应用反相高效液相色谱内标法测定血清尿酸

Determination of uric acid in human serum using a revered-phase high perfermance liquid chromatography

目的评价以5-氟尿嘧啶(5-FU)作内标的反相高效液相色谱(HPLC)测定血清尿酸(UA)的方法,拟初步推荐其作为血清UA测定的候选参考方法;并应用此方法评价常规酶比色法测定血清UA的结果偏倚。方法对测定血清UA的反相HPLC内标法进行方法学评价;通过测定有证参考物质(SRM)、参加国际临床化学与检验医学联合会(IFCC)参考实验室考察活动,验证此方法的准确性及可靠性。以反相HPLC内标法为比较方法,以6种常用检测系统中的酶比色法作为实验方法,对61份血清样本进行测定。组成6个比对组,对每组比对数据作散点图、偏倚图,计算线性方程和相关系数(R2),进行偏倚评估。结果反相HPLC内标法测定UA线性范围达1190μmol/L,批内变异系数(CV)<2.1%,日间CV<3.9%,绝对回收率为96.2%~101.0%,平均相对回收率为95.1%~99.4%。测定SRM的平均相对偏倚为-8.29%;测定IFCC参考实验室考察活动提供的样本,其结果与同位素稀释质谱(ID-MS)法的相对偏倚范围为-3.45%^-1.26%;在Beckman、Roche、Dade、Vitros封闭系统及Beckman、Roche开放系统中的酶比色法分别与反相HPLC内标法比较,各组中2种方法均相关良好(R2=0.9969、0.9972、0.9952、0.9967、0.9973、0.9947),在UA为120、470L、630μmol/L时,预期相对偏倚范围分别为-14.6%~4.3%、-0.4%~8.0%、0.8%~8.8%,其中Beckman封闭系统测定结果与反相HPLC内标法的一致性好,Roche封闭系统低值偏低,Dade和Vitros封闭系统高值偏高,封闭系统比开放系统结果略好。结论拟推荐以5-FU为内标的反相HPLC法作为血清UA测定的候选参考方法。酶比色法在不同检测系统中所测结果存在一些差异。

Objective To evaluate the revered-phase high performance liquid chromatography （HPLC） with 5- fiuoronracil （5-FU） as an internal standard for serum uric acid （UA） determination, mad to recommend this method as a candidate reference method. To evaluate biases of results of serum uric acid determination by uricase-peroxidase-trinder methods according the revered-phase HPLC result. Methods The methodology of the revered-phase HPLC was evaluated. The result accuracy of serum UA by the revered-phase HPLC was verified according to determine a certified reference material （SRM） and the Survey RELA KS （1/06） serum sample of RELA 2006 4th IFCC Ring Trial for Reference Laboratories. The revered-phase HPLC as a comparison method and the uricase methods with closed-system including Beckman, Roche, Dade, Vitros and opened-system including Beckman, Roche as a test methods. 61 samples of serum UA were determined by two methods respectively, which formed six comparison groups. The scatter figures, bias figures, linear equations and correlation coefficients of comparison data in each group were made and the biases of measurement results were evaluated. Results The linear range of HPLC was up to 1 190 p^mol/L. The intraassay coefficient of variation （CV） and interassay CV of HPLC were 〈 2. 1% and 〈 3.9% , respectively. The absolute recoveries were 96.2%-101.0% and the average relative recoveries were 95.1%-99.4%. The average relative bias was -8. 29% for the determination of UA level in SRM by HPLC. Compared with result of isotope dilution mass spectrometry,the relative biases were -3.45% to -1.26% when determined the samples of IFCC by HPLC. There was good correlation between the comparison method （ HPLC ） and the test method （ closed-system including Beckman, Roche,Dade,Vitros and opened-system including Beckman） （ R^2 = 0. 996 9,0. 997 2, 0. 995 2,0. 996 7,0. 997 3, 0. 994 7 ）. When the levels of UA were 120,470,630 μmol/L,the predicted relative bias ranges were - 14.6% 4.3%, - 0.4% -8.0% ,0.8% -8.8% , respectively. There was good consistency between Beckman closed-system and reveredphase HPLC. The results of Roche closed-system in the low value range were lower. The Dade and Vