LC-MS/MS method for simultaneous quantification of lopinavir and ritonavir in human plasma

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of lopinavir and ritonavir in human plasma.  Analytes were separated from plasma by a  combination of alkalinized protein precipitation and liquid-liquid extraction with ethyl acetate.  Chromatographic separation was performed on a Agilent ZORBAX Eclipse XDB-C₁₈ column with the mobile phase consisted of methanol-0.1% formic acid in water (80∶20).  A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive ion mode.  Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 629.6 → 155.2, m/z 721.4 → 268.2, and m/z 515.2 → 276.2 for lopinavir, ritonavir and telmisartan (internal standard), respectively.  The method showed a good linearity in a concentration range of 62.5 − 10 000 ng·mL⁻¹ for lopinavir, and 12.5 − 2 000 ng·mL⁻¹ for ritonavir.  The lower limits of quantification were 15 pg·mL⁻¹ and 8 pg·mL⁻¹ for lopinavir and ritonavir, respectively.  The intra- and inter-day precision was less than 15% and the absolute recovery was above 75%.  This method was selective and rapid, sensitive for investigating blood drug concentrations in clinics.