Rapid Sample Preparation and High Performance Liquid Chromatographic Determination of Total and Unbound Serum Disopyramide

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Abstract
A rapid sample preparation procedure, which requires no solvent extraction or concentration, for the high performance liquid chromatographic (HPLC) determination of disopyramide is described. The chromatography is performed on a C-18 radial compression .mu.-Bondapak column and detection by absorbance at 254 nm with a run time of 12 min. The mobile phase is 10 mM sodium acetate (pH 4.5)/acetonitrile (3:1 vol/vol). For the total drug assay, 50 .mu.l 30% (wt/vol) trichloroacetic acid is added to 500 .mu.l serum, which causes the precipitation of protein. Following centrifugation, 100 .mu.l of supernatant is mixed with 25 .mu.l of internal standard (25 .mu.g/ml, ethyl p-aminobenzoate), and 50 .mu.l of this mixture is injected into the HPLC. Unbound disopyramide is separated from protein-bound drug by filtration with an Amicon Centrifree filter, which removes 99.6% of protein and does not retain disopyramide. To 100 .mu.l of this filtrate is added 25 .mu.l of internal standard, and 50 .mu.l is injected into the HPLC. The assay is linear to at least 20 .mu.g/ml. The total drug assay shows an average recovery of 93.0% with an average coefficient of variation (CV) of 3.4%. The unbound drug assay shows an average CV of 4.1%. The percentage of free drug in a sample containing 4.85 g/dl protein varies from 68.0 to 83.5% at concentrations of 2.5-10 .mu.g/ml, which illustrates the concentration-dependent nature of the protein binding, and the need to measure the unbound fraction of drug. Of 31 drugs tested for interference, none were found to interfere.