The Validation of Plasma Darunavir Concentrations Determined by the HPLC Method for Protease Inhibitors

Abstract

Darunavir (DRV), a new protease inhibitor (PI), is used to treat human immunodeficiency virus (HIV) type-1. According to in vitro experiments, DRV was active against HIV-1 with PI resistance mutations and against PI resistant clinical isolates. [1][2][3][4] This drug is expected to be effective in antiretroviral treatment-experienced patients, such as those possessing HIV-1 strains which are resistant to more than one PI. [5][6][7][8] Bouche et al. recently determined plasma DRV concentrations using liquid chromatography-tandem mass spectrometry (LC/MS/MS). 9) However, as LC/MS/MS equipment is very expensive and unavailable in conventional hospital laboratories, development of alternate methods is necessary.We have already developed a simple HPLC method for simultaneous quantitative determination of seven HIV protease inhibitors and efavirenz. 10) We expect DRV can be measured using this method because amprenavir, whose chemical structure is quite similar to DRV, was successfully measured.In this study we aimed to validate the measurement of plasma DRV concentrations using the HPLC method. This is the first report where plasma DRV concentration has been measured using this HPLC method. MATERIALS AND METHODSStandard Solutions and Chemicals DRV was supplied by Tibotec Pharmaceuticals Ltd. (Eastgate Village, Eastgate, Little Island, Co Cork, Ireland). The internal standard (IS), 6,7-dimethyl-2,3-di(2-pyridyl)-quinoxaline, was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Stock solutions of DRV and IS were prepared by dissolving accurately weighed amounts of each reference compound in water/ ethanol (50 : 50, v/v) to yield concentrations of 259 mg/ml for DRV, and 588 mg/ml for IS. These stock solutions were stored at Ϫ80°C and thawed until the day of analysis. The stock solution was diluted in drug-free plasma to yield concentrations of 0.13, 1.30, 2.59, 5.18 and 10.36 mg/ml for DRV. All other chemicals and solvents were of analytical grade and have been described in our previous report. 10)Chromatography The HPLC system consisted of a Waters pump (model 515), a 717 plus autosampler, and a 2487 dual l absorbance detector coupled to the Empower TM software (Waters, Milford MA, U.S.A.). The analytical column was a Radial-Pak Nova-Pak C 18 column (4 mm, 8ϫ100 mm, Waters) protected by Guard-Pak Inserts Nova-Pak C 18 precolumn. Absorbance was measured at 205 nm and separations were performed at 30°C. The mobile phase consisted of 39% 50 mM phosphate buffer (pH 5.9), 22% methanol and 39% acetonitrile. The assay run time was 30 min with a flow rate of 1.8 ml/min. Drugs were quantified by measuring the peak areas under the chromatograms. The other equipment and methodology used in this study have been described in our previous report. 10)Sample Preparation Two milliliters of ethyl acetate/nhexane (50 : 50, v/v) containing the IS (3.55 mg/ml) and 1 ml of 0.5 M sodium carbonate were added to a 500 ml plasma sample. The mixture was vortexed and then centrifuged at 3500ϫg for 5 min. The organic layer was separated and e...

A Potent HIV Protease Inhibitor, Darunavir, Does Not Inhibit ZMPSTE24 or Lead to an Accumulation of Farnesyl-prelamin A in Cells

“…c affinity constant for the binding of the drug to wild-type HIV-1 protease, measured by surface plasmon resonance (36). d Trough and maximum drug concentrations in the plasma (31,32,46). e 50% inhibitory activity against mouse ZMPSTE24 (12).…”

Characteristics, Complexation and Analytical Methods of Darunavir

“…We performed a literature search on methods for analysis of darunavir and were found to detect the drug in tablets by thin layer chromatography (TLC), UV spectrophotometry, infrared spectroscopy (IR) and high performance liquid chromatography (HPLC) [21,22], in plasma by HPLC [3,15], ultra efficiency liquid chromatography coupled to mass spectrometry (UHPLC-MS) [23] and HPLC coupled with mass spectrometry (HPLC-MS) [4]. It was found the HPLC-MS method for simultaneous detection of darunavir and other antiretroviral drugs in plasma [2,[24][25][26][27][28] and in blood, saliva and tissue [29].…”

“…Its spectrum is very broad and it is effective against all subtypes of HIV-1 and HIV-2. It is also more active against the most resistant HIV compared to other protease inhibitors [3,4,15], and its robustness against the known mechanisms of HIV resistance is also superior to other available protease inhibitors.…”

“…Darunavir, the newest protease inhibitors, is a non-peptide synthetic analogue of amprenavir [1], which was approved by the U.S.FDA in June 2006 and in February 2007 the European Commission approved its marketing [2]. It is effective in patients with experiments in antiretroviral treatments, such as those with HIV-1 strains that are resistant to more than one protease inhibitors [3]. Darunavir ( Fig.…”

Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC–MS–MS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

“…A number of methods have been reported for the analysis of efavirenz alone in plasma, and in combination with other antiretroviral agents including HPLC [16][17][18][19] and LC-MS-MS [20][21][22]. To date, no pharmacokinetic data of efavirenz metabolites with efavirenz after oral administration of efavirenz has been reported in the literature.…”