Determination of lamivudine/stavudine/efavirenz in human serum using liquid chromatography/electrospray tandem mass spectrometry with ionization polarity switch

A high-performance liquid chromatography/tandem mass spectrometry (LC-MS-MS) method with ionization polarity switch was developed and validated in human serum for the determination of a lamivudine (3TC)/stavudine (d4T)/efavirenz combination HIV therapy. Solid phase extraction (SPE) was used to extract these anti-HIV drugs and internal standard aprobarbital. A gradient mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer with pH adjusted to 4.5 using glacial acetic acid was utilized to separate these drugs on a hexylsilane column (150 x 2.0 mm i.d.). The total run time between injections was 18 min. The precursor and major product ions of these drugs were monitored on a triple quadrupole mass spectrometer in the multiple reactions monitoring (MRM) mode. Ionization polarity was switched in the middle of the LC run allowing these anti-HIV drugs with different physicochemical properties to be detected simultaneously. The effect of ion suppression from human serum was studied and no interference with the analysis was noted. The method was validated over the range of 1.1-540 ng/mL for 3TC, 12.5-6228 ng/mL for d4T and 1.0-519 ng/mL for efavirenz. The method was shown to be accurate, with intra-day and inter-day accuracy less than 14.0% and precise, with intra-day and inter-day precision less than 13.1%. The extraction recoveries of all analytes were higher than 90%.