高效液相色谱法测定草酸钙结石大鼠尿中草酸含量

目的 建立高效液相色谱法(HPLC)检测大鼠尿液中草酸含量。方法 将SPF级雄性健康Wistar大鼠24只采用随机数字表法分为空白对照组(n=12)和乙二醇组(n=12)。空白对照组给予去离子水喂养,每天以1 ml生理盐水灌胃;乙二醇组给予1%乙二醇自由饮水喂养,每天2%氯化铵2 ml灌胃。造模28 d后收集两组大鼠24 h尿液,采用HPLC法测定尿中草酸含量,并与铬酸钾氧化甲基红催化光度法的结果进行比较。HPLC法采用色谱柱Aglient 5TC-C₁₈(250×4.6 mm,5 μm),以甲醇(0.1 mol/L)、醋酸铵水溶液(15∶85)为流动相,流速为1.2 ml/min,紫外检测波长为314 nm,柱温20 ℃。结果 大鼠尿液中高、低浓度草酸标准曲线分别为y=5909.1x+378730,R²=0.9984和y=7810.5x-16635,R²=0.9967;最低检测浓度为5 μg/ml,大鼠尿液中草酸高浓度线性范围为62.50~2000.00 μg/ml,低浓度线性范围为6.25~100.00 μg/ml。平均回收率为95.1%,日内及日间精密度分别为3.4%~10.8%和3.8%~9.4%。HPLC法和铬酸钾氧化甲基红催化光度法均显示乙二醇组大鼠尿草酸浓度和24 h尿草酸含量显著高于空白对照组尿草酸浓度:(736.35±254.52)μg/ml比(51.56±36.34)μg/ml,(687.35±234.53)μg/ml比(50.24±42.34)μg/ml;24 h尿草酸含量:(11.23±4.12)mg比(0.87±0.45)mg,(9.89±3.55)mg比(0.77±0.65)mg;P均＜0.01];两种方法检测出的尿草酸浓度和24 h尿草酸含量差异无统计学意义(P均＞0.05)。结论 HPLC法简单快速、回收率高、精密度好,与铬酸钾氧化甲基红催化光度法检测结果一致,适用于大鼠尿中草酸含量的测定。

Determination of Urine Oxalate Level in Rats with Renal Calcium Oxalate Calculus by High-performance Liquid Chromatography

Objective To establish a method of high-performance liquid chromatography(HPLC)for determining the urine oxalate levle in rats with renal calcium oxalate calculus. Methods Totally 24 SPF Wistar healthy male rats were randomly divided into control group(n=12)and ethylene glycol(EG)group(n=12). Rats in EG group were administered intragastrically with 2% ammonium chloride(AC)2 ml/rat per day+1% ethylene glycol(EG),along with free access to drinking water.The control group was fed with deionized water,along with the intragastric administration of normal saline(1 ml per day). Twenty-eight days after modelling,the 24-hour urine samples were collected,and the urine oxalic acid levels were determined using HPLC and the results were compared with those of catalytic spectrophotometry using oxidation of methyl. During the HPLC,the samples were separated on Aglient 5TC-C₁₈(250×4.6 mm,5 μm),eluted with mixture of methanol(0.1 mol/L)and ammonium acetate(15∶85)at 1.2 ml/min,and detected at 314nm,with the column temperature being 20 ℃. Results The standard curves of high and low concentrations of oxalic acid were y=5909.1x+378730,R²=0.9984 and y=7810.5x-16635,R²=0.9967,respectively. The lowest detectable concentration in this method was 5 μg/ml. The linear high concentration range of oxalate stood at 62.50-2000.00 μg/ml,and the linear low concentration range of oxalate stood at 6.25-100.00 μg/ml. Its average recovery was 95.1%,and its within-day and day-to-day precisions were 3.4%-10.8% and 3.8%-9.4%. Both HPLC and catalytic spectrophotometry showed significantly higher urinary oxalic acid concentration and 24 h urine oxalate level in EG group compared with the control group urinary oxalic acid concentration:(736.35±254.52)μg/ml vs.(51.56±36.34)μg/ml,(687.35±234.53)μg/ml vs.(50.24±42.34)μg/ml;24 h urine oxalate level:(11.23±4.12)mg vs.(0.87±0.45)mg,(9.89±3.55)mg vs.(0.77±0.65)mg;all P＜0.01]. No statistically significant difference was observed in the results of urinary oxalate concentration and 24 h urine oxalate level between HPLC and potassium chromate oxidation of methyl red spectrophotometry(all P＞0.05). Conclusion HPLC is a simple,rapid,and precise method in detecting urine oxalate level in rats with renal calcium oxalate calculus,with high recovery rate.