UniGE, Life Sciences Mass Spectrometry 1 Quantification of endogenous and exogenous metabolites in small samples using parallel narrow bore to capillary LC with fast polarity switching MRM G. Hopfgartner 1 , K. Watanabe 1,2 and E.Varesio 1 1). Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences EPGL, University of Lausanne, University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland 2) Global Application Development Center Shimadzu Corporation, Kyoto, Japan EBF 7th Open Meeting, Diversity of the Bioanalytical Techniques November 20, 2014, Barcelona, Spain
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UniGE, Life Sciences Mass Spectrometry 1
Quantification of endogenous and exogenous metabolites in small samples using parallel
narrow bore to capillary LC with fast polarity switching MRM
G. Hopfgartner1, K. Watanabe1,2 and E.Varesio1
1). Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences EPGL, University of Lausanne, University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland
2) Global Application Development Center Shimadzu Corporation, Kyoto, Japan
EBF 7th Open Meeting, Diversity of the Bioanalytical Techniques
November 20, 2014, Barcelona, Spain
UniGE, Life Sciences Mass Spectrometry 2
Life is a Dynamic System !
Metabolome B(t2) Lipidome B(t2) Proteome B(t2)
Aging Metabolome A(t1)
Lipidome A(t1) Proteome A(t1)
Disease or Toxicological exposure
Metabolome D Lipidome D Proteome D
Cure
Metabolome A’ Lipidome A’ Proteome A’
Metabolome Y Lipidome Y Proteome Y
Aging Death
or Change in Lifestyle
UniGE, Life Sciences Mass Spectrometry 3
Metabolites: Large Chemical Space, Large Dynamic Range, Different MS Response Factors!
QUAL/QUAN Data Idependant Acquisition Mode (DIA/MS)
m/z
%
UniGE, Life Sciences Mass Spectrometry 5
Challenges with Pharmacometabolomics
• Multi-components assays for drugs, endo and exo metabolites
• Limited samples volumes • Large dynamic range • Large number of sample to be analyzed
Use of parallel LC systems Tune Sensitivity with different LC column internal diameter
UniGE, Life Sciences Mass Spectrometry 6
LC-SRM/MS Assay for multiple Metabolites
The constrains: • Need of different mechanisms of retention • Multiple types of biological matrices (plasma, tissue,..) • ESI in positive and negative mode
Parallel LC systems with column-switching QqQ and fast polarity switching
UniGE, Life Sciences Mass Spectrometry 7
The Analytes (n= 75)
49 analytes detected in positive mode 36 analytes detected in negative mode
UniGE, Life Sciences Mass Spectrometry 8
Dual UHPLC Column-Switching System with SCX and SAX trapping columns
LC-MS 8050
pH = 2.5
pH = 6.9
UniGE, Life Sciences Mass Spectrometry 9
Analytical Sequence
UniGE, Life Sciences Mass Spectrometry 10
LC-SRM/MS Pause time 10 msec, dwell time 15 msec (LCMS 8050 - 50 transitions)
UniGE, Life Sciences Mass Spectrometry 11
LC-SRM/MS Pause time 2 msec, dwell time 2 msec (LCMS 8050 - 50 transitions)
Gain in Sensitivity 0.3 mm versus 2.1 mm i.d. columns and Matrix Effects
Expected theoretical gain = 49 fold
*Only singly charge SRM transition n=3, CV< 10%
UniGE, Life Sciences Mass Spectrometry 27
Conclusions • In mass spectrometry based pharmacometabolomics
several dimensions needs to be take in account in particular the chemical space (m/z, -/+ detection and response factors).
• As chromatographic performance is important fast acquiring MS are mandatory with fast polarity switching
• Dual LC systems are an elegant way to increase throughput or to expend the numbers of analytes to monitor.
• Column i.d. reduction still is a promising approach when limited sample is available and to achieve good sensitivity. However, differences is MS response at different flow regimes with standards or extracts can be observed and should be further investigated.
UniGE, Life Sciences Mass Spectrometry 28
Acknowledgments
UniGE, Life Sciences Mass Spectrometry 29
University of Geneva Bandar Alghanem Aivett Bilbao Pena Tobias Bruderer Sandrine Cudré Chantal Grivet Sandra Jahn Andras Kiss Eliane Kuehn Jonathan Sidibé Ying Zhang
Shimadzu Neil Loftus AB Sciex Ron Bonner Yves J.C. Le Blanc
Acknowledgments
UniGE, Life Sciences Mass Spectrometry 30
Important Dates
Opening of abstract submissions and registration: 1 Octobre 2014 Closing of abstract submissions for oral contributions 5 January 2015 Closing of poster abstract submissions and Early Bird registration 1 April 2015 www.hplc2015-geneva.org