High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma

Mesalazine (5-aminosalicylic acid, 5-ASA), an anti-inflammatory agent for the treatment of inflammatory bowel diseases, is metabolized in organism to the principal biotransformation product, N-acetyl-5-ASA. Some other phase II metabolites (N-formyl-5-ASA, N-butyryl-5-ASA, N-β-d-glucopyranosyl-5-ASA) have also been described. 5-ASA is a polar compound and besides it exhibits amphoteric properties. The extraction of this compound from biomatrices and its chromatographic analysis is complicated. In order to improve the reliability of the determination of parent 5-ASA, a derivatization of 5-ASA together with 4-ASA (added to samples as a precursor of I.S.-2) was involved into the method. More lipophilic N-propionyl-5-ASA and N-propionyl-4-ASA (I.S.-2) were obtained using propionic anhydride. These derivatives were well extractable together with N-acyl-5-ASAs (metabolites) and N-acetyl-4-ASA (I.S.-1). As the first internal standard (I.S.-1) was used for the evaluation of extracted N-acyl-metabolites, the second internal standard (I.S.-2) served for the evaluation of both derivatization and extraction steps of parent drug 5-ASA. Based on these reasonings, new HPLC bioanalytical method for the determination of 5-ASA and its metabolites in blood plasma was developed and validated. The sample preparation step consists of the deproteination of plasma by HClO4 and the above-mentioned derivatization of ASAs followed by liquid–liquid extraction of all N-acyl-ASA-derivatives. Chromatographic analyses were performed on a 250-4 mm column containing Purospher RP-18 e, 5 μm (Merck, Darmstadt, Germany) with a precolumn (4-4 mm). The column effluent was monitored using both UV photodiode-array (λ = 313 nm) and fluorescence detectors (λexc. = 300 nm/λemiss. = 406 nm) in tandem. The identity of individual N-acyl-ASAs in the extracts from biomatrices was verified by characteristic UV-spectra and by HPLC/MS experiments. The whole analysis lasted 23 min at the flow rate of 1 ml min−1. LLOQ (LOD) was estimated 126 (20) pmol ml−1 of plasma for N-acetyl-5-ASA and 318 (50) pmol ml−1 of plasma for N-propionyl-5-ASA. The validated HPLC method was applied to pharmacokinetic studies of mesalazine in humans and animals.