High-performance liquid chromatographic assay of tolbutamide and carboxytolbutamide in human plasma

A high-performance liquid chromatographic method was developed for the simultaneous measurement of tolbutamide and its major metabolite, carboxytolbutamide, in plasma. The assay involves the ether extraction of 1 ml of plasma, using chlorpropamide and an internal standard. The extract is dried, the residue is taken up in acetonitrile, and 5 micro l is injected into a reversed-phase column. The mobile phase consisted of 35% acetonitrile and 65% 0.05 M phosphoric acid buffer (pH 3.9). A fixed-wavelength detector was set at 254 nm. The sensitivity limits for the tolbutamide and carboxytolbutamide assay were 2 and 0.1 microgram/ml, respectively. The ratio of carboxytolbutamide to tolbutamide in plasma obtained from a subject given a 500-mg tolbutamide tablet was 1:20.