bio analytical application of lc-ms

Bioanalytical Applications of Liquid Chromatography Mass Spectrometry

Dulal Mahavir MohatoDepartment of Chemical technology

Dr. Babasaheb Ambedkar Marathwada University, Aurangabad (M.S) India

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

H3O+ H2O

M

H2O H2O

MH+

MS

Heat

Heat

760 torr

N2

N2

Heated pneumatic nebulizerLC/MS interface

Corona discharge needle2-6 kV

gas+

vaper

Gas phase ion-molecular reaction , IMR

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

Fail sample preparation can cause: Interference Extraction efficiency variation Ionization suppression/enhancement

Adequate sample preparation is a key aspect of quantitative bioanalysis and can often be the bottlenecks during high-throughput analysis.

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

NH4Br NH4Cl NH4F

Neural hydroxyl drugs Anionic Adduct Ions

Anionic attachment-ESI

• Neutrals exhibit unsatisfied response in ESI-MS.

• Chemical derivatization complicate the analytical process.

5 ul/min 3 ppm 0.2 mM NH4F

150 160 170 180 190 200 210 220 230 240 250 260 270m/z0

100

%

MEPH-F-2-1 1 (0.022) Sm (Mn, 2x0.50) Scan ES- 3.13e7201

221202


150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300m/z0

100

%

GUAI-F-2-1 1 (0.022) Sm (Mn, 2x0.50) Scan ES- 2.48e7217

237218255


350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550m/z0

100

%

SV-F-2-1 1 (0.022) Sm (Mn, 2x0.50) Scan ES- 6.44e6457437

399438 458

493463469


260 280 300 320 340 360 380 400 420 440 460 480 500 520m/z0

100

%

PODO-F-2-1 1 (0.022) Sm (Mn, 2x0.50) Scan ES- 8.78e6433

255

453

434 445

459


130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280m/z0

100

%

INOSITOL-F-2-1 1 (0.022) Sm (Mn, 2x0.50) Scan ES- 1.41e7199

179219

221

mephenesin

guaifenesin

simvastatin

[M+FHF]-

ESI-MS

[M+FHF]-

[M-H]-

Mephenesin, MW=182.22

Guaifenesin, MW=198.22

Simvastatin, MW=418.57

Podophyllotoxin, MW=414.41Inositol, MW=180.16

[MF]-

[MF]-

[MF]-

[MF]-

[M+FHF]-

[MF]-

[M+FHF]-

[M+FHF]-

[M+FHF]-

[M+FHF]-


50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220m/z0

100

%

MEPH-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50) Daughters of 201ES- 8.87e6107


50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250m/z0

100

%

GUAI-F-2--D-1 1 (0.022) Sm (Mn, 2x0.50) Daughters of 217ES- 9.20e6123


100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0

100

%

SV-F-2-1-D-1 1 (0.022) Sm (Mn, 2x0.50) Daughters of 437ES- 1.10e6399

115


250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450m/z0

100

%

PODO-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50) Daughters of 433ES- 1.31e6383

413


50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z0

100

%

INOSITOL-F-2-D-1 1 (0.022) Sm (Mn, 2x0.50) Daughters of 199ES- 5.03e6179

107

181

197

123

399

283

413383

179

199

115

mephenesin

guaifenesin

simvastatin

podophyllotoxin

inositol

[M-H]-

[M-H]-

[M-H]-

[M-H]-

[M-H]-

417

[M-H]- X-H+

(proton bonded mixed dimmers of anions)

Cai, Y.; Cole, R. B. Anal. Chem. 2002, 74, 985-991

1.00 2.00 3.00 4.00 5.00Time0

100

%

48

100

%

79

100

%

1.00 2.00 3.00 4.00Time0

100

%

65

100

%

72

100

%

5 ng/ml

5 ng/ml

0.05 ng/ml

Blank plasma

0.05 ng/ml

Blank plasma

201>107 m/z

201>107 m/z

201>107 m/z

217>123 m/z

217>123 m/z

217>123 m/z

(a)

(b)

(a) Mephenesin (b) guaifenesin

0.5 ml plasma, liq-liq, post-infusion of 0.2 mM NH4F

Hydrophilic Interaction Liquid Chromatography (HILIC)

- It was introduced by Alpert (1990) and later used by Strege in tandem withMS 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.

Macroporous StructureAllows rapid flow (up to 9mL/min) at low pressuresEach 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.

Mesoporous StructureCreates large surface areaThe 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.

Excellent performance with minimal HPLC system stress

Turbulent Flow Chromatography

Allows direct injection of biological samples into an MS/MS system.

http://www.cohesivetech.com/technologies/turboflow/index.asp

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.

The large interstitial spaces between the column particles and the high linear mobile phase velocity creates turbulence within the TurboFlow column.

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.

