Why we need and use LCMS

Inam's Dummy - Use of LCMS

1. LC/MS
Why is it the fastest growing
analytical technique ?

2. Discussion topics
◗Evolution of LC/MS
◗Advantages of API
◗Why should I use LC/MS ?
◗LC/MS markets

3. 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

4. Soft ionisation (gives the molecular weight)
Sensitive (low pg amounts routinely)
Robust, simple, run routinely 24 hr/dayWide range of flow rates (nanospray to analytical)
Wide range of applications (drugs, proteins)
Wide range of industries
Advantages of API

5. API Publications
0
100
200
300
400
500
600
700
800
900
1000
1991 1992 1993 1994 1995 1996 1997
Halket JM and Down S, LC/MS Update, HD Science, Nottingham

6. LC/MS for shorter analysis times
Example :
MS vs photo diode array detector for the
analysis of steroids

7. 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
progesterone
4.6
deoxycorticosterone
methyltestosterone
1.0 mL/Min
60/40 MeOH/Water/1% HOAC
4.6 mm X 3.3 C18 column
0.8
Steroid standards, 25ng injected

8. hydrocortisonedeoxycorticosterone
methyltestosterone progesterone
PDA spectra of standard steroids

9. Relative Abundance
331
deoxycorticosterone
Relative Abundance
363
hydrocortisone
Relative Abundance
303
methyltestosterone
Relative Abundance
315
progesterone
MS Spectra of steroids

10. What happens if we speed up the
chromatography ?
(from 60/40 to 90/10 MeOH/Water)
LC/MS for shorter analysis times

11. Detection by UV
Steroids coelute
No distinguishing UV spectra

12. hydrocortisone
300
320
340
360
380
400
m/z
Relative Abundance
303
315
363
331
progesterone
methyltestosterone
deoxycorticosterone
Detection by MS
363
331
315
303

13. 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

14. 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.

15. Thermo Separation Products
Frequently used analgesics
CH
3
O
H
3
C
OH
O
Naproxen
N
N
O
O
H
3
C
Phenylbutazone
H
N
HO O
Cl
Cl
CH
3
Meclofenamic Acid
N
H
N
CH
3
CF
3
COOH
Flunixin

16. 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
N
H
N
CH
3
CF
3
COOH

17. 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 11.0
Time (min)
100
0
10
20
30
40
50
60
70
80
90
Relative Abundance
100 120 140 160 180 200 220 240 260 280 300 320 340
m/z
0
20
40
60
80
100
297

18. Why should I use LC/MS ?
◗Higher sample throughput
–$
◗Shorter method development
–$
◗Better sensitivity
– environmental, legislation
◗Unequivocal ID
–safety

19. LC/MS markets
Who uses LC/MS?

20. LC/MS in the pharmaceutical market
◗Drug discovery
– molecular weight, structural
– open access
– combinatorial chemistry
◗Metabolism
– structural identification and quantification of
metabolites
◗Toxicology
– quantitation

21. 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

22. LC/MS in pharmaceutical related markets
◗Contract research organisations
– quantitation, pre-clinical & clinical trials
– structural studies
◗Generic drug companies
– QC and production

23. LC/MS in the biotechnology market
◗Protein characterisation
– molecular weight, (3D structure)
◗Proteomics
– rapid peptide sequencing
– post translational modifications
◗QC
– confirm sequence & impurities

24. LC/MS in the biotechnology market
◗Nucleotides
– molecular weight, sequence
◗Carbohydrates
– molecular weight, sequence

25. LC/MS in the agrochemical market
◗Compound discovery
◗Metabolism
◗Toxicology
◗Pharmacokinetics
◗QC and production

26. LC/MS in industrial markets
◗Organometallics
– structure
◗Detergents
– QC, competitors products
◗Polymers
– molecular weight, structure

27. LC/MS in the environmental market
◗Water
– ID and quantitation of pollutants
◗Food
– chemical contaminants
– natural toxins
◗Animal feeds
– contaminants, illegal substances

28. 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

29. LC/MS in clinical markets
◗Replace immuno assays
◗Drugs of abuse

30. LC/MS in academia
◗Related to all of the above
◗Fundamental research
◗Teaching

31. Predicted market for LC/MS systems
0
200
400
600
800
1000
1200
1985 1990 1995 2000 2005 2010 2015 2020
Instrument sales ($ million)
LC/MS
1997-2007
Willoughby R, Sheenan E and Mitrovich S, “A Global View of
LC/MS”, Global View Publishing (1998)

32. 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