Simultaneous Analysis of Ascorbic Acid , Uric Acid and Xanthine in Human Urine by Column Tandem Technique

Ascorbic acid, uric acid and xanthine in human urine are important markers of many diseases. A method for simultaneous detection of ascorbic acid , uric acid and xanthine in human urine by high performance liquid chromatography based on column tandem technique was established. The separation of these three compounds was the key to simultaneously analyze the three compounds to a certain extent because the conventional liquid chromatography could not effectively separate these highly polar substances simultaneously. The Innoval ASB C-18 was coupled with the hydrophilic interaction chromatography ( HILIC) column to trigger off a dual-column series separation mode. The separation performance of the two columns was complementary to achieve baseline separation of the three target analytes. The results showed that a single HILIC column could not achieve the baseline separation of the three analytes. The C-18 column also showed very weak retention to the three analytes. The retention time was so early that the target analyte peaks might be interfered by the solvent peaks. Only the tandem of these two columns could achieve the baseline separation for the three analytes. Ultimately, an analytical method for simultaneous analysis of ascorbic acid, uric acid and xanthine in human urine with the C-18 column tandem HILIC column was established. The correlation coefficient was good in the linear range of 0. 005-20 mg/L , and limits of detection ( LODs ) of ascorbic acid , uric acid , and xanthine were 1. 06 mu g/L, 0. 94 mu g/L and 0. 46 mu g/L, respectively. The three target analytes could be found in actual human urine by the proposed method. The contents of ascorbic acid, uric acid and xanthine were 0. 47-4. 70 , 6. 91 -8. 01 and 0. 10-0. 19 mg/L, respectively. The recoveries of the spiked urine samples were 86. 2% -117. 9% , and the relative standard deviation ( RSD) was less than 9. 3%. The established high performance liquid chromatography based on column tandem analytical method was simple, fast and efficient, and could be used for separation and simultaneous determination of ascorbic acid , uric acid and xanthine in human urine.