Estimation of 5-aminosalicylic acid and its metabolite in human serum by front-face fluorometry: a simple and sensitive method.

Salicylazosulfapyridine (SASP), commonly used in the treatment of inflammatory bowel disease, breaks down in the colon into sulfapyridine and 5-aminosalicylic acid (5-ASA), the active moiety of SASP. We report a sensitive method to measure 5-ASA and its known major metabolite acetyl 5-ASA (Ac-5-ASA) directly from the serum without any extraction procedure. Using front-face fluorometry, 5-ASA and Ac-5-ASA were detected at the excitation wavelength of 310 nm with emission maxima at 475 nm and 440 nm, respectively. Standard curves were obtained by adding known amounts of 5-ASA and Ac-5-ASA to several individual and pooled human sera. Presence of sulfapyridine (0 to 20 micrograms/ml) and SASP (0 to 15 micrograms/ml) in the serum did not interfere with the assays. Five microliters of acetic anhydride was added to the serum to convert all 5-ASA to Ac-5-ASA. The difference in the spectrum before and after addition of acetic anhydride represented the concentration of free 5-ASA. The values thus estimated were within 1% of the expected readings from the standard curves. This assay was compared with the organic extraction method for the determination of free and acetylated 5-ASA in sera of patients given olsalazine (azodisalicylate). The results demonstrate that direct analysis of the sera by front-face fluorometry enables us to measure 5-ASA and Ac-5-ASA at levels as low as 0.1 micrograms/ml in serum, making this method at least 10-fold more sensitive than the current available extraction methods.