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.
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy. Electronic address: isabella.squellerio@ccfm.it.
Isabella
I
Squellerio
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy. Electronic address: benedetta.porro@ccfm.it.
Benedetta
B
Porro
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy. Electronic address: paola.songia@ccfm.it.
Paola
P
Songia
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy. Electronic address: fabrizio.veglia@ccfm.it.
Fabrizio
F
Veglia
Università degli Studi di Milano, Dipartimento di Scienze Farmacologiche e Biomolecolari, Via Balzaretti 9, 20100 Milan, Italy. Electronic address: donatella.caruso@unimi.it.
Donatella
D
Caruso
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy; Università degli Studi di Milano, Dipartimento di Scienze Farmacologiche e Biomolecolari, Via Balzaretti 9, 20100 Milan, Italy. Electronic address: elena.tremoli@ccfm.it.
Elena
E
Tremoli
Centro Cardiologico Monzino-IRCCS, Via Parea 4, 20138 Milan, Italy; Università degli Studi di Milano, Dipartimento di Scienze Cliniche e di Comunità, Via Parea 4, 20138 Milan, Italy. Electronic address: viviana.cavalca@unimi.it.
Viviana
V
Cavalca
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000032:analysis / Q000097:blood*; D000328:Adult; D016718:Arachidonic Acid / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013929:Thromboxane B2 / Q000032:analysis / Q000097:blood*
D016718:Arachidonic Acid; D013929:Thromboxane B2; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid
12(S)-hydroxyeicosatetraenoic acid; Mass spectrometry; Platelet; Serum; Thromboxane B(2)