9M), good linearity was obtained in the range of 62.5-2000 ng/mL
The works has been accepted by “DNA and Cell Biology”
Determination of the urinary markers of
occupational exposure to toluene
Toxicology Letters 147 (2004) 177–186
Benzylmercapturic acid is superior to hippuric acid and o-cresol as a
urinary marker of occupational exposure to toluene
O. Inoue a, E. Kannoa, K. Kasai a, H. Ukai b, S. Okamotob, M. Ikedab,∗
Simplified Biotransformation of Toluene
The analytical methods of urinary hippuric acid, creatinine, o-cresol
and benzylmercaturic acid have been established in our laboratory.
1.
The urinary hippuric acid, creatinine were determined with a
HPLC-UV method reported by IOSH (IOSH83-A209).
2.
The urinary o-cresol was determined with a in house
developed/validated HPLC-FL method.
3.
The urinary benzylmercaturic acid was determined with a in
house developed/validated HPLC-MS/MS method.
LC-UV chromatogram of hippuric acid and creatinine in Urine
Hippuric acid
Creatinine
LC-FL chromatogram of o-cresol in Urine
p-cresol
o-cresol
LC-MS/MS chromatogram of BMA in Urine
blank urine
100
4.03
2781
13C6 BMA
m/z 258>129 ES-
%
0
100
4.04
191
BMA
m/z 252>123 ES-
%
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Time
6.50
Both 13C6 and Methyl BMA were synthesized and had been
examined as the internal standard for the determination of BMA in
urine sample.
It was proved, the use of isotope internal standard allowed the use of
water as the blank matrix.
Thank You For Your
Attention !