Automated determination of drugs in blood samples after enzymatic hydrolysis using precolumn switching and...

Enzymatic hydrolysis of blood samples with subtilisin-A releases protein-bound drugs and permits the repeated (10-50 times) injection of up to 1-ml volumes on short (2-30 mm) precolumns without appreciable build-up of pressure or loss of performance of the precolumn. The principle of fully automated serum and plasma analysis is demonstrated with the drug secoverine as a model compound. After enzymatic hydrolysis of the sample with an equal volume of a 1 mg/ml solution of subtilisin-A for 15 min at 55 degrees C, the model compound is preconcentrated using a microprocessor-controlled column switching unit. Separation occurs in a reversed-phase liquid chromatographic system using a CN-type stationary phase and a buffered aqueous dioxane solution as mobile phase. Detection is done by UV spectrophotometry or fluorometrically after post-column ion-pairing reaction with dimethoxyanthracenesulphonate. The relative standard deviation of the procedure is less than +/- 6% (n = 10).