Application of a New UPLC–MS/MS Method for the Analysis of Salicylic Acid and Its Metabolites, Gentisic Acid and Salicyluric Acid, in Rat Serum and Urine

Salicylates have analgesic, antipyretic, anti-inflammatory, and antiplatelet effects and play a preventive role in the development of preeclampsia. The pharmacological effects of salicylates are closely related to their concentrations, which depend mainly on salicylate intake. A new UPLC–MS/MS method was developed for the determination of salicylates, including salicylic acid (SA) and its metabolites: gentisic acid (GA) and salicyluric acid (SCA), in rat serum and urine. The analytes were separated using a Luna Phenyl-Hexyl analytical column and a mobile phase consisting of 0.1% formic acid in water and acetonitrile. MS detection was performed using a triple quadrupole mass spectrometer with electrospray ionization in negative mode for SA and GA, and in positive mode for SCA. The lower limit of quantification was 0.005 µg mL−1 for SA and 0.02 µg mL−1 for GA and SCA in serum, and 0.02 µg mL−1 for SA and 0.2 µg mL−1 for GA and SCA in urine. The inter- and intra-day precision of the method, expressed as relative standard deviation, ranged from 2.77 to 17.1% in serum and from 1.37 to 16.5% in urine. The accuracy of the method was 91.2–118.0% in serum and 80.4–115.0% in urine. The extraction recoveries were between 58.3 and 79.6%. The validated method was successfully applied to the analysis of these compounds in the serum and urine of twelve preeclamptic rats fed a standard diet, half of which also received acetylsalicylic acid (ASA) (10 mg kg−1 of diet). It was observed that the urinary excretion profile of salicylates was ASA dose-dependent.