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

• An LC–MS/MS assay to quantify TXB2 and 12(S)-HETE is described.
• This method allows the quantification of both analytes in a small volume of serum.
• In healthy subjects TXB2 is 159 ± 15 ng/ml and 12(S)-HETE is 831 ± 50 ng/ml (mean ± SE).
• Female sex, age, platelet count and TXB2 are independent predictors of 12(S)-HETE.

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 0.244 ng/ml and 0.976 ng/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.

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