Quantitative analysis of diclazuril in animal plasma by liquid chromatography/electrospray ionization mass spectrometry.

A novel, sensitive and specific method for the quantitative determination of diclazuril in animal plasma using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with negative ion detection is presented. Extraction of the samples was performed with a rapid deproteinization step using acetonitrile. Chromatography of diclazuril and the internal standard was achieved on a Nucleosil ODS 5-microm column, using a gradient elution with 0.01 M ammonium acetate and acetonitrile. To obtain the highest sensitivity and specificity possible, the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1-100 ng/mL and 100-2000 ng/mL showed good linear correlation (r >or= 0.9991, goodness-of-fit coefficient <or=7.0%). The trueness and within-run precision at 50 and 500 ng/mL did not exceed 8.8 and 10.5%, respectively. The between-run precision for the analysis of quality control samples at 50 and 1000 ng/mL was within 11.7%. The most striking advantage of the proposed method was the high sensitivity and specificity achieved. A limit of quantification of 1 ng/mL was obtained, for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3:1, the limit of detection was calculated to be 0.03 ng/mL. The method has been successfully used for the quantitative determination of diclazuril in plasma samples from treated sheep, demonstrating the usefulness of the developed method for application in the field of pharmacology and pharmacokinetics.