Automated analysis of indapamide in drug-rodent food mixtures

Indapamide, an antihypertensive agent, is an aryl sulfonamide that inhibits carbonic anhydrase in vitro but not in vivo. An assay was developed for indapamide in drug-rodent food mixtures that utilizes this inhibitory effect. Indapamide was extracted from the mixtures with methanol, and an aqueous dilution of the extract was sampled by a continuous-flow system. In the system, the drug was extracted with butanol and then back-extracted into alkali. This solution was neutralized, buffered, and mixed with bovine erythrocyte carbonic anhydrase. The substrate, p-nitrophenyl acetate, was added, the solution was incubated, and the amount of p-nitrophenol formed was measured. The assay was sensitive to 20 micrograms of indapamide/g of food, and 20 unknown samples could be analyzed per hour on the continuous-flow system. It is possible that the method could be extended to the analysis of other toxicological test substances that inhibit carbonic anhydrase in vitro.