Determination of Total Salicylates, Phenacetin and/or Total N-Acetyl-p-aminophenol, and Caffeine in Biological Samples and Analgesic Formulations

Abstract

An analytical method was developed for the determination of phenacetin (apparent phenacetin), total salicylate (aspirin, salicylic acid, and its conjugates), total N-acetyl-p-aminophenol (N-acetyl-p-aminophenol and its conjugates), and caffeine in biological samples. Two 1-ml. samples are required. The first sample, after being acid hydrolyzed (2 N HCl, 2 hr., 121°) to convert total salicylate to free salicylic acid and total N-acetyl-p-aminophenol to p-aminophenol, is extracted with chloroform to remove caffeine and salicylic acid. p-Aminophenol in the aqueous phase is estimated by its reaction with phenol and sodium hypobromite. Salicylic acid extracted from the chloroform with sodium bicarbonate solution is estimated by reacting it with either Folin-Ciocalteu reagent or ferric nitrate. The caffeine, remaining in the chloroform, is estimated from its absorbance at 276nm. In the second sample, phenacetin is hydrolyzed (8 N HCl, 6 hr., 121°) to p-aminophenol (total p-aminophenol), which is estimated as before. Time-dependent plasma and kidney levels of caffeine, phenacetin, total N-acetyl-p-aminophenol, and total salicylate, following the oral administration of a single dose (500 or 900 mg./kg. body weight) of aspirin-phenacetin-caffeine mixture to rats, were determined. The method also was adapted to the analysis of aspirin-phenacetin-caffeine powders.