高效液相色谱法测定尿液中有机酸含量的方法学评价及临床应用

Methodology Evaluation and Clinical Application on Determination of the Contents of Oxalate and Citrate in Urine by High Performance Liquid Chromatography

目的:建立高效液相色谱法(high performance liquid chromatography,HPLC)测定尿液中有机酸含量的方法学并应用于临床。方法:应用HPIC技术分析健康人及尿结石患者尿液中草酸和枸橼酸的含量。色谱条件:色谱柱LC-C_(18)(250 mm×4.6 mln,5μm),保护柱LC-C_(18)(4.6 mm×12.5 mm,5μm)。流动相为0.25%磷酸二氢钾(含2.5 mmol/L四丁基磷酸氢铵和2.65×10^(-5)mol/L二乙胺四乙酸二钠,pH2.0),检测波长210 nm,流速1.0 ml/min,柱温25℃,进样量20μl。选择上述色谱条件计算曲线下面积测定草酸和枸橼酸含量。结果:草酸与枸橼酸的标准曲线分别为A_(oxa)=12528.4C_(oxa)-641.9(r=0.9990,n=5)和A_(cit)=1607.5C_(cit)-13.8(r=0.9990,n=5);最低检测限分别为0.4μg/ml和0.8μg/ml;线性范围分别为1.563～100μg/ml和3.125～200μg/ml;平均回收率分别为96.6%和95.7%;日内及日间精密度分别小于13.5%和8.3%。二水草酸钙组的尿草酸高于一水草酸钙组和对照组(P<0.05),一水草酸钙组的尿枸橼酸低于二水草酸钙组和对照组(P<0.05)。结论:HPLC用于检测人24h尿液中的草酸和枸橼酸方法简单。一水草酸钙结石的形成与结石抑制物枸橼酸的缺乏有关,二水草酸钙结石的形成与高尿草酸有关。

Objective：To establish a method of simultaneous determining the contents of oxalate and citrate in urine by high performance liquid chromatography （HPLC）, and apply it in clinic. Methods：We detected contents of oxalic acid and citric acid in urinary sample of healthy people and patients with urinary calculi by HPLC. Chromatographic conditions： LC-C18（250 mm× 4.6 mm, 5 um） column and LC-C18 （4.6 mm× 12.5 mm, 5 um） guard column; an aqueous solution containing 0. 25 % potassium dihydrogen phosph-ate （2.5 mmol/L tetrabutylammonium hydrogensulphate and 2. 65 × 10^-5mol/L EDTA at pH 2.0 ） was used as mobile phase. The detection wavelength was 210 nm, flow rate 1 . 0 ml / min. Flow rate was at 25℃, injection volume was 20 ul. Selected the above- mentioned chromatographic conditions, and calculated area under the curve, detected the content of oxalic acid and citric acid. Results： The standard curve of oxalic and citric respectively： Acxx= 12 528.4Coxa --641.9 （r= 0. 9990,n=5）and Acit=1 607.5Ccit- 13.8（r=0. 9990,n=5）, the detection limit respectively： 0. 4ug/ml and 0. 8 ug/ml, the linear range respec-tively： 1. 563-100 ug/ml and 3. 125-200ug/ml, its average re-covery respectively.96. 6% and 95.7%, its RSD of intra 2day and inter 2day respectively less than 13.5% and 8. 3%. Urine oxalic acid of calcium oxalate dehydrate （COD） group were higher than that of the calcium oxalate monohydrate （COM） group or the control group （P〈0.05）, Urine citric acid of the COM group was lower than that of the COD group or control group（P〈0.05）. Conclusions： HPLC is simple and sensitive method for quantifying oxalate and citrate in human urine. The lack of citric acid as an inhibition is one of the main reasons for forming COM. The formation of COD is related to the high level of urine oxalic acid.