Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction.
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.
Wang
Perry G
PG
Drug Analysis Department, Abbott Laboratories, Abbott Park, IL 60064, USA. perry.wang@abbott.com
Wei
Jack S
JS
Kim
Grace
G
Chang
Min
M
El-Shourbagy
Tawakol
T
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005663:Furans / Q000737:chemistry; D006801:Humans; D061466:Lopinavir; D013058:Mass Spectrometry / Q000379:methods*; D000432:Methanol / Q000737:chemistry; D011744:Pyrimidinones / Q000097:blood* / Q000302:isolation & purification; D015203:Reproducibility of Results; D019438:Ritonavir / Q000097:blood* / Q000302:isolation & purification; D012997:Solvents / Q000737:chemistry; D014867:Water / Q000737:chemistry
D005663:Furans; D011744:Pyrimidinones; D012997:Solvents; D014867:Water; D061466:Lopinavir; C018674:tetrahydrofuran; D019438:Ritonavir; D000432:Methanol