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New assays in development:

• darunavir assay, which will include atazanavir, efavirenz, lopinavir, saquinavir, and ritonavir
• nevirapine, lamivudine and stavudine

Delavirdine and its Metabolite

The procedure involves protein precipitation of 200 mL of sample with 400 mL of acetonitrile containing internal standard (U-88822 10 mg/ml, Pharmacia & Upjohn Laboratories, Kalamazoo, Michigan). After centrifugation, the supernatant was diluted 1:2 with 10 mM potassium phosphate buffer (pH 6.0), then directly injected onto the chromatographic system and eluted with a mobile phase consisting of 10 mM potassium phosphate (pH 6.0) and acetonitrile in a ratio of 2:1 at a flow rate of 1.5 ml/min. The HPLC column used was a Zorbax SB-CN (MAC-MOD Analytical Inc., Chadds Ford, PA) with a 5 mm particle size. The compounds were measured with fluorescence detection (Hitachi F1080 Fluorescence Spectrophotometer) with excitation at 302 nm and emission filtered at 425 nm. Quantitation of the compounds was performed by the Millenium 2000 Chromatography Manager (Waters, Milford, MA), a program which calculates peak height ratios relative to the internal standard for unknown specimens, and determines unknown values by comparing these ratios to a standard curve determined from plasma calibration standards. The standard curve is formed by a linear regression of peak height ratio versus concentration (c) with a weighting of 1/c2. The measurable concentration ranges were 0.210 to 91.8 mM for delavirdine and 0.174 to 71.7 mM for N-DLV. The interday percent coefficient of variation for the delavirdine quality controls were 6.5% (0.366 mM), 2.6% (3.85 mM) and 5.3% (35.9 mM), while those for N-DLV were 7.3% (0.359 mM), 2.7% (3.79%) and 5.8% (35.9 mM).