The evaluation and application of a radioimmunoassay for the measurement of diphenoxylic acid, the major metabolite of diphenoxylate hydrochloride (Lomotil), in human plasma.

Antibodies to diphenoxylic acid, the pharmacologically active metabolite of Lomotil, were successfully used to develop a precise and specific radioimmunoassay for the measurement of diphenoxylic acid in human plasma. The observed cross-reaction of the antiserum with Lomotil (23.5%) and p-hydroxy diphenoxylic acid (2.9%) was not considered to affect significantly the accuracy of the direct determination of diphenoxylic acid in plasma from human volunteers after ingestion of Lomotil tablets. Within-day and between-day coefficients of variation were better than 3 and 6%, respectively, over the concentration range of 3.4 to 255 ng ml-1. Comparable precision could be achieved at 2 ng ml-1 by doubling the volume of sample analyzed. The assay was used to measure plasma concentrations of diphenoxylic acid in 12 human volunteers for up to 24 hr after ingestion of Lomotil (10mg) tablets. Plasma diphenoxyllic acid levels rose to a mean (SE) maximum level of 87.8 (2.7) ng ml-1 3.3 (0.3) hr after dosing. By 24 hr after dosing plasma levls had decreased to 14.26 (1.67) ng ml-1. The appearance and elimination of plasma diphenoxylic acid could be described by a biexponential function. The appearance half-life was calculated to be 0.82 (0.09) hr, and the elimination half-life was 7.24 (0.73) hr.