DETERMINATION OF ASPIRIN AND SALICYLIC-ACID IN HUMAN PLASMA BY COLUMN-SWITCHING LIQUID-CHROMATOGRAPHY USING ONLINE SOLID-PHASE EXTRACTION

A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plas ma. Blood samples are taken into chilled tubes containing a fluoride a nticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 mu L aliquot of the sample is injec ted directly onto the HPLC system. The C-18 extraction column is washe d with acidified water for 2 min, after which time the compounds are r emoved by back-flushing directly onto the analytical column (C-8 Nucle osil, 5 mu m, 250 mm x 4.6 mm). The now rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV ab sorbance at 225 nm. The mobile phase is a mixture of water-methanol-ac etonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 mu g/mL for aspirin and 0.25-15.00 mu g/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 mu g/mL and a limit of detection of 0.04 mu g/mL. Extensive stability tests have been carried out, and validation studi es reveal the method to be reproducible and repeatable. Excellent reco veries from plasma obviate the need for an internal standard. The proc edure is easier to execute and requires less sample handling than meth ods currently described in the literature. It has been successfully ap plied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of as pirin.