DETERMINATION OF P-AMINOBENZOIC ACID AND ITS METABOLITES IN RABBIT PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

A simple, accurate and sensitive high-performance liquid chromatograph ic method with fluorescence detection was used for measuring plasma co ncentrations of p-aminobenzoic acid (PABA) and its three metabolites: p-acetaminobenzoic acid (PAABA), p-aminohippuric acid (PAHA) and p-ace taminohippuric acid (PAAHA). A Cosmosil MS-C-18 column (250x4.6 mm, 5 mu m) was used under temperature control at 40 degrees C. The mobile p hase was H2O-CH3CN-CH3COOH (100:3:1, pH 4.0) with a flow-rate of 1.5 m l/min. The excitation and emission wavelengths for fluorescence detect ion were set at 270 and 350 nm, respectively. Plasma samples (200 mu l ) were acidified by the addition of 150 mu l of 1 M HClO4 solution con taining salicylic acid (SA) as the internal standard. After centrifuga tion, 30 mu l of the supernatant were injected onto the column. Using this method, PABA and its three metabolites could be determined within 25 min. Within the investigated concentration ranges of PABA (0.1-50 mu g/ml), PAABA (0.2-50 mu g/ml), PAHA (0.1-50 mu g/ml) and PAAHA (0.5 -50 mu g/ml), good linearity (r>0.99) for the standard curves was obta ined. The validation of this method showed coefficient of variance (C. V.) that was well below 15% for all compounds. After intravenous (i.v. ) administration of PABA (20 mg/kg) to rabbits (n=7), PABA followed a one-compartment open model elimination with a half-life of 10.90+/-1.0 3 min. The mean half-lives for PAABA, PAHA and PAAHA were 24.61+/-6.42 , 12.8+/-6.04 and 11.27+/-2.77 min, respectively.