Method for Determination of Aspirin and Salicylic Acid in Rat Whole Blood by High Pressure Liquid Chromatography

A method has been developed to simultaneously determine aspirin and salicylic acid in rat whole blood. Aspirin and salicylic acid are extracted from acidified whole blood into a 50/50 v/v ethylacetate/butylchloride organic solvent system containing internal standard (meta-anisic acid). Following controlled evaporation of the organic extract under partial vacuum, the dried residue is reconstituted with mobile phase. Chromatography is ion suppression reverse phase on a 5 μm octyldecasiland column with detection by UV absorbance at 280 nm. In this method, the procedure of blood centrifugation for plasma preparation is eliminated. Therefore, the blood volume required is decreased and the sensitivity of analysis is considerably increased. Concentrations of aspirin as low as 0.5 mcg/ml and salicyclic acid as low as 1.5 mcg/ml can be quantitated in as little as 100 μl of whole blood. Aspirin is one of the most extensively used drugs. A method of high pressure liquid chromatography (HPLC) for the analysis of aspirin and its metabolites in human plasma and urine was developed in our laboratory in the late 1970s and revised in the early 1980s1,2. With modifications to improve the sensitivity and reproducibility, this method can be applied to measure aspirin and salicylic acid in rat whole blood. Therefore, the studies of kinetics and metabolism of aspirin in the rat are both possible and the extrapolation of these results to humans can be meaningful. The HPLC method described herein has been employed to measure aspirin (ASA) and salicylic acid (SA) in more than 500 rat whole blood samples collected during a pharmacokinetic study.