LC/MS has become the fastest growing analytical technique due to its advantages over other methods like its ability to provide molecular weight information through soft ionization, high sensitivity, robustness, wide range of applications across industries, and ability to handle various flow rates. It has evolved greatly from early interfaces like moving belt and dynamic FAB to widely used techniques like atmospheric pressure ionization. The technique is used across various markets like pharmaceuticals, biotechnology, environmental testing, and forensics due to its high sample throughput, short method development time, sensitivity, and ability to unambiguously identify compounds. The LC/MS market is predicted to continue growing rapidly through 2020.

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. 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)

4. 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

5. 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

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

7. Steroid standards, 25ng injected
600
0.8 1.0 mL/Min
hydrocortisone 60/40 MeOH/Water/1% HOAC
4.6 mm X 3.3 C18 column
500
deoxycorticosterone
400
1.9
300
progesterone
200
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

8. PDA spectra of standard steroids
deoxycorticosterone hydrocortisone
methyltestosterone progesterone

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

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

11. Detection by UV
Steroids coelute
No distinguishing UV spectra

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

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

15. Thermo Separation Products
Frequently used analgesics
C H3
OH
O N
H3C O N
O
O
Naproxen H3C
Phenylbutazone
HO O C H3
Cl
H H
N C H3 N N C F3
Cl COOH
Meclofenamic Acid Flunixin

16. Is this peak Flunixin ?
It has the correct retention time by UV
254 nm
CH3
180
H
N N CF3
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

17. LC/MS confirms it to be Flunixin
Flunixin Full Scan MS
100
90
Relative Abundance
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
100 120 140 160 180 200 220 240 260 280 300 320 340
m/z

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
1200
LC/MS
1000
1997- 2007
Instrument sales ($ million)
800
600
400
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)

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