A tableted enzymic reagent for salicylate, for use in a discrete multiwavelength analytical system (Paramax)

A fully enzymic reagent for determination of salicylate in serum has been developed for use in the Paramax analytical system. The assay, run as an equilibrium determination, is based on the reaction of salicylate with NADPH and oxygen in the presence of salicylate hydroxylase (EC 1.14.13.1) to form catechol, NADP+, carbon dioxide, and water. The reaction is complete within 7 min, after which time the resulting absorbance change at 340/405 nm is measured. The sample:reagent ratio is 1:60 (5 microL of sample in a 300 microL final reaction volume). A single 30-mg tablet contains all of the reactants with tableting excipients. The use of NADPH eliminates interferences from reactions involving NADH. The large sample:reagent ratio, high sensitivity, and choice of bichromatic wavelengths minimize sample error. Results are linearly related to salicylate concentration to 1500 mg/L. Precision (CV) is 1.7% at 540 mg/L and 2.4% at 280 mg/L.