Development and validation of HPLC/UV-procedures of efavirenz determination in biological fluids

Abstract

Efavirenz is a non-nucleoside reverse transcriptase inhibitor with a number of side effects manifested by psychiatric symptoms. There are cases of acute poisoning due to administration of efavirenz, including cases of suicide attempts; therefore, efavirenz may be approved as a toxic compound in forensic toxicology. Aim. To apply the MiLiChrome® A-02 HPLC-analyzer system for efavirenz quantitative determination in biological fluids and perform validation of the procedures developed. Results and discussion. Three HPLC/UV-procedures of efavirenz determination in blood and urine have been proposed. Validation of all procedures developed has been performed by such parameters as specificity, recovery, linearity, accuracy and precision in the variants of the methods of calibration curve and standard. The results of analysis have shown the absence of peaks with the retention time, which is coincident with the efavirenz retention time, on the chromatograms of blank-samples for all variants of procedures of the analyte isolation. All procedures of sample preparation show the high efficiency of efavirenz isolation both for blood and urine (at the level of 90 %). All procedures studied are characterized by the acceptable parameters of linearity, within-run and between-run accuracy and precision. Experimental part. Sample preparation of blood and urine was carried out in three ways – 1) liquid-liquid extraction with the mixture of chloroform and 2-propanol (80 : 20); 2) 2-propanol extraction and salting-out with ammonium sulfate; 3) acetonitrile extraction and salting-out with ammonium sulfate. The chromatographic conditions were as follows: the column – Ø2 × 75 mm, ProntoSIL 120-5-C18 AQ, 5 μm; temperature – 40 °С; the flow rate – 100 μl/min; Eluent A – 0.2 M LiClO4 – 0.005 M HClO4; Eluent B – acetonitrile; the elution mode – linear gradient; detection – UV, 247 nm; the volume of injection – 2 μl. Conclusions. The set of HPLC-procedures of efavirenz quantitative determination in blood and urine has been developed. Validation of the procedures developed has been performed.

Keywords

• efavirenz
• high-performance liquid chromatography
• blood
• urine
• sample preparation
• validation