Specific radioimmunoassay for amitriptyline and nortriptyline in plasma

A specific radioimmunoassay (RIA) for the determination in plasma of the widely used tricyclic antidepressant amitriptyline (AT) and its major metabolite nortriptyline (NT) has been developed employing 3H-AT as the radioligand and a rabbit antiserum to a bovine serum albumin conjugate of N-succinyl-nortriptyline. Although the antiserum cross-reacts almost equally well with AT and NT, specificity is achieved by selective extraction of each compound from plasma at a different pH. A unique aspect of the assay is that at no time during the entire extraction procedure is the AT or NT taken out of solution. Both compounds are back extracted from the organic phase into 0.1 N HC1 and the acid fraction subjected to RIA directly. The method has a limit of sensitivity of about 2 ng/ml using a 0.5 ml sample of plasma. Satisfactory agreement was obtained for plasma levels of AT and NT when determined by the RIA and a specific GC/MS procedure. The correlation coefficients were 0.89 and 0.98 for AT and NT, respectively. THE RIA has been used to measure steady-state levels of AT and NT in man after chronic administration of AT and following a single oral 75 mg dose. The method also lends itself for the specific determination of NT alone in subjects receiving therapeutic doses of NT.