Liquid chromatography-tandem mass spectrometry for simultaneous measurement of thromboxane B2 and 12(S)-hydroxyeicosatetraenoic acid in serum

Arachidonic acid (AA) is metabolized in human platelets by two main pathways: via cyclooxygenase (COX-1) to prostaglandins and thromboxane (TX)A₂ and via 12-lipoxygenase (12-LOX) to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). While COX products are known to regulate platelet reactivity, the role of 12-LOX metabolites is still controversial. To better understand the platelet enzymatic pathways, we developed a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of both platelet metabolites in human serum. After the addition of deuterated d₄-TXB₂ and d₈-12(S)-HETE as internal standards and the solid-phase extraction of serum samples, analytes were resolved using reversed-phase C₁₈ column and quantified using negative ion electrospray ionization-tandem mass spectrometry. Intra and interassay imprecisions were less than 10% for both analytes. The lower limits of quantification were 0.244ng/ml and 0.976ng/ml for TXB₂ and 12(S)-HETE, respectively. This method was applied to measure platelet metabolites in healthy subjects (n=35). LC-MS/MS allows rapid, simultaneous, sensitive and accurate quantification of both platelet AA products in human serum with a small sample volume required and a minimal sample preparation.