R-COOH R-COO-

Deprotonation

R-COO-PFB R-COO-

[M - H ]-

[M – PFB]-

Negative APCI

Electron Capture

Negative APCINegative ESI

Matrix Effect: APCI < ESI Sensitivity: APCI- < ESI-

Flufenamic acid MW = 281.23

Thioctic acid MW = 206.23

Estradiol MW = 272.39

(c)

(d)

[M-181]-

[M-H]-

80%MeOH, 0.5 ml/min

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350m/z0

100

%

280

281

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350m/z0

100

%

280

281

STD(f)

(e)

[M-181]-

[M-181+32]-

[M-H+32]-

90%MeOH, 0.3 ml/min

200 220 240 260 280 300 320 340 360 380 400m/z0

100

%

303

271

304

220 240 260 280 300 320 340 360 380 400m/z0

100

%

303

271

[M-H]-

PFB-APCI

Flufenamic acid-PFB

Flufenamic acid-STD

Estradiol-PFB

Estradiol-STD

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.

(a)

(b)

[M-181]-

[M-H]-

[M-H+32]-

MeOH/CAN/Water= 60/20/20, 0.5 ml/min

180 190 200 210 220 230 240 250 260 270 280 290 300 310m/z0

100

%

205

237

207

180 190 200 210 220 230 240 250 260 270 280 290 300 310m/z0

100

%

205

Thioctic acid-PFB

Thioctic acid-STD

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50Time0

100

%

4.363.18 3.75

Thioctic Acid-PFB

Thioctic Acid-STD

45.2 ppb SIM (m/z 205)

AP-

1.50 2.00 2.50 3.00 3.50 4.00 4.50Time0

100

%

3.152.632.13

1.28

Flufenamic acid-PFB

Flufenamic Acid-STD

2.65 ppb SIM (m/z 280)

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 4.50Time0

100

%

2.72

2.161.68 5.9 ppb, SIM (m/z 303)

Estradiol-PFB

Estradiol-STD

PFB-APCI

Mobile Phase: 80%CH3OH(aq), 1 ml/min.Corona: 20 A, Probe Temp: 600 oC

10-25 fold enhancement

FlunitrazepamMW = 313.29

(b)

(a)

295 300 305 310 315 320 325 330 335m/z0

100

%

314.1

315.2

290 295 300 305 310 315 320 325 330 335 340m/z0

100

%

295 300 305 310 315 320 325 330 335m/z0

100

%

314.1

315.2

295 300 305 310 315 320 325 330 335m/z0

100

%

313.1

314.1

[MH]+

[MH]+

[M]

(d)

(c)

ES-3.86e7

ES+1.23e8

AP-2.57e8

AP+1.27e8

Flunitrazepam (5 g/ml) 80%ACN(aq) 40 µl/min。

Flunitrazepam。

Matrix Effect

Matrix effect is a phenomenon observed when the signal of analyte can be either suppressed or enhanced due to the co-eluting 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 post-

spiked 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

LC Pump API-MS

Sample Loop (10 µL)

Syringe Pump

(Standard Solution)

(Plasma Extracts)

Valco T

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 “matrix-

affected” 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 Determination of Unknown Leads in Mouse Plasma by LC-MS/MSMouse Plasma by LC-MS/MS

Two pharmaceutical compounds were analyzed by LC-ESI-MS/MS without the structure information.

STD 1.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00Time0

100

%

32

100

%

0

100

%

DCB-02-22 MRM of 3 Channels ES+ 366.1 > 132

8.96e4

DCB-02-22 MRM of 3 Channels ES+ 285.1 > 153.8

4.00e3


1.38e4

By using 20 µL plasma sample, 1 ng/ml sensitivity was obtained for both compounds.

STD 100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00Time0

100

%

0

100

%

0

100

%

DCB-02-24 MRM of 3 Channels ES+ 366.1 > 132

9.18e4


1.10e5


2.08e5

Unknown 1

Unknown 2

IS

Unknown 1

Unknown 2

IS

Usually, quite limited sample volume is available for animal samples.

09-May-2006 09:34:06CV=31

200 400 600 800 1000 1200 1400 1600 1800 2000m/z0

100

%

PL 1 (0.176) Sm (Mn, 2x0.50) Scan ES+ 1.12e7779.47

584.91

468.03

381.53

353.37143.08

260.88

674.58 1167.95

784.98899.11

09-May-2006 09:43:23CV=31 CE60

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800m/z0

100

%

PL MS2-779 1 (0.176) Sb (1,40.00 ) Daughters of 779ES+ 3.62e6110.06

86.22

84.08

120.23

177.15

176.83 285.25195.15223.45 286.13

343.10

Determination of Specific Polypeptide in Fish and Rat Plasma by LC-ESI-MS/MS

m/z 779 = [M+3H]3+

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).

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.

[M+2H]2+

[M+4H]4+

m/z 110 was selected for quantization.

Fig. 1

Fig. 2

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,∗

Determination of the urinary markers of occupational exposure to toluene

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-FL chromatogram of o-cresol in Urine

p-cresol

o-cresol

LC-UV chromatogram of hippuric acid and creatinine in Urine

Hippuric acidCreatinine

blank urine

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50Time0

100

%

0

100

%

4.032781

4.04191

13C6 BMAm/z 258>129 ES-

BMA m/z 252>123 ES-

LC-MS/MS chromatogram of BMA in Urine

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

bio analytical application of lc-ms

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