Automated liquid–liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid–liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2 mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10 mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5 mL/min. The linear dynamic range was established over the concentration range 1.1–213.1 ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5 min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10 mg extended release methoxsalen formulation under fasting condition.