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

Abstract

We developed a rapid and sensitive method for determining efavirenz, 8-hydroxyefavirenz, and 8,14-dihydroxyefavirenz in human plasma simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Three compounds and ritonavir, an internal standard, were extracted from plasma using ethyl acetate in the presence of 0.1 M sodium carbonate after incubation of b-glucuronidase (500 U). After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:20 mM ammonium acetate, 90:10, v/v) and injected onto a reversed-phase C 18 column. The isocratic mobile phase was eluted at 0.2 mL min -1 . The ion transitions monitored in multiple reaction-monitoring mode were m/z 314 ? 244, 330 ? 258, 346 ? 262, and 721 ? 296 for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The retention time is 1.93, 1.70, 1.52, and 1.82 min for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The coefficients of variation of the assay precision were less than 10.7%, and the accuracy was 90-111%. The lower limits of quantification (LLOQ) were 5 ng mL -1 for efavirenz and 8-hydroxyefavirenz. This method was used to measure the plasma concentrations of efavirenz and its metabolites from healthy volunteers after a single 600 mg oral dose of efavirenz. This analytical method is a very rapid, sensitive, and accurate to determine the pharmacokinetic profiles of efavirenz including its metabolites.

Rifampin enhances cytochrome P450 (CYP) 2B6-mediated efavirenz 8-hydroxylation in healthy volunteers

“…A previously published liquid chromatography–tandem mass spectrometry (LC–MS/MS) [API 3000, Applied Biosystems, Foster City, CA, USA; equipped with an electrospray ionization interface] method [35] was slightly modified to quantify efavirenz, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz concentrations in plasma and urine. Plasma or urine samples (200 µL) were incubated with 2 mL of 0.2 M sodium acetate buffer (pH 5.0) and 100 µL of 10,000 unit β-glucuronidase at 37 °C for 17 h. After adding the internal standard (20 µL of 5 µg/mL ritonavir) and alkalinized with 1 mL sodium carbonate buffer (0.1 M Na 2 CO 3 /NaHCO 3 , pH = 9.4), the sample was extracted with 5 mL ethyl acetate.…”

A simple, rapid, economical, and practical method for the determination of efavirenz in plasma of Chinese AIDS patients by reverse phase high-performance liquid chromatography with ultraviolet detector

“…The method established in this paper, by RP-HPLC with UV detector, has a lower requirement for equipments compared to reported methods (17)(18)(19)(20)(21)(22)(23)(24)(25). In our method, diazepam was used as internal standard because of its cheapness, accessibility, and well-separated with efavirenz.…”

“…Up to now, several methods for the determination of efavirenz in plasma using LC-MS (17,18), LC-MS-MS (19,20), MALDI-TOF/IOF (21), HPLC-PAD (22)(23)(24)(25), etc., have been published. These methods may not be widely used in underdeveloped countries because of the inaccessibility and high costs of laboratory equipments.…”

Efavirenz and Metabolites in Cerebrospinal Fluid: Relationship with CYP2B6 c.516G→T Genotype and Perturbed Blood-Brain Barrier Due to Tuberculous Meningitis

“…The samples were evaporated to dryness at room temperature in a stream of nitrogen. The samples were then incubated at 37°C for 2 h with 400 μl of a solution containing 200 units of β-glucuronidase from Helix pomatia in 0.2 M sodium acetate buffer (pH 5) ( 27 ). The samples were subsequently alkalinized with 20 μl of potassium carbonate buffer (0.1 M, pH 9.4) and extracted with 3 ml of a mixture of organic solvents, ethyl acetate-hexane (60:40, vol/vol).…”