Validation of an assay for voriconazole in serum samples using liquid chromatography-tandem mass spectrometry.

A simple and rapid liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the analysis of voriconazole has been developed. For comparison, serum voriconazole was measured using HPLC and bioassay. For the HPLC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of serum to 40 microL of 0.1 M zinc sulfate solution. Proteins were precipitated by adding 100 microL acetonitrile containing ketoconazole as internal standard. After vigorous mixing and centrifugation, 3 microL of the supernatant was injected into the HPLC-MS/MS system. An HPLC system was used to elute a C18 cartridge (2 mm x 4 mm) at 0.6 mL/min with a step gradient of 50% to 100% methanol containing 2 mM ammonium acetate and 0.1% (vol/vol) formic acid. The column was maintained at 55 degrees C, and the retention times were voriconazole 1.50 minutes and ketoconazole 1.47 minutes. Cycle time was 3 minutes, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: voriconazole m/z 350.0 > 224.1 and ketoconazole m/z 531.1 > 489.1. Within- and between-batch CVs were < 5% and < 8%, respectively, over the range 0.38 to 15.3 mg/L. The lower limit of quantification was 0.1 mg/L. Regression analysis showed HPLC-MS/MS = 1.06 +/- 0.02 (HPLC-UV) - 0.07 +/- 0.1, R2 = 0.95, n = 99.