Loading…
A highly efficient, high-throughput lipidomics platform for the quantitative detection of eicosanoids in human whole blood
We have developed an ultra-performance liquid chromatography–multiple reaction monitoring/mass spectrometry (UPLC–MRM/MS)-based, high-content, high-throughput platform that enables simultaneous profiling of multiple lipids produced ex vivo in human whole blood (HWB) on treatment with calcium ionopho...
Saved in:
Published in: | Analytical biochemistry 2013-02, Vol.433 (2), p.181-188 |
---|---|
Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Summary: | We have developed an ultra-performance liquid chromatography–multiple reaction monitoring/mass spectrometry (UPLC–MRM/MS)-based, high-content, high-throughput platform that enables simultaneous profiling of multiple lipids produced ex vivo in human whole blood (HWB) on treatment with calcium ionophore and its modulation with pharmacological agents. HWB samples were processed in a 96-well plate format compatible with high-throughput sample processing instrumentation. We employed a scheduled MRM (sMRM) method, with a triple–quadrupole mass spectrometer coupled to a UPLC system, to measure absolute amounts of 122 distinct eicosanoids using deuterated internal standards. In a 6.5-min run, we resolved and detected with high sensitivity (lower limit of quantification in the range of 0.4–460pg) all targeted analytes from a very small HWB sample (2.5μl). Approximately 90% of the analytes exhibited a dynamic range exceeding 1000. We also developed a tailored software package that dramatically sped up the overall data quantification and analysis process with superior consistency and accuracy. Matrix effects from HWB and precision of the calibration curve were evaluated using this newly developed automation tool. This platform was successfully applied to the global quantification of changes on all 122 eicosanoids in HWB samples from healthy donors in response to calcium ionophore stimulation. |
---|---|
ISSN: | 0003-2697 1096-0309 |
DOI: | 10.1016/j.ab.2012.10.022 |