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KMID : 0614020090240010077
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Journal of Pharmaceutical Sciences (C.N.U.)
2009 Volume.24 No. 1 p.77 ~ p.82
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LC/MS/MS Analyasis of Efavirenz, Indinavir, Lopinavir, Ritonavir in HIV-Infected Patients
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Chae Jung-Woo
Bae Kyung-Jin
Baek In-Hwan
Seo Jeong-Won
Lee Byung-Yo
Lee Eun-Joo
Nam Jin-Kyung
Kang Won-Ku
Kwon Kwang-Il
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Abstract
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Efavirenz indinavir and kaleta (co-formulation of lopinavir and ritonavir) are important antiretroviral drugs which have been proved to be human immunodeficiency virus (HIV) protease inhibitors and reduced the morbidity and mortality associated with HIV-1 infection. A brief and fast high performance liquid chromatography coupled with electrospray mass spectrometry (LC-MS/MS, API 4000) method for the determination of 4 anti-retroviral agents (efavirenz, lopinavir, indinavir, ritonavir) in human plasma was developed and validated. A simple protein precipitation method was used on 100μl of human plasma. And internal standard solution (10 ng/ml methaqualone) 1ml and reconstitution solution (MeOH) 1ml were added. After vortexing for 30 s and centrifuging at 13,200rpm for 10min, 2μl of supernatant was injected into the column (XTerra MS C_(18) column, 2.1mm × 50mm 3.5㎛ particle size). The mobile phase consisted of MeOH and 0.1% formic acid in water (80:20 , v/v). The chromatogram was run for 1.5 min at a flow rate of 300μl/min. A triple quadrupole mass spectrometer was operated in a positive ion mode (lopinavir, indinavir, ritonavir) and negative mode (efavirenz), simultaneously and multiple reaction monitoring (MRM) was used for drug quantification. The precursor-to-product ion transitions of m/z 316→69 (efavirenz) and 629→447 (lopinavir) and 614→421 (indinavir) and 721→296 (ritonavir)were used to measure and quantify the analyte. The limit of quantitation (LOQ) was 50 ng/ml (efavirenz, indinavir, ritonavir) and 100 ng/ml (lopinavir). The weighted (l/y²) calibration curve was linear over human plasma range 50∼5000ng/ml (efavirenz), 100∼20000ng/ml (lopinavir), 50∼10000ng/ml (indinavir), 50∼2000ng/ml (ritonavir), correlation coefficient(r²) of 4 antiretroviral agents were higher than 0.998. Accuracies and intra-run precisions ranged within 86.60 and 113.29%, 1.06 and 11.16% for all 4 drugs analysed. This analytical method used to determine these drugs was fast and easy to perform, with minimal sample preparation, and without compromising precision and accuracy. The developed method was successful to determine antiretroviral agents in human plasma, and proved suitable for clinical pharmacokinetic study.
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KEYWORD
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efavirenz, indinavir, ritonavir, lopinavir, pharmacokinetic, LC/MS/MS
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