HPLC-UV method for the quantitation of nevirapine in biological matrices following solid phase extraction

Nevirapine (VIRAMUNE) is a non-nucleoside reverse transcriptase inhibitor with activity against human immunodeficiency virus type 1 (HIV-1), currently marketed for the treatment of HIV-1 infected adults. A reverse phase HPLC-UV method was optimized and validated for the determination of nevirapine in human plasma, serum, milk and cerebrospinal fluid. The analyte was extracted from 250 microl of biofluid using a bonded silica solid phase extraction column, and resolved chromatographically on a reversed-phase, 15x0.46 cm i.d. 5 microm particle Supelco LC-8 analytical column with an isocratic mobile phase of 63% phosphate buffer (0.025 M, pH 6.0) with 1-butanesulfonic acid as anion-pair reagent: 21.5% methanol: 15.5% acetonitrile. The peaks were detected at a flow rate of 1.0 ml min(-1), at a wavelength of 280 nm, with a run time of 10 min. The assay was linear over a range of 25 to 10000 ng ml(-1). This method has been used for the clinical development of nevirapine.