A highly sensitive ultra performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) technique for quantitation of protein free and bound efavirenz (EFV) in human seminal and blood plasma

Abstract

A combined UPLC-tandem mass spectrometric (UPLC-MS/MS) technique has been validated for quantitation of protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and seminal plasma. The analytical method possesses capabilities for concentration measurements of EFV ranging from 0.5 ng/ml to 10,000 ng/ml with an accuracy (%dev) of −5.2% to 8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients of variation (r 2 ) > 0.98. The method employs a racemic fluorinated analog of EFV (F-EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC column via gradient elution with detection via negative ion multiple reaction monitoring (MRM). EFV and F-EFV, respectively, were detected via the following MRM transitions: m/z 314.0 > 244.1 and m/z 298.0 > 227.9. The time required for the analysis of each sample was 8.0 minutes. The analytical technique is capable of a reliable detection limit of ~15-20 femtomoles of EFV injected on column.

Protein-Free Efavirenz Concentrations in Cerebrospinal Fluid and Blood Plasma Are Equivalent: Applying the Law of Mass Action To Predict Protein-Free Drug Concentration

“…Concentrations of EFV were determined using individual ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assays performed using an AB Sciex QTRAP 5500 (AB Sciex, Foster City, CA) interfaced with an Acquity UPLC (Waters Inc., Milford, MA). EFV samples were analyzed by a modified version of a previously described method (27). Briefly, samples were extracted using a liquid-liquid extraction method of 600 l of 50 mM ammonium formate and 900 l of a solution of hexanes and ethyl acetate (1:1).…”

Compartmentalization and Antiviral Effect of Efavirenz Metabolites in Blood Plasma, Seminal Plasma, and Cerebrospinal Fluid

“…EFV and EFV metabolites were detected and quantified using a UPLC-MS/MS assay performed on an AB Sciex QTRAP 5500 (AB Sciex, Foster City, CA) interfaced with an Acquity UPLC (Waters Inc., Milford, MA). EFV and EFV metabolite samples were analyzed according to a modification of a previously established method (Avery et al, 2010). EFV and EFV metabolites were resolved using a reverse-phase UPLC column (2.1 Â 50 mm; Acquity UPLC BEH C18) with a flow rate of 0.5 mL/min by gradient elution: mobile phase A (MPA) of 0.1% formic acid in water; mobile phase B (MPB) of 0.1% formic acid in acetonitrile.…”

“…The assay was linear from 0.5-500 ng/ml with assay characteristics consistent with the U.S. Food and Drug Administration Guidance for Industry (2001), where all calibration standards and quality controls were linear with a percentage deviation and coefficient of variation of #15%. A fluorinated analog of EFV (F-EFV) was employed as the internal standard, as previously described elsewhere (Avery et al, 2010). EFV, 8-OH EFV, 7-OH EFV, 8,14-OH EFV, and F-EFV were detected via the MRM transitions: m/z 314.0 .…”

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.…”