Determination of Uric Acid and Purine Compounds in Human Plasma
|School||Chongqing Medical University|
|Keywords||uric acid xanthine allopurinol high performance liquidchromatography liquid chromatography/isotope dilution massspectrometry|
Uric acid is the final metabolite of purine and nucleic acids. Theconcentration of uric acid is20-60mg/L in plasma of nomals, excessiveproduction of uric acid may lead to gout, renal disease, hypertension andcoronary heart disease. Enzymatic methods are affected by variousendogenous substances (such as the plasma xanthine, ascorbic acid andfree radical). It is necessary to develop a accurate and reliable method todetermine uric acid. A high performance liquid chromatography and liquidchromatography/isotope dilution mass spectrometry have been developedand validated to determine uric acid and purine compounds in plasma.PartⅠ Simultaneous determination of six purines in plasma byhigh-performance liquid chromatographyObjective: To develop and validate a simple HPLC method todetermine simultaneously uric acid (UA), xanthine (Xan), hypoxanthine(Hyp), adenine(Ade), guanine(Gua) and allopurinol(Oyp) in plasma ofnormals and the patients with Leukemia and liver cirrhosis.Methods:The separation was performed on Phenomenex C18column(250mm×4.6mm,5μm), with50mmol/L ammonium acetate （pH4.65） -methanol (99:1,v/v) as mobile phase isocratic elution, and measuredabsorbance at254nm.Results: The resolution of uric acid, hypxanthine, guanine, xanthine，allopurinol，adenine were5.39,1.64,3.56,5.45and5.39, respectively.Uric acid had good linear relationship in the range of0.5-20μg/mL, andthe correlation coefficient was0.9997; hypoxanthine, adenine, xanthineand allopurinol had good linear relationships in the range of0.005-0.3μg/mL, and the correlation coefficients were higher than0.9993; guaninehad good linear relationships in the range of0.015-0.3μg/mL, and thecorrelation coefficient was0.9971.Conclusion: There are significant differences in the concentration ofuric acid, hypoxanthine and xanthine in plasma between the nomals and thepatients with leukemia or liver cirrhosis, while adenine and guanine do notdiffer significantly.Part ⅡSimultaneous determination of uric acid,xangthine andallopurinol in plasma by liquid chromatography/isotope dilution massspectrometryObjective: To develop and validate a liquid chromatography/isotopedilution mass spectrometry method to determine uric acid,xanthine,allopurinol in palsma.Methods:[1,3-15N2] uric acid and [1,3-15N2] xanthine were used asinternal standards, the plasma proteins were precipitated by acetonitrle, A triple quadrupole mass spectrometer with electrospray ionization sourcewas used for monitoriing the target ions(negative mode,[1,3-15N2]uricacid M/Z:169,uric acid M/Z:167,[1,3-15N2]xanthine M/Z:153,xanthineM/Z:151, allopurinol M/Z：135) with selected ion monitoring mode (SIM),peak height ratio of isotope internal standard and target compounds wasused for quantifaction.Results: Uric acid had good linear relationship in the range of25-1028μmol/L, and the correlation coefficient was0.9989; xanthine andallopurinol had good linear relationships in the range of10-400μmol/L,and the correlation coefficients were higher than0.9951.we determinesimultaneously the uric acid, xanthine and allopurinol in plasma of fiveidentical hyperuricemia by HPLC and liquid chromatography/isotopedilution mass spectrometry method,and compared the result of twomethods. The results are in agreement.Conclusion: The high performance liquid chromatography methodcan be used to determine the purine substances of plasma.