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. 2014 Apr;4(2):153-158.
doi: 10.1016/j.jpha.2013.11.003. Epub 2013 Dec 12.

Determination of gouty arthritis' biomarkers in human urine using reversed-phase high-performance liquid chromatography

Lei-Wen Xiang  1   2 , Jing Li  1 , Jin-Ming Lin  1 , Hai-Fang Li  1
Affiliations

Determination of gouty arthritis' biomarkers in human urine using reversed-phase high-performance liquid chromatography

Lei-Wen Xiang et al. J Pharm Anal. 2014 Apr.

Abstract

Creatinine, uric acid, hypoxanthine and xanthine are important diagnostic biomarkers in human urine for gouty arthritis or renal disease diacrisis. A simple method for simultaneous determination of these biomarkers in urine based on reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detector was proposed. After pretreatment by dilution, centrifugation and filtration, the biomarkers in urine samples were separated by ODS-BP column by elution with methanol/50 mM NaH2PO4 buffer solution at pH 5.26 (5:95). Good linearity between peak areas and concentrations of standards was obtained for the biomarkers with correlation coefficients in the range of 0.9957-0.9993. The proposed analytical method has satisfactory repeatability (the recovery of data in a range of creatinine, uric acid, hypoxanthine and xanthine was 93.49-97.90%, 95.38-96.45%, 112.46-115.78% and 90.82-97.13% with standard deviation of <5%, respectively) and the limits of detection (LODs, S/N≥3) for creatinine, uric acid, hypoxanthine, and xanthine were 0.010, 0.025, 0.050 and 0.025 mg/L, respectively. The established method was proved to be simple, accurate, sensitive and reliable for the quantitation of gouty arthritis' biomarkers in human urine samples. The ratio of creatinine to uric acid was found to be a possible factor for assessment of gouty arthritis.

Keywords: Creatinine; Gouty arthritis; High-performance liquid chromatography; Hypoxanthine; Uric acid; Xanthine.

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Figures

None
Graphical abstract
Fig. 1
Fig. 1
Chemical structures of uric acid, creatinine, hypoxanthine and xanthine.
Fig. 2
Fig. 2
Effect of the ratio of organic phase. (The flow rate was 0.8 mL/min, the column temperature was 25 °C, the UV detection wavelength was 210 nm, and the injection volume was 20 μL. 1, 2, 3 and 4 stand for creatinine, uric acid, hypoxanthine and xanthine, respectively. A and C are mixed standard while B and D are actual urine sample. The HPLC mobile phases were methanol/NaH2PO4 buffer solution (10:90) and methanol/NaH2PO4 buffer solution (5:95)).
Fig. 3
Fig. 3
A time course of the concentration of creatinine, uric acid, hypoxanthine and xanthine in human urine.
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