A high-performance liquid chromatographic method for the determination of hypoxanthine, xanthine, uric acid and allantoin in serum.
A method was developed for the simultaneous determination of hypoxanthine, xanthine, uric acid and allantoin based on isocratic reversed-phase chromatography. This HPLC-method additionally allows the direct determination with UV-detection of inosine-5'-phosphate, uridine, thymine, orotic acid, allopurinol and oxipurinol, besides hypoxanthine, xanthine and uric acid in the same chromatographic run. Allantoin elutes in this system near the void volume and a fraction is collected covering the retention time range for this substance. After hydrolysis allantoin is converted to glyoxylate-2,4-dinitrophenylhydrazone, rechromatographed and detected at 360 nm. The coefficient of variation for this method does not exceed 5.0% for a serum concentration of 0.3 mumol/l hypoxanthine and is not greater than 5.3% for a xanthine concentration of 0.3 mumol/l serum. Recoveries were 90-110% for both hypoxanthine and xanthine. The determination of uric acid had an imprecision and inaccuracy not exceeding 1.45% in the concentration range of 103-568 mumol/l. Due to the more complex procedure required for the determination of allantoin, the coefficient of variation between days was 13.6% for a sample containing 0.8 mumol/l allantoin and the recoveries for this analyte were in the range of 86-93%. Reference ranges (mean +/- SD) determined on 171 serum samples from healthy adults were 12.7 +/- 6.6 mumol/l for hypoxanthine, 3.3 +/- 1.4 mumol/l for xanthine, and 15.7 +/- 7.9 mumol/l for allantoin. No significant age or sex dependence was observed. Uric acid concentrations were 320 +/- 55 mumol/l serum for men and 206 +/- 55 mumol/l for women.
Kock
R
R
Institute of Clinical Chemistry and Pathobiochemistry, Medical Faculty, University of Technology Aachen.
Delvoux
B
B
Greiling
H
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000481:Allantoin / Q000097:blood*; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000097:blood*; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014527:Uric Acid / Q000097:blood*; D019820:Xanthine; D014970:Xanthines / Q000097:blood*
D007042:Hypoxanthines; D014970:Xanthines; D019820:Xanthine; D014527:Uric Acid; D019271:Hypoxanthine; D000481:Allantoin