Comparison of extraction procedures for assessment of matrix effect for selective and reliable determination of atazanavir in human plasma by LC–ESI-MS/MS

A comparative study with three conventional extraction techniques namely protein precipitation (PP), liquid–liquid extraction (LLE) and solid phase extraction (SPE) has been demonstrated to assess the magnitude of matrix interference by post-column analyte infusion and post extraction analyte spiking for the determination of atazanavir from human plasma. Severe ion suppression observed in PP and to a lesser extent in LLE was circumvented by SPE on LiChrosep Sequence extraction cartridge. Based on these observations a selective, rugged and high throughput SPE-LC–MS/MS method has been developed for reliable determination of atazanavir in human plasma. The chromatographic separation was achieved on a Hypersil Gold C18 (50 mm × 4.6 mm, 5 μm) analytical column using 5 mM ammonium formate in water:methanol (10:90, v/v) as the mobile phase under isocratic conditions. The method was validated over a wide dynamic concentration range of 10–6000 ng/mL. The mean relative recovery and absolute matrix effect across quality controls were 84.9 and 93.2%, respectively. The precision value for relative matrix effect between eight different lots of plasma, expressed as %CV of the slopes of the calibration lines was 2.41. The stability of atazanavir under different storage conditions varied from −8.4 to 5.4%. The method was successfully applied to a bioequivalence study of 300 mg atazanavir capsule formulation in 24 healthy Indian males under fasting condition.

► A reliable SPE-LC–ESI-MS/MS is proposed for determination of atazanavir in human plasma.
► A comparative study with PP, LLE and SPE is demonstrated to show the magnitude of ion suppression.
► Matrix effect assessment is done by post-column analyte infusion and post extraction spiking methods.
► The method is practically free from matrix interference based on relative matrix effect in different lots of plasma.
► The application is demonstrated by a bioequivalence study in healthy volunteers and incurred sample reanalysis.