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

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