Liquid Chromatography – tandem Mass Spectrometry An Introduction for Beginners Steven Pauwels Jessa Hospital, 7 th of May 2013
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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
Presentator
Presentatienotities
Begin immunosuppresiva: vaak voor TDM/tox: nog geen immunoassay, specificiteit
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
Presentator
Presentatienotities
http://www.hitachi-hitec.com/global/science/lc/lc_basic_1.html
www.restek.com
www.phenomenex.com
www.wikipedia.com
www.restek.com
www.restek.com
Demystifying Mass Spectrometry
www.wikipedia.com
www.wikipedia.com
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q F
F
www.wikipedia.com
www.wikipedia.com
www.shimadzu.com
www.shimadzu.com
www.shimadzu.com
Presentator
Presentatienotities
Shimadzu
www.planetorbitrap.com
Raida M et al. J Am Soc Mass Spectrom 1999
Presentator
Presentatienotities
Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate Manfred Raidaa, , , Peter Schulz-Knappea, Gabriele Heinea, Wolf-Georg Forssmanna a Lower Saxony Institute for Peptide Research, Hannover, Germany Received 10 December 1997 Revised 21 July 1998 Accepted 22 August 1998 Available online 15 July 1999 http://dx.doi.org/10.1016/S1044-0305(98)00117-2, How to Cite or Link Using DOI Permissions & Reprints
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
Presentator
Presentatienotities
Mass Spectrometry and Protein Analysis Science 14 April 2006: �vol. 312 no. 5771 212-217
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
Presentator
Presentatienotities
www.molecularstation.com
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
www.bioanalytics.com
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