Kaletra is an important antiretroviral
drug, which has been developed by Abbott Laboratories. It is composed of
lopinavir (low-pin-a-veer) and
ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (
HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1
infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of
lopinavir and ritonavir in human plasma, in which
lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of
hexane:
ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v)
acetonitrile: 2 mM
ammonium acetate aqueous
solution containing 0.01%
formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of
tetrahydrofuran (THF),
methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for
drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for
lopinavir and 11-3,100 ng/mL for
ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both
lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.