Setting up a 2D-LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6-oxo-PGF1α and TXB2 as markers for hemostasis assessment

6-oxo-PGF(1 alpha) and TXB2 are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1 alpha) and TXB2 metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB2 anomers for a robust quantitation measurement. After a protein precipitation, 100 mu l of the supernatant (corresponding to 50 mu l of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1 alpha) and and a LOQ of 25 pg/ml for TXB2. Copyright (C) 2008 John Wiley & Sons, Ltd.