Use of Derivatization for LC- MS/MS Analysis in the Clinical Lab

Transcription

1 Use of Derivatization for LC- MS/MS Analysis in the Clinical Lab Asian Pacific Conference on Chromatography & Mass Spectrometry January 2010 Alan L. Rockwood and Mark M. Kushnir ARUP Laboratories, Dept. of Pathology, University of Utah School of Medicine

2 Outline Preliminaries Background Definitions Etc. Application examples to illustrate pros, cons, and other issues Sensitivity MS/MS (tandem MS) quality Separation (LC) quality Compound class analysis

3 Purpose of derivatization Modify physical or chemical properties Improve analysis detection, quantitation, separation, identification

4 FISH: Non-classical Derivatization Fluorescently labeled DNA sequences Target specific DNA sequences Targeted sequences visualized on chromosomes Source of figure: Wikimedia Commons. Gnu public license

5 Derivatization in GC-MS and LC-MS GC-MS Very common Improve volatility Improve thermal stability LC-MS Less common than in GC and GC-MS Less often needed Avoid if possible Desirable in specific cases

6 Why derivatize in LC-MS? Improve ionization efficiency Improve fragmentation pattern (sometimes) Shift molecular weight of parent ion Improve separation properties (sometimes) Enable/improve quantitative analysis Class-specific detection Analyte confirmation

7 Why avoid derivatizing in LC-MS? Extra work, time, cost, and (possibly) error rate Supplier issues Yield (efficiency, isomer formation, side product formation) Poorer chromatography (sometimes) Poorer fragmentation patterns (sometimes)

8 Application examples/points to illustrate Keto-steroids Ionization efficiency LC separation issues Methylmalonic acid Fragmentation pattern Class-dependent ionization efficiency Hydroxy-steroids Ionization efficiency Fragmentation pattern issues Newborn screening Class-selective analysis Peptides Semi-quantitative analysis

9 Application: keto-steroids Oxime derivative Improved ionization efficiency (sensitivity) Poorer LC separation - sometimes Compound confirmation (possibly)

10 Oxime formation (testosterone) Keto-steroid (testosterone) OH OH O N NH 2 OH hydroxylamine OH Oxime derivative

11 Protonation (ionization) OH H + OH N OH H + N OH

12 Sensitivity enhancement 1.5.E+06 Nonderivatized Oxime 1.0.E E+05 Intensity, cps Te 11DC 17OHProgest 17OHPregn Pregn

13 Chromatography of testosteroneoxime derivative XIC of +MRM (17 pairs): 304.2/112.1 amu from Sample 19 (Precision 4-2) of Data reinj.wiff (Turbo Spray) Max. 5.9e4 cps. 7.0e4 6.5e4 Split peaks 6.0e4 5.5e4 5.0e4 OH 4.5e4 4.0e4 OH Intensity, cps 3.5e4 3.0e4 HO N 2.5e4 2.0e4 1.5e4 1.0e4 Isomers N OH Time, min

14 Difficulties and approaches More difficult peak integration Approach: modify LC method to merge peaks Approach: live with it Often results in poorer separation of related compounds May require better LC separation

15 Application: methylmalonic acid Dibutyl ester O O More selective ionization HO OH Improved MS/MS spectrum CH 3

16 Biosynthetic Pathway Methylmalonyl-CoA racemase Propionyl-CoA ATP + CO 2 Propionyl-CoA carboxylase ADP + P i (S)-Methylmalonyl-CoA Methylmalonyl-CoA mutase (Coenzyme B 12 ) (R)-Methylmalonyl-CoA X Succinyl-CoA MMA

17 Methylmalonic acid derivatization O O HO C CH C OH + 2 C H 3 OH CH 3 O O H 3 C O C CH C O CH 3 CH H 2 O

18 Dibutyl esters of di-carboxylic acids more sensitive than monocarboxylic acids 4.E+07 3.E+07 2.E+07 Q1 M+1 Q1 M+17 1.E+07 0.E+00 MMA 2-OH-glutaric Adipic Suberic Sebacic 1,12 Dodecanoic Ethylmalonic Dimethylmalonic 2-OH-3-methylvaleric 2-OH-isovaleric 2-oxyisovaleric 2-OH-butyric

19 4.E+07 3.E+07 2.E+07 1.E+07 0.E+00 Q1 M+1 Q1 M+17 MMA 2-OH-glutaric Adipic Suberic Sebacic 1,12 Dodecanoic Ethylmalonic Dimethylmalonic 2-OH-3-methylvaleric 2-OH-isovaleric 2-oxyisovaleric 2-OH-butyric Esters of di-carboxylic acids

20 MMA 2-OH-glutaric Adipic Suberic Sebacic 1,12 Dodecanoic Ethylmalonic Dimethylmalonic 2-OH-3-methylvaleric 2-OH-isovaleric 2-oxyisovaleric 2-OH-butyric 4.E+07 3.E+07 2.E+07 1.E+07 0.E+00 Q1 M+1 Q1 M+17 Esters of mono-carboxylic acids

21 Non-selective MS/MS spectrum (underivatized) e6cps O HO C CH CH 3 C O O - Intensity, % Intensity, % MMA m/z e6cps Succinic acid m/z Free Acid Ionization in the Negative Ion Mode

22 Selective MS/MS of derivatives [MH - 2 C 4 H 8 ] e7 cps MMA Intensity, % [MH - C 4 H 8 ] Parent ion [MH - 2 C 4 H 8 -H 2 O] m/z 2.70e7 cps SA Intensity, % 50 [MH - C 4 H 8 -H 2 O] Parent ion m/z

23 Application: hydroxy-steroids Dansyl chloride derivatization Improved ionization efficiency (sensitivity) Poorer MS/MS spectrum

24 Dansyl chloride derivatization Dansyl chloride Reaction site Cl H Estradiol Derivative

25 Ionization of dansyl derivatives H +

26 Dissociation of dansyl derivative of estradiol m/z 171 m/z 506 parent ion m/z 156 H + Non-specific daughter ions

27 Non-specific product Ions of dansyl derivative of estradiol m/z 171 Product ions from dansyl-end of ion. m/z 156

28 Difficulties and approaches Lower specificity of MS/MS spectrum Improve extraction Improve LC

29 Application: newborn screening Butyl ester derivative MS/MS only Class sensitive compound detection Acyl carnatines (fatty acids/organic acids) Amino acids Scan functions Precursor ion Constant neutral loss

30 Acylcarnitines derivatization and fragmentation RCOO H (CH 3 ) 3 N CH 2 CH CH COOH Butylation RCOO H (CH 3 ) 3 N CH 2 CH CH COOC 4 H 8 H CID - RCOOH -(CH 3 ) 3 N -C 4 H 8 [ CH 2 CH CH COOH ] + (m/z 85) Slide courtesy of Mike Morris, Waters Corp ] +

31 Normal vs. MCAD deficiency 100 C2 carnitine precursor ion scans Normal C16 carnitine % C3 carnitine Elevated C8 carnitine MCAD Deficiency % C6 carnitine C10:1 carnitine m/z Slide courtesy of Mike Morris, Waters Corp

32 Application: relative peptide qantitation using derivatization ICAT Cysteine ITRAQ & MTRAQ Primary amines

33 ICAT reagents Light ICAT reagent (hydrogen labels) Heavy ICAT reagent (deuterium labels) Adapted from

34 ICAT quantitation control sample test sample test sample control sample label with ICAT mass tags MS scan -> quantitation MS/MS scan -> identification combine samples d8 ratio ~ 3:1 overnight trypsin digest d0 peptide extraction analysis by mass spectrometry

35 Conclusions Use derivatization only when advantages outweigh disadvantages Sensitivity MS/MS quality Separation quality Special applications Compound class analysis Quantitation of peptides Etc.

36 Additional reading Sensitivity enhancement in liquid chromatography/atmospheric pressure ionization mass spectrometry using derivatization and mobile phase additives. Songmei Gao, Zong-Ping Zhang, H. T. Karnes. J. Chromatography B, 825 (2005) Handbook of Analytical Derivatization Reactions, by Daniel Knapp (Mostly oriented to GC-MS)

37 Acknowledgements Bingfang Yue, PhD David Crockett, MS ARUP Institute for Clinical and Experimental Pathology Additional source materials as listed on slides