Setting up a 2D‐LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6‐oxo‐PGF_1α and TXB₂ as markers for hemostasis assessment

Abstract

6-oxo-PGF(1alpha) and TXB(2) 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(1alpha) and TXB(2) 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 TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl 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(1alpha), and and a LOQ of 25 pg/ml for TXB(2).

A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites

“…Notably, DIOL column (Fig. 4b) gave two peaks for the enantiomers of TXB 2 , which has also been reported with C18 column by Casetta et al 20 while FP gave a single peak. The AA-metabolites containing alcoholic moieties exhibited the greatest retention on the DIOL and 2-PIC columns as these columns are known to have somewhat similar retentions.…”

“…As AA-metabolites are prone to oxidation, antioxidants such as BHT and COX inhibitors (indomethacin) were incorporated into the extraction process to limit any alteration of AA-metabolites during the process. 20 In general, our sample preparation technique was fast, reproducible and cost effective and maintained the native nature of the analytes. According to some previous reports, the extraction follows enrichment of analytes by SPE to remove all endogenous interfering substances.…”

“…LOQ were 0.5 ng mL −1 (equivalent of 1.5 pg on column) for PGE 2 , PGD 2 , PGF 2α , LTB 4 and 2.5 ng mL −1 (equivalent to 7.5 pg on column) for 6KetoPGF 1α which are comparable or better as compared to already existing LC-MS/MS methods for similar analytes (Table 2). 4,20,24 Precision and accuracy were assessed by replicate analysis (n = 4) of spiked plasma samples at three different concentrations and data are presented in Table 3. The intra-and inter-day precisions were between 1.2% and 13.5% for all the analytes and the accuracy was within ±10% excluding 6KetoPGF 1α .…”

A method for the direct injection and analysis of small volume human blood spots and plasma extracts containing high concentrations of organic solvents using revered-phase 2D UPLC/MS

“…31,32 Over the last few years 2D chromatography has seen a resurgence in interest for both comprehensive chromatography of complex samples and targeted analysis of peptide therapeutics in a complex mixture. 33,34 In this LC format the analysis can be performed in several different modes including trap-elute, forward flush, backward-flush and heart cutting. 35 These approaches can deliver greater specificity to the analysis yielding more precise and accurate results.…”

Intermittent hypoxia-activated cyclooxygenase pathway: role in atherosclerosis

“…Urinary 11-dTXB 2 of patients, TXB 2 and 6-keto-PGF-1a in supernatants of mice were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described [9,22]. The limits of quantitation of TXB 2 and 6-keto-PGF1a were 14 pg?mL -1 and 7.25 pg?mL -1 , respectively, and their inter-and intra-assay coefficients of variation were ,12% for both analytes.…”