Sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18® solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery® C18, 5 μm, 150×2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)–acetonitrile–methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC–MS–MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.