大鼠血清尿酸液相色谱-紫外检测新方法的建立及比较研究

Quantification of Uric Acid of Rat Serum by Liquid Chromatographyultraviolet Detection and Its Comparison Study

目的建立更准确、灵敏的液相色谱紫外(liquid chromatography-ultraviolet,LC-UV)检测大鼠血清尿酸的方法,并将该方法与市售试剂盒的检测结果进行比较,为氧嗪酸钾诱导的大鼠高尿酸血症模型中血清尿酸含量的准确测定提供新方法。方法采用向SPF级雄性SD大鼠腹腔注射氧嗪酸钾(300 mg/kg)的方法建立高尿酸血症大鼠模型,对照组给予等量的0.5%羧甲基纤维素钠溶液。经眼眶后静脉丛采血,离心获得大鼠血清样品,经0.1%三氟乙酸-乙腈(含内标3,4-二羟基苄胺氢溴酸盐)沉淀后,取上清液进样分析。采用Waters XBridge HILIC色谱柱(150 mm×4.6 mm,3.5µm)对尿酸进行分离,使用乙腈(含0.5%甲酸与2 mmol/L甲酸铵)作为有机相,甲醇溶液(甲醇∶水=1∶1,含0.5%甲酸与2 mmol/L甲酸铵)作为水相,进行等度洗脱,于290 nm波长处进行检测。使用活性炭处理的血清样品作为空白生物基质,用于方法学验证。分别使用所建立的LC-UV方法与两种市售试剂盒(尿酸酶法和磷钨酸法)检测高尿酸血症模型大鼠的血清尿酸水平,并对3种检测方法的准确度进行比较。结果大鼠血清中的尿酸在10~200µg/mL质量浓度内线性关系良好(R>0.999),定量下限为10µg/mL,准确度为-2.17%~2.21%,批内精密度为0.52%~1.95%,批间精密度为3.04%~4.90%,提取回收率为83.12%~89.91%。在氧嗪酸钾诱导的大鼠高尿酸血症模型中,市售的磷钨酸法试剂盒测定结果显著高于LC-UV法测定结果,市售的尿酸酶法试剂盒测定结果显著低于LC-UV法测定结果,但LC-UV方法的加样回收率结果最佳(95.90%~99.96%)。结论所建立的LC-UV方法能够准确测定大鼠血清中尿酸含量,与市售试剂盒相比具有更高的准确度,推荐作为氧嗪酸钾诱导的大鼠高尿酸血症模型中血清尿酸含量的检测方法。

Objective To establish a more accurate and sensitive liquid chromatography-ultraviolet(LCUV)method for the determination of uric acid in rat serum,and compare the results with those of commercial kits,providing a new method for the accurate determination of uric acid in the rat hyperuricemia model induced by potassium oxonate.Methods A hyperuricemia model was established by intraperitoneal injection of potassium oxonate(300 mg/kg)into SPF-grade male SD rats,and the control group was administered an equal amount of 0.5%sodium carboxymethylcellulose solution.Blood samples were collected from the posterior orbital venous plexus and centrifuged to obtain serum samples.After precipitation with 0.1%trifluoroacetic acid-acetonitrile(containing the internal standard 3,4-dihydroxybenzylamine hydrobromide),the supernatant was injected for analysis.Uric acid was separated on a Waters XBridge HILIC column(150 mm×4.6 mm,3.5µm)using acetonitrile(containing 0.5%formic acid and 2 mmol/mL ammonium formate)as the organic phase and methanol solution(methanol∶water=1∶1,containing 0.5%formic acid with 2 mmol/L ammonium formate)as the aqueous phase for isocratic elution and detection at 290 nm.Serum samples treated with activated carbon were used as substitute matrices for the methodological verification.Serum uric acid levels in rats with potassium oxonate-induced hyperuricemia were measured using the established LC-UV method and commercially available kits(uricase and phosphotungstic acid methods),and the accuracies of the three methods were compared.Results Serum uric acid showed a good linear relationship(R>0.999)at mass concentration of 10–200µg/mL in rats,the lower limit of quantification was 10µg/mL,the accuracy ranged from-2.17%to 2.21%,the intra-batch precision ranged from 0.52%to 1.95%,the inter-batch precision ranged from 3.04%to 4.90%,and the extraction recovery ranged from 83.12%to 89.91%.In the rat model,the results obtained using the commercially available phosphotungstic acid method kit were significantly higher than those of the LC-UV method,and those obtained using the commercially available uricase method kit were significantly lower than those of the LC-UV method,but the LC-UV method showed the best recovery of the spiked sample(95.90%–99.96%).Conclusion The LC-UV method developed in this study can determine the concentration of uric acid in rat serum with higher accuracy than commercially available kits and is recommended for the determination of serum uric acid in the rat model of hyperuricemia induced by potassium oxonate.