LC/MS Why is it the fastest growing analytical technique ? Discussion topics ◗ Evolution of LC/MS ◗ Advantages of API ◗ Why should I use LC/MS ? ◗ LC/MS markets Evolution of LC/MS interfaces 1970s to Present Moving belt interface (EI and CI, library searchable) Dynamic FAB (low flow rates, very fiddly) TSP ionisation (first widely used LC/MS interface) Atmospheric Pressure Ionisation (ESI and APCI) Advantages of API Soft ionisation (gives the molecular weight) Sensitive (low pg amounts routinely) Robust, simple, run routinely 24 hr/day Wide range of flow rates (nanospray to analytical) Wide range of applications (drugs, proteins) Wide range of industries API Publications 1000 900 800 700 600 500 400 300 200 100 0 1991 1992 1993 1994 1995 1996 Halket JM and Down S, LC/MS Update, HD Science, Nottingham 1997 LC/MS for shorter analysis times Example : MS vs photo diode array detector for the analysis of steroids Steroid standards, 25ng injected 600 1.0 mL/Min 60/40 MeOH/Water/1% HOAC 4.6 mm X 3.3 C18 column 0.8 hydrocortisone 500 deoxycorticosterone 400 1.9 300 progesterone 4.6 200 methyltestosterone 100 2.7 0 -100 0.0 0.5 1.0 1.5 2.0 2.5 Minutes 3.0 3.5 4.0 4.5 5.0 5.5 PDA spectra of standard steroids deoxycorticosterone hydrocortisone methyltestosterone progesterone MS Spectra of steroids 363 Relative Abundance deoxycorticosterone Relative Abundance 331 315 methyltestosterone Relative Abundance Relative Abundance 303 hydrocortisone progesterone LC/MS for shorter analysis times What happens if we speed up the chromatography ? (from 60/40 to 90/10 MeOH/Water) Detection by UV Steroids coelute No distinguishing UV spectra Detection by MS 303 363 methyltestosterone 331 Relative Abundance progesterone 315 315 deoxycorticosterone 363 303 331 hydrocortisone 300 320 340 360 m/z 380 400 LC/MS shorter run times ◗ Changing from 6 to 2 min / sample means : – 10 samp / hr – 80 samp / day – 20,000 samp / yr 30 samp / hr 240 samp / day 60,000 samp /yr Did This Horse Win The Race Legally ? A wide variety of drugs are dosed to horses to enhance performance during racing. Thermo Separation Products Frequently used analgesics C H3 OH H3C O O N O N O H3C Naproxen Phenylbutazone HO O H N Cl Cl Meclofenamic Acid C H3 N H N C H3 C O OH Flunixin C F3 Is this peak Flunixin ? It has the correct retention time by UV 254 nm 180 N mAU 160 H N CH3 CF3 COOH 140 Flunixin ? 120 100 80 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Minutes 5.2 5.4 5.6 5.8 6.0 6.2 6.4 LC/MS confirms it to be Flunixin Relative Abundance Flunixin Full Scan MS 100 90 80 70 60 50 40 30 20 10 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Time (min) 297 100 80 60 40 20 0 100 120 140 160 180 200 220 m/z 240 260 280 300 320 340 Why should I use LC/MS ? ◗ Higher sample throughput – $ ◗ Shorter method development – $ ◗ Better sensitivity – environmental, legislation ◗ Unequivocal ID – safety LC/MS markets Who uses LC/MS ? LC/MS in the pharmaceutical market ◗ Drug discovery – molecular weight, structural – open access – combinatorial chemistry ◗ Metabolism – structural identification and quantification of metabolites ◗ Toxicology – quantitation LC/MS in the pharmaceutical market ◗ Pharmacokinetics – quantitation, sensitivity,precision and accuracy – pre-clinical and clinical studies ◗ Formulation – structural, degradation products ◗ QC & Production – quantitation – ID of impurities & unexpected peaks LC/MS in pharmaceutical related markets ◗ Contract research organisations – quantitation, pre-clinical & clinical trials – structural studies ◗ Generic drug companies – QC and production LC/MS in the biotechnology market ◗ Protein characterisation – molecular weight, (3D structure) ◗ Proteomics – rapid peptide sequencing – post translational modifications ◗ QC – confirm sequence & impurities LC/MS in the biotechnology market ◗ Nucleotides – molecular weight, sequence ◗ Carbohydrates – molecular weight, sequence LC/MS in the agrochemical market ◗ Compound discovery ◗ Metabolism ◗ Toxicology ◗ Pharmacokinetics ◗ QC and production LC/MS in industrial markets ◗ Organometallics – structure ◗ Detergents – QC, competitors products ◗ Polymers – molecular weight, structure LC/MS in the environmental market ◗ Water – ID and quantitation of pollutants ◗ Food – chemical contaminants – natural toxins ◗ Animal feeds – contaminants, illegal substances LC/MS in the forensic market ◗ Scene of crime – illegal substances, toxic agents ◗ Horse race doping – illegal substances ◗ Explosives ◗ Drugs of abuse – urine, hair, banknotes LC/MS in clinical markets ◗ Replace immuno assays ◗ Drugs of abuse LC/MS in academia ◗ Related to all of the above ◗ Fundamental research ◗ Teaching Predicted market for LC/MS systems 1200 LC/MS Instrument sales ($ million) 1000 1997- 2007 800 600 400 200 0 1985 1990 1995 2000 2005 2010 2015 Willoughby R, Sheenan E and Mitrovich S, “A Global View of LC/MS”, Global View Publishing (1998) 2020 Conclusion ◗ LC/MS is an established technique ◗ Market is growing rapidly ◗ LC/MS moving out of the specialised labs into every department ◗ Wide variety of LC/MS analyser types ◗ Non-specialist users