Internally standardized determination of anipamil in human plasma by means of high-pressure liquid chromatography

A high pressure liquid chromatographic method with internal analogue standardization for the determination of anipamil in plasma is described. The method comprises extraction from plasma diluted with citrate buffer pH 3 using n-heptane/isoamylalcohol (95/5, v/v), distribution between this organic phase and a methanol/citric acid mixture, and quantification by means of fluorescense detection after HPLC separation using a reverse phase. When using 1 ml plasma the lower limit of detection is 0.2 ng/ml. Under routine conditions the limit of determination is estimated at 1 ng/ml, with higher numbers of replicates and with a predetermined precision limit of 15% concentrations as low as 0.6 ng/ml can be determined reliably. The variation coefficients drop from about 10% in the low range to about 5% at 2 ng/ml or more. The determination of anipamil is not impaired by its N-nor-compound, which is to be expected as metabolite. Neither do other potential metabolites of anipamil with very similar chromatographic behaviour interfere with its quantification.