title: Determination of efavirenz, a selective non-nucleoside reverse transcriptase inhibitor, in human plasma using HPLC with post-column photochemical derivatization and fluorescence detection.
abstract: Methods for the quantitative determination of efavirenz in human plasma and the qualitative assessment of the stereochemical integrity of efavirenz in post-dose human plasma samples are described. After the addition of an internal standard, plasma samples were extracted with hexane-methylene chloride (65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile phase. Upon exposure to UV light, the analyte was found to form fluorescent products; the major fluorescent product was isolated and identified as a substituted quinoline. Thus, the plasma extracts were analyzed via HPLC with post-column photochemical derivatization and fluorescence detection. Reverse phase chromatography was used for the quantitative assay, whereas chromatography with a column containing a chiral stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment. The quantitative assay has been validated in the concentration range of 50-1000 ng/ml using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and 104% of nominal over the range of the assay. The selective detection method reduces the likelihood of interference by co-administered medications or endogenous species. The stereochemical configuration of efavirenz was confirmed to remain intact in post-dose human plasma samples. The quantitative method has been successfully utilized to support a study in which a possible drug interaction between co-administered HIV protease inhibitors and efavirenz was evaluated.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: C Z
authors.initials: CZ
authors.lastname: Matthews
authors.forename: E J
authors.initials: EJ
authors.lastname: Woolf
authors.forename: R S
authors.initials: RS
authors.lastname: Mazenko
authors.forename: H
authors.initials: H
authors.lastname: Haddix-Wiener
authors.forename: C M
authors.initials: CM
authors.lastname: Chavez-Eng
authors.forename: M L
authors.initials: ML
authors.lastname: Constanzer
authors.forename: G A
authors.initials: GA
authors.lastname: Doss
authors.forename: B K
authors.initials: BK
authors.lastname: Matuszewski
mesh_terms: D000480:Alkynes; D048588:Benzoxazines; D002851:Chromatography, High Pressure Liquid; D003521:Cyclopropanes; D006801:Humans; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D010078:Oxazines / Q000097:blood* / Q000493:pharmacokinetics; D010777:Photochemistry; D010782:Photolysis; D011786:Quality Control; D018894:Reverse Transcriptase Inhibitors / Q000097:blood* / Q000493:pharmacokinetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
chemical_list: D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D007202:Indicators and Reagents; D010078:Oxazines; D018894:Reverse Transcriptase Inhibitors; C098320:efavirenz