High-pressure liquid chromatographic determination of salicylsalicylic acid, aspirin, and salicylic acid in human plasma and urine.

A high-pressure liquid chromatographic method was developed for the separation and quantitation of salicylsalicylic acid (I), aspirin (II), and salicylic acid (III) in human plasma and urine. The method for plasma involves the selective extraction of I--III and an internal standard, alpha-phenylcinnamic acid, into methylene chloride from acidified plasma, followed by evaporation of the organic phase and dissolution of the residue in methanol. A 25-microliter aliquot is analyzed on a reversed-phase column with UV detection. Urine is treated similarly with hexane as the extracting solvent. If 300 nm is used as the monitoring wavelength, the assays of I and III are linear over the concentration range of 1--150 microgram/ml in both plasma and urine. If 280 nm is used as the monitoring wavelength, II can be quantitated along with I and III; however, detector sensitivities of I and III are two to three times greater at 300 nm. The accuracy and precision of the methods for I--III are adequate for clinical pharmacokinetic studies. Following therapeutic doses of I in humans, the method was applied successfully to the determination of I and III in plasma and urine.