ABSTRACT
OBJECTIVE: To establish an isobutyryl derivatization combined with UPLC-MS/MS method for the quantitative analysis of 5-aminosalicylic acid (5-ASA) in human urine. METHODS: Urine sample was analyzed by UPLC-MS/MS after pretreatment with isobutyryl derivatization and precipitation with acetonitrile. The separation was performed on the ACQUITY UPLC BEH-C18 column (2.1 mm×100 mm,1.7 μm) at 40 ℃ with injection volume of 3 μL. The mobile phase was acetonitrile-5 mmol•L-1 ammonium formate containing 0.075% formic acid (20:80) at a flow rate of 0.4 mL•min-1. Electrospray ionization (ESI) source was applied and operated in multiple reaction monitoring (MRM) mode with negative ionization using the transitions of m/z 222.0→178.1 and m/z 206.1→162.0 for isobutyl 5-ASA and isobutyl 3-amino benzoic acid (3-ABA, IS), respectively. RESULTS: The method was specific and sensitive. It exhibited good linearity over the concentration of 0.100-30.0 μg•mL-1 for 5-ASA with the LLOQ of 0.100 μg•mL-1, extraction recovery of 101.7%-115.4% and normalized matrix factor of 1.000-1.025. The intra- and inter-day precisions were both less than 15%. CONCLUSION: The method is specific, simple, accurate and stable for quantitative analysis of 5-ASA in human urine.