Automated analysis of chlorthalidone

Chlorthalidone inhibition of the enzymatic hydrolysis rate of p-nitrophenyl acetate by bovine erythrocyte carbonic anhydrase was used as a basis for chlorthalidone determination in plasma and urine. For urinary samples, a completely automated, continuous flow system was developed to extract the samples and perform the enzymatic reaction. Over 100 samples per day could be assayed by one person. The assay had a sensitivity of 0.5 micrograms/ml and thus could determine urinary concentrations after a therapeutic chlorthalidone dose. To determine plasma concentrations after a therapeutic dose, a manual extraction procedure was used in combination with a second continuous flow system for the enzymatic reaction. This system was optimized to detect the lowest chlorthalidone concentration allowed by the enzymatic inhibition constant and could detect 25 ng/ml.