Liquid Chromatography – tandem Mass Spectrometry

An Introduction for Beginners

Steven Pauwels Jessa Hospital, 7th of May 2013

Why show interest?

Tacrolimus Proficiency testing

0

5

10

15

20

25

30

35

40

45

1999 2010 2013

% LC MS

www.bioanalytics.co.uk

www.pubmed.org

2004 2012

6772 analyses

Immunosuppresives

11927 analyses

Immunosuppresives, Antiepileptic,

Antifungal, Endocrine

?

?

Steroids Antibiotics?

Metabolites? Cancer therapy?

~ 0,1% van analyses

Evolution UZ Leuven

Grebe SK, Singh RJ. Clin Biochem Rev 2011

van den Ouweland JMW, Kema IP. J Chromatography B 2012

Liquid Chromatography in a Nutshell

Mobile phase: -RP: polar -NP: apolar

Stationary phase: -RP: apolar -NP: polar

www.wikipedia.com

www.restek.com

www.phenomenex.com

www.wikipedia.com

www.restek.com

www.restek.com

Demystifying Mass Spectrometry

www.wikipedia.com

www.wikipedia.com

v

q F

F

www.wikipedia.com

www.wikipedia.com

www.shimadzu.com

www.shimadzu.com

www.shimadzu.com

www.planetorbitrap.com

Raida M et al. J Am Soc Mass Spectrom 1999

Q IT TOF FT OT FT ICR

Resolution - +/- + ++ +++

Mass Accuracy (ppm)

- - ++ ++ +++

Acquisition Speed (Hz)

+ + +++ +/- -

m/z range - + +++ + ++

Linearity ++ + + +/- +/-

Price +++ ++ +/- +/- -

Domon B, Aebersold R. Science 2006

Holcapek et al. J Chromatography B 2012

Ionization: the Missing Link

Grebe SK, Singh RJ. Clin Biochem Rev 2011

Grebe SK, Singh RJ. Clin Biochem Rev 2011

Vogeser M, Seger C. Clin Chem 2010

Holcapek et al. J Chromatography B 2012

Practical Example

• Develop a LC-MS/MS method for X

• How to start?

Sample Preparation

• Dilute and shoot • Protein precipitation • SPE • Liquid-Liquid • IAC

Purity Cost Time

After Google

• Tuning • Optimalisation/Amelioration • Validation (extended)

– also ME – IS – Linearity – LOQ – Interferences

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