The role of LC-MS in Toxicology

Transcription

1 The role of LC-MS in Toxicology Kjell Mortier, Tom Benijts, Riet Dams, Willy Lambert Laboratory of Toxicology, Ghent University, Belgium

2 Overview Introduction: comparison with GC-MS LC-DAD Interface characteristics Different types of MS Applications in toxicology Opiates / Cocaine / LSD Amphetamines / designer drugs Cannabinoids Multicomponent analysis Difficulties General conclusions

3 Toxicological Analysis Screening tests Identification Confirmation Elimination Quantitation Interpretation Spot tests Immunologic Limitations: Specific drugs/ classes of drugs Chromatography Spectral info and t r GC-MS / HPLC-DAD Scientific lit. Lib. search

4 GC-MS and HPLC-DAD Advantage Disadvantage GC-MS: HPLC- DAD: Specific Sensitive Large libraries Polar Thermolabile Polar, derivatization Thermolabile High MW compounds Less specific Less sensitive HPLC-MS: IDEAL COMBINATION? No derivatization Sensitive Specific, chromatographic separation not mandatory Thermolabile, polar and high Mw compounds

5 Interfaces: hyphenation of LC to MS LC Interface MS LC Analyte in liquid High liquid flow Earlier Interfaces DLI Moving Belt Particle Beam Thermospray MS Ion in gas phase Works in vacuum API interfaces Electrospray (or Ionspray) APCI Sonic spray ionisation APPI

6 API interfaces: general properties LC Interface MS Only volatile buffers at low C (typically?10 mm) e.g. formic acid, acetic acid, ammonia Better ionization with high % organic phase Preferred solvents: water, methanol, AcCN Basic substances: positive mode (voltages) API primarily result [M + H] + (no fragmentation) Acid substances: negative mode (opposite V) API primarily result [M - H] - (no fragmentation) For maximum sensitivity: a lot of optimization of buffer species, ph, buffer C, type interface, analyte, solvent type, solvent flow

7 Electrospray Or with coaxial N 2 flow : Ionspray Electric field on capillary sprayer tip Ionization in liquid phase Concentration sensitive miniaturization Nano-ES : protein, peptide analysis Multiple charged ions Capillary electrophoresis Soft ionization technique : little fragmentation High mol. mass range by multiple charging Thermolabile / highly polar compounds

8 Electrospray mechanism (M-8000, Merck-Hitachi) Aperture 1 Assistant gas heater Cover plate N 2 LC-flow Desolvator ESI-probe tip at high voltage +/- 0-4 kv

9 Electrospray mechanism (molecular) LC-flow Ion separation Droplet Gas-phase ions Micro-droplet /- 0-4 kv

10 Atmospheric Pressure Chemical Ionization Higher flows compatible Ionization in gas phase (analyte / solvent) Heated nebulizer ( C) High ionization efficiency Most applications: Mw < 1000 Da Medium to low polarity molec. (not thermolabile) Pesticides, drugs, steroids High flow rates - standard columns Less susceptible to matrix suppression

11 APCI mechanism (M-8000, Merck-Hitachi) Nebulizer Desolvator Cover plate Aperture C 180 C LC-flow +/- 0-4 kv Corona needle

12 Sonic Spray Ionization Coaxial N 2 Flow at sonic velocity (~1 Mach) No need for high heating No need for electric field Advantages: Thermolabile compounds Little in source fragmentation Flow rates: ml/min. SSI spectra comparable to ESI spectra

13 SSI mechanism (M-8000, Merck-Hitachi) N 2 (3 to 5 l/min) Cover plate Aperture 1 LC-column Ion analysis Fused silica capillary Super Sonic spray

14 Different types of Mass Analysers Single quadrupole instruments Triple quadrupole instruments Ion trap intruments (quadrupole) time-of-flight detectors Choice depends on: applications requirements funding 1. Single quadrupole instruments Cheapest Single quadrupole Selected ion monitoring: SIM (with loss of information) Scan mode (with loss of sensitivity) Few spectral information, no collision cell Isobaric interferences possible Low resolution, e.g. 1 Da Major application: molecular mass determination pure samples required

