Simultaneous determination of lopinavir and three ester prodrugs by LC-MS/MS in lysates of BeWo cells

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionization (ESI) mode has been validated for the simultaneous determination of lopinavir (LPV) and three prodrugs, succinic acid ester of lopinavir (SLPV), glutaric acid ester of lopinavir (GLPV), and diglycolic acid ester of lopinavir (DLPV) in BeWo cell lysate. A methanolic precipitation was employed after addition of an internal standard ritonavir (RTV), followed by centrifugation, separation of the supernatant liquid, evaporation, and reconstitution. A reversed-phase Phenomenex C18 column was used for separation of lopinavir and three prodrugs with a mobile phase which comprises methanol (solvent A), acetonitrile (solvent B), and 50 mM ammonium acetate buffer adjusted to pH 5.5 with acetic acid (solvent C), with isocratic elution (A:B:C = 34:34:32, v/v). The detection of target compounds was conducted using multiple reaction monitoring (MRM) with the following transitions of m/z 729 → 447, 745 → 563, 743 → 429, 629 → 447 and 721 → 296 to measure SLPV, DLPV, GLPV, LPV and RTV (I.S.), respectively. The calibration ranges were determined using quadratic regression over concentration ranges of 7-743 ng/mL (DLPV), 7.5-745 ng/mL (GLPV), 7-729 ng/mL (SLPV) and 6-189 ng/mL (LPV). The reverse calculated residuals for LPV and these prodrugs were all less than 15% different from nominal (range). The method we established was fully validated for accuracy (≤15% different from nominal) and precision (≤15% RSD). The results proved that the method is accurate and specific, and also this method has been successfully applied to test the uptake of lopinavir and prodrugs into BeWo cells.