Chlorthalidone analysis using carbonic anhydrase inhibition

Chlorthalidone was analyzed in the concentration range of 0.1-3.0 microgram/ml with a precision of +/- 0.05 microgram/ml. Chlorthalidone inhibition of the enzymatic hydrolysis rate of p-nitrophenyl acetate by bovine erythrocyte carbonic anhydrase was used as a basis for the determination. The amount of p-nitrophenol formed was measured by monitoring the absorbance at 400 nm, and its formation rate was proportional to the chlorthalidone concentration. The mixing of the enzyme, substrate, and sample, the incubation of the reaction mixture, and the recording of the absorbance were automated. A survey of urine samples from 26 normal human subjects did not reveal any endogenous substances that interfered with the assay. Analyses of urine samples from six subjects after oral administration of 100 mg of chlorthalidone indicated rapid absorption and a biphasic elimination. The alpha-phase half-life was 1.5 hr, and the beta-phase half-life was 35 hr.