Development and validation of a high-throughput and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of oseltamivir phosphate and its oseltamivir carboxylate metabolite in human plasma for pharmacokinetic studies.

A rapid, sensitive and rugged solid-phase extraction ultraperformance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for determination of oseltamivir phosphate (OP) and oseltamivir carboxylate (OC) in human plasma. The procedure for sample preparation includes a simple SPE extraction procedure coupled with a Chromatopack C(18) column (50 × 3.0 mm, i.d., 3.0 µm) with isocratic elution at a flow-rate of 0.600 mL /min and acyclovir was used as the internal standard. The analysis was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. Using 500 µL plasma, the methods were validated over the concentration ranges 0.92-745.98 and 5.22-497.49 ng/mL for OP and OC, with a lower limit of quantification of 0.92 and 5.22 ng/mL. The intra- and inter-day precision and accuracy of the quality control samples were within 10.1%. The recovery was 68.72, 70.66 and 71.59% for OP, OC and IS, respectively. Total run time was only 1.0 min. The method was highly reproducible with excellent chromatography properties.