15 Different types of Mass Analysers 2. Triple quadrupole instruments More expensive QQQ met collision cell Spectral information is obtained Less interference Very sensitive in MRM Less sensitive in full scan Major application: quantitiative analysis of known compounds (very popular in pharmaceutical companies) Q1 Q2 Q3 Parent Ion Selection CID- Process Fragment Analysis

16 Different types of Mass Analysers 3. Quadrupole ion trap instruments Cap Ring Cap Relatively cheap Ions are stored and sequentually ejected from the trap A lot of spectral information MS n Ideal for identification Sensitive in full scan Major application: Identification and quantification

17 Different types of Mass Analysers 4. Quadrupole Time-of-Flight instruments Most expensive Very sensitive in full scan Very high mass resolution (0.01 Da accuracy is no problem) Not ideal for quantitative work Major applications: proteomics, genomics, research, identification, quantification Q1 Q2 TOF Parent Ion Selection CID- Process Fragment Analysis

18 Mass Analysers in Toxicology Looking for unknown analytes: full scan spectrum is required Identification or confirmation necessary Theoretically: Ion-trap and QTOF seem to be the preferred techniques For quantification of known target compounds: Theoretically: Triple quad seems to be the preferred technique

19 Applications OPIATES Relevant compounds : Heroin, 6-MAM, morph, accod, cod, M-3-G, M-6-G, DHM, DHC Interface : Majority APCI and ESI (TSP 94 & 96) Mass analysis : Q, SIM or QQQ, MRM Advantages / complications : M-3-G and M-6-G can be analyzed Fragmentation into glucuronic acid / aglycon Time programmed extractor voltage increase

20 Large difference in polarity (M-3-G? heroin) Gradient necessary: Influences ionization process Reproducible reference spectra difficult to obtain? Flow programming Identification (unequivocal) Molecular ion inadequate CID using LC-MS/MS Sensitivity LC-MS equal or better than GC-MS (from 0.5ng/ml, with simple extraction method)

21 NON-CLASSICAL OPIATES : Papaverine, noscapine Differentiation non-prescription / prescribed heroin use by urine analysis AC, C, C-6-G, P, N : Non prescription AC : very specific P : short t 1/2 N : most prevalent, long t 1/2 LC-APCI, positive ionization, SIM LOD : ng/ml

22 HEROIN IMPURITY PROFILING: AC, C, heroin, 6-MAM, Morph., N, P LC-SSI-Ion Trap, positive ionization LOD : 2-20 ng/ml Less sensitive but sufficient (+scan) Monolithic column (Chromolith ) Gradient elution : H 2 O/AcCN Flow : 5 ml/min - split 1/20

23 COCAINE Relevant compounds : cocaine,benzoylecgonine, cocaethylene, in blood, urine, hair, saliva Interface : Majority APCI and ESI (TSP 96) Mass analysis : Q, SIM or QQQ, MRM Advantages/complications : Fragmentation well studied Transition for MS/MS well defined No derivatization (polar metabolites, long t 1/2 ) Deuterated IS or 2 -methylcoc, 2 -methylbe Thermolabile coc-n-oxide can be analyzed No artefactual formation of pyrolysis products

24 High speed chomatography possible Need for TOF detector instead of scanning instruments Analysis of cocaine possible in difficult matrices (Hair, low volumes) LOQ: most applications 10ng/ml Challenges : Method including more coc related compounds Identical masses common fragments coc, norce; BE, norcoc Chrom. separation reproducible t r How to extract them all? Less specific extraction ion suppression

25 LSD Relevant compounds : LSD, nor-lsd, 13-, 14-OH LSD, Interfaces / mass analysis : ESI & APCI / Q, SIM; QQQ, MRM Challenges : Low expected levels Extensively metabolized Photosensitive, thermally unstable LC >>> GC Adsorption to glass or GC colums LC-MS(/MS): method of choice Advantages / complications : Fragmentation well known new metabolites identified (2-oxo-LSD)

26 Sample prep: often immunoaffinity extraction : saturation problem but very low LOD (2.5pg/ml) Also SPE extraction Detection limits : range: 0.1 to 0.5 ng/ml (LSD, nor-lsd) Conclusion LC-MS/MS is the method of choice and is already highly optimized

27 AMPHETAMINE DESIGNER DRUGS Relevant compounds : Amph., methamph., MDMA, MDEA, MDA, MBDB Interface/mass analysis : ESI & APCI / Q, SIM, QQQ MRM, Q-TOF Advantages / complications / challenges : Chiral compounds Stereospecific differentiation relevant Small structural differences and new drugs: MS ESI and APCI have both been used LODs: most vary around ng/ml

28 Our Lab: Identification of PMA (paramethoxyamphetamine) in postmortem samples (blood, urine and tissue). Sonic spray ionisation Validated method Quantitative: PMA, MDMA, Amph, MDA MS/MS: full scan spectrum for ID LOD: ng/ml

29 CANNABINOIDS Relevant compounds : THC, THC-COOH, 11-OH-THC, glucuronide Limited applications : Urine analysis (only 2 reports) THC-COOH quantitative THC-COOH glucuronide qualitative Deconjugation still performed Analysis in + mode No derivatization for thermolabile THC-COOH, LOQ around 10 ng/ml

30 MULTICOMPONENT ANALYSIS Relevant compounds : different classes of drugs simultaneously quantified multidrug use still target compound analysis Drugs of abuse in saliva All quantified in one run (Q-TOF analysis) : Amphetamine Methamphetamine MDA MDMA (XTC) MDEA (EVE) Morphine Codeine 6-monoacetylmorphine Benzoylecgonine Cocaine

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32 LC-MS screening Data dependent acquisition (automatic function switching) MS full scan run (no fragmentation) switches to MS/MS automatically (when peak above treshold value is detected) Very interesting in toxicology Problem: what treshold Sensitivity better when further in gradient Some matrix compounds have high C Some drugs have low toxic C

33 Screening Difficulties Literature: A lot of target compound analysis Few screening for unknowns Compound library LC-MS spectra Large universal libraries not available yet ( GCMS) Difficulties in uniformity of spectra Adduct formation: M + Na + or K + instead of H + Gives wrong idea of M with unknowns LC-MS fragmentation still not fully understood (radicals and neutral molecules can be lost)

34 Difficulties Quantification Matrix suppression Less efficient ionization of analyte in the presence of matrix material typical ESI process Leads to decreased analyte response and errors in quantification Solution: Labelled internal standards (not always available not applicable in toxicology)

35 Beginning General conclusions Toxicologic relevant compounds used for demonstrating LC-MS capabilities Now Ideal (expensive) detector High sample throughput Less efficient separation allowed Matrix ionization suppression! New possibilities polar metabolites intact glucuronides

36 Most applications : Target compound analyses Less suited for profiling purposes API interface becomes standard : Persuading hesitating toxicologists Other sources (SSI) specific advantages

37 Future Database of ESI-generated spectra 600/1200 compounds +,-, different fragmentation energy large variability in intensity/fragmentation pattern Standardization needed Application shift from target analysis Screening for unknowns

38 Other research groups: Henion, JAT, 1996, 20, 27 (LSD) Bogusz et al., JAT 2001, 431 (Opiates) Our research group: Mortier et al., RCM 2001, 15, 1773 (Saliva analysis) Mortier et al. RCM 2002, 16, 865 (PMA determination). Dams et al., Forensic Sci. Int. 2001, 123, 81 (Impurity profiling) Dams et al. Anal. Chem (accepted for pub.) Dams et al. RCM 2002, 16, 1072 (2002). Laboratory website: Cited papers

39 Cited papers Interesting review papers about LC-MS in Toxicology:? P. Marquet, Therapeutic drug monitoring 2002, 24, 255? Van Bocxlaer et al. Mass Spectrom. Rev. 2000, 19, 165? Marquet et al. J. Chromatogr. B 1999, 733, 93? H. Maurer, J. Chromatogr. B 1998, 713, 3? M. Bogusz, J. Chromatogr. B 1999, 733, 65? Tatsuno et al. J. Anal. Toxicol. 1996, 20, 281? Hoja et al. J Anal. Toxicol. 1997, 21, 116