why we need and use lcms

LC/MS

Why is it the fastest growinganalytical technique ?

Discussion topics

◗ Evolution of LC/MS

◗ Advantages of API

◗ Why should I use LC/MS ?

◗ LC/MS markets

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)

Evolution of LC/MS interfaces

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

Advantages of API

API Publications

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800

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1000

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

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

-100

0

100

200

300

400

500

600

hydrocortisone

1.9

2.7

progesterone4.6

deoxycorticosterone

methyltestosterone

1.0 mL/Min60/40 MeOH/Water/1% HOAC4.6 mm X 3.3 C18 column

0.8

Steroid standards, 25ng injected

hydrocortisonedeoxycorticosterone

methyltestosterone progesterone

PDA spectra of standard steroids

Rela

tive

Abu

ndan

ce331

deoxycorticosterone

Rela

tive

Abu

ndan

ce

363hydrocortisone

Rela

tive

Abu

ndan

ce

303methyltestosterone

Rela

tive

Abu

ndan

ce

315progesterone

MS Spectra of steroids

What happens if we speed up the chromatography ?

(from 60/40 to 90/10 MeOH/Water)

LC/MS for shorter analysis times

Detection by UV

Steroids coelute

No distinguishing UV spectra

hydrocortisone

300 320 340 360 380 400m/z

Rel

ativ

e A

bund

ance

303

315

363

331

progesterone

methyltestosterone

deoxycorticosterone

Detection by MS

363

331

315

303

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 RaceDid This Horse Win The Race

LegallyLegally ??

A wide variety of drugs are dosed to horses to enhance performance during racing.

Thermo Separation ProductsFrequently used analgesics

CH3

OH3C

OH

O

Naproxen

NN

O

O

H3CPhenylbutazone

HN

HO O

Cl

C l

CH3

Meclofenamic Acid

NHN

CH3

CF3

COOH

Flunixin

It has the correct retention time by UV

Is this peak Flunixin ?

Minutes

mAU Flunixin ?

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

80

100

120

140

160

180

254 nm254 nm

NHN

CH3

CF3

COOH

LC/MS confirms it to be Flunixin

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 11.0Time (min)

100

0102030405060708090

Rel

ativ

e Ab

unda

nce

100 120 140 160 180 200 220 240 260 280 300 320 340

m/z

0

20

40

60

80

100

297

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

0

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800

1000

1200

1985 1990 1995 2000 2005 2010 2015 2020

Inst

rum

ent s

ales

($ m

illio

n)

LC/MS

1997- 2007

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