Modification of the 4-(p-nitrobenzyl) pyridine assay for measurement of melphalan and mechlorethamine in plasma

A sensitive, reproducible spectrophotometric assay for mechlorethamine and melphalan was developed through modifications in the use of 4-(p-nitrobenzyl) pyridine as the chromogenic reagent. Key factors that led to enhanced color development and consistency of results were: (1) protein precipitation with 4 degrees C perchloric acid; (2) control of the pH of the reaction with acetate buffer; (3) precise timing of color development in alkali; and (4) sample reading at the appropriate wavelength. The modifications allowed measurement of mechlorethamine and melphalan levels in perfusate samples obtained from isolated rat liver perfusions. Half-lives calculated from perfusate decay curves after addition of mechlorethamine at 50 microgram/ml and 10 microgram/ml were 15.9 min and 15.0 min respectively. The perfusate half-life after addition of melphalan at 5 microgram/ml was 44.2 min. Plasma levels of mechlorethamine were not detectable by this assay after intrapleural administration of 15 mg to one patient but were detectable after intravenous administration of 20 mg to a second patient.