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 chromatographic method with fluorescence detection was used for measuring plasma concentrations of p-aminobenzoic acid (PABA) and its three metabolites: p-acetaminobenzoic acid (PAABA), p-aminohippuric acid (PAHA) and p-acetaminohippuric acid (PAAHA). A Cosmosil MS-C₁₈ column (250X4.6 mm, 5μm) was used under temperature control at 40°C. The mobile phase was H₂O-CH₃CN-CH₃COOH (100:3:1, pH 4.0) with a flow-rate of 1.5 ml/min. The excitation and emission wavelengths for fluorescence detection were set at 270 and 350 nm, respectively. Plasma samples (200 μl) were acidified by the addition of 150 μl of 1 M HClO₄ solution containing salicylic acid (SA) as the internal standard. After centrifugation, 30 μl 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 μg/ml), PAABA (0.2-50 μg/ml), PAHA (0.1-50 μg/ml) and PAAHA (0.5-50 μg/ml), good linearity (r>0.99) for the standard curves was obtained. 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 ± 1.03 min. The mean half-lives for PAABA, PAHA and PAAHA were 24.61 ± 6.42, 12.81 ± 6.04 and 11.27 ± 2.77 min, respectively.