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A simple, specific, and high throughput liquid chromatography tandem mass spectrometry method is developed for the determination of tenofovir, a nucleotide reverse transcriptase inhibitor, in human plasma using adefovir as internal standard. Plasma samples are prepared by solid-phase extraction of the analyte and internal standard using Waters Oasis MCX cartridges (1 cc, 30 mg). The chromatographic separation is achieved on a reversed-phase Chromolith, C18 analytical column (100 mm × 4.6 mm, 5 µm) under isocratic conditions. The mobile phase consists of 0.5% formic acid in water and acetonitrile (90:10, v/v) to give a run time of 1.8 min. The protonated precursor → product ion transitions for tenofovir and IS are monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The fragmentation pathways for tenofovir are studied by varying the collision energy (5–55 V) using nitrogen as CAD gas. A linear dynamic range of 3.1–1002.0 ng/mL is established using 0.2 mL plasma sample. The method is fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, recovery, stability, and dilution integrity. It is applied to a bioequivalence study in 43 human subjects after oral administration of 300 mg tablet formulation under fasting conditions.

Rapid and Specific Liquid Chromatographic Tandem Mass Spectrometric Determination of Tenofovir in Human Plasma and its Fragmentation Study