Identification and determination of salicylic acid and salicyluric acid in urine of people not taking salicylate drugs

Background Salicylic acid (SA) is present in the serum of people who have not taken salicylate drugs. Now we have examined the urine of these subjects and found that it contains SA and salicyluric acid (SU), We have established the identities of these phenolic acids and determined their concentrations. Methods and Results The acidic hydrophobic compounds of urine were separated using high-performance liquid chromatography (HPLC) and were detected and quantified electrochemically. Two approaches were used to establish the identity of SA and SU. First, the retention times (R-t) of the substances extracted and those of SA and SU were compared under two sets of chromatographic conditions; the R-t of the compounds suspected to be SA and SU and those of the authentic substances were very similar under both sets of conditions. Second, the unknown substances, isolated by HPLC, were treated with acetyl chloride in methanol and compared with the methyl esters of SA and SU by using gas chromatography-mass spectrometry; the unknown compounds after esterification had very similar mass spectra and gas chromatographic R-t to those of methyl salicylate and methyl salicylurate, The median (n=10) urinary concentration of SA was 0.56 mumol/L (range 0.07-0-89 mumol/L) and that of SU was 3.20 mumol/L (range 1.32-6.54 mumol/L). SA and its major urinary metabolite, SU, were found in the urine of all of the 10 people examined.