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 1997 Halket JM and Down S, LC/MS Update, HD Science, Nottingham
LC/MS for shorter analysis times Example : MS vs photo diode array detector for the analysis of steroids
Steroid standards, 25ng injected 600 500 400 0.8 hydrocortisone 1.9 deoxycorticosterone 1.0 ml/min 60/40 MeOH/Water/1% HOAC 4.6 mm X 3.3 C18 column 300 200 progesterone 4.6 100 methyltestosterone 2.7 0-100 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Minutes
PDA spectra of standard steroids deoxycorticosterone hydrocortisone methyltestosterone progesterone
MS Spectra of steroids 331 deoxycorticosterone 363 hydrocortisone Relative Abundance Relative Abundance Relative Abundance 303 methyltestosterone 315 progesterone Relative Abundance
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 315 progesterone deoxycorticosterone 363 331 315 303 hydrocortisone 300 320 340 360 380 400 m/z
LC/MS shorter run times Changing from 6 to 2 min / sample means : 10 samp / hr 30 samp / hr 80 samp / day 240 samp / day 20,000 samp / yr 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 CH 3 OH H 3 C O O O N N Naproxen H 3 C O Phenylbutazone HO O H N Cl CH 3 N H N CH 3 CF 3 Cl COOH Meclofenamic Acid Flunixin
Is this peak Flunixin? It has the correct retention time by UV 254 nm 180 N H N CH3 CF 3 160 COOH mau 140 Flunixin? 120 100 80 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 Minutes
LC/MS confirms it to be Flunixin Relative Abundance 100 90 80 70 60 50 40 30 20 10 0 Flunixin Full Scan MS 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) 100 297 80 60 40 20 0 100 120 140 160 180 200 220 240 260 280 300 320 340 m/z
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 Instrument sales ($ million) 1200 1000 800 600 400 LC/MS 1997-2007 200 0 1985 1990 1995 2000 2005 2010 2015 2020 Willoughby R, Sheenan E and Mitrovich S, A Global View of LC/MS, Global View Publishing (1998)
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