Simultaneous determination of Pyragrel, a novel anti-thrombotic agent, and its two primary metabolites in plasma by HPLC-MS/MS.

Pyragrel is a novel thromboxane A2-synthetase inhibitor for the treatment of cerebral infarction, and it is currently being investigated in phase I clinical trials. This paper reports the first reliable LC-MS/MS method for the simultaneous determination of Pyragrel and its two main metabolites, M1 and M2, in human plasma. All analytes were extracted from human serum using liquid-phase extraction and separated on a Zorbax EcLipse XDB C18 column using isocratic elution with a mobile phase composed of methanol, water and formic acid (65:35:0.1, v/v/v). Determination of the analytes was achieved by tandem-mass spectrometry with positive electrospray ionization. The multiple reaction monitoring transitions under positive electrospray ionization were performed at m/z 329.0 → m/z 135.9 for Pyragrel, m/z 303.1 → m/z 135.0 for M1, m/z 331.2 → m/z 135.0 for M2, and 482.2 → m/z 258.0 for IS, respectively. The following parameters were validated: specificity, recovery, matrix effects, carry-over, linearity, sample stability under a variety of storage and handling conditions, and stock solution stability. The validated method has been successfully applied to an initial pharmacokinetic study in healthy volunteers following intravenous administrations of 60 mg of Pyragrel, and this method will facilitate further studies involving more comprehensive identification of the metabolic profile of Pyragrel and the appropriate dosage regimen.