Ultra-performance liquid chromatographic method for simultaneous quantification of HIV non-nucleoside reverse transcriptase inhibitors and protease inhibitors in human plasma

Abstract

Um método rápido para a determinação de seis inibidores de protease (indinavir, amprenavir, saquinavir, atazanavir, lopinavir e ritonavir) e de dois inibidores não-nucleosídicos da transcriptase reversa (efavirenz e nevirapine), empregando cromatografia líquida de ultra-eficiência com detector de arranjo de diodos foi desenvolvido e validado. Após extração liquido-líquido de 0,5 mL de plasma com metil-tert-butil éter, os analitos foram separados em uma coluna ACQUITY UPLC BEH ® C18 (2,1 × 150 mm, d.p. 1,7 µm), eluída com um gradiente de tampão fosfato trietilamônio pH 3.0 e acetonitrila. O tempo total de análise cromatográfica foi de 9,5 min. As curvas de calibração foram lineares entre 0,1 a 10,0 µg mL -1. O limite inferior de quantificação foi 0,1 µg mL -1 para todos os fármacos. A exatidão esteve entre 94,9 e 103,5%. Os coeficientes de variação intra e inter-dias foram inferiores a 7,7% para todos os analitos. Os rendimentos de extração foram superiores a 88,2%.A fast ultra-performance liquid chromatographic with diode-array detection method has been developed and validated for the determination of six protease inhibitors (indinavir, amprenavir, saquinavir, atazanavir, lopinavir, and ritonavir) and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). After liquid-liquid extraction of 0.5 mL plasma with methyl-tert-butyl ether, the analytes were separated on a ACQUITY UPLC BEH ® C18 column (2.1 × 150 mm, p.d. 1.7 µm) column eluted with a gradient of acetonitrile and triethylammonium phosphate buffer 5 mmol L -1 pH 3.0. The total run time was 9.5 min. Calibration curves were linear in the range 0.1 to to 10.0 µg mL -1. The lower limit of quantitation was 0.1 µg mL -1 for all drugs. Accuracy ranged from 94.9 to 103.5%. Both interday and intraday coefficients of variation were less than 7.7% for all analytes. The extraction yields were greater than 88.2%. Keywords: antiretroviral drugs, ultra-performance liquid chromatography, therapeutic drug monitoring, UPLC-DAD IntroductionAntiretroviral therapy for human immunodeficiency virus (HIV) infections usually consists of combinations of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and, more recently, entry inhibitors and integrase inhibitors. 1 The currently approach to the therapy of HIV infection is the so-called "highly active antiretroviral therapy" (HAART), which is based on the combination of several drugs in the daily dosing regimen. The usual HAART scheme consists of one or more NRTIs, one or more PIs and one NNRTI.2 Several reports had demonstrated the relationship between plasma drug concentrations and clinical effects, either toxicity or efficacy, for compounds of the NNRTI and PI groups. [3][4][5][6][7][8] Therefore, these drugs are prone for therapeutic drug monitoring (TDM) programs and, considering the necessity of interlaboratory comparison of results for the use of consensual therapeutic target levels, reliable analytical methods mu...

Related factors to atazanavir plasma levels in a cohort of HIV positive individuals with undetectable viral load

“…Ultra performance liquid chromatography (UPLC) with diode array detection was the method used to determine ATV plasma concentrations. 11 After the liquid–liquid extraction of 0.5 mL of plasma with methyl- tert -butyl ether, the analytes were separated on a ACQUITY UPLC BEH ® C18 column (2.1 mm × 150 mm, p.d. 1.7 μm) eluted with a gradient of triethylammonium phosphate buffer pH 3.0 and acetonitrile.…”

“…In that context, higher plasma levels of the drug is one of the key factor that might contribute to the development of toxicity. 10 , 11 Therapeutic drug monitoring can be a useful weapon for dose adjustment in order to avoid concentrations that are out of the therapeutic range, thus preventing toxicity. 5 , 12 , 13 The purpose of this study was to assess the factors associated with plasma concentrations of ATV in HIV infected individuals with undetectable viral load in order to try to prevent toxicities attributed to this drug.…”

Ultra-performance liquid chromatographic method for measurement of voriconazole in human plasma and oral fluid

“…18 However, the coupling of UPLC to PDA detection allows fast analysis while keeping the advantages and robustness of UV detection modes, at a moderate cost. 19 In the present work, we validated a novel and fast UPLC-PDA assay for TDM of VRC in human plasma and oral fluid samples, after a simple liquid-liquid extraction.…”

Stability Indicating High Performance Thin Layer Chromatographic Method for Determination of Atazanavir and Ritonavir in Combined Tablet Dosage Forms

“…Ritonavir (RTV), 2, 4, 7, 12-Tetraazatridecan-13oic acid, 10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-5-thiazolylmethyl ester [5S-(5R*,8R*,10R*,11R*)] is a potent cytochrcome P -450 (CYP)3A inhibitor and usually used as pharmacokinetic booster for other protease inhibitor including Atazanavir, thereby providing increased plasma level of Atazanavir 2 . Literature survey reveals High Performance Liquid Chromatographic (HPLC) [3][4][5][6][7][8][9] , LC-MS 10 and Ultra Performance Liquid Chromatography (UPLC) 11 methods for determination of ATV as single and in combination with other drugs in human plasma. Also Spectrophotometric method for degradation studies of ATV in dosage form has been also reported 12 .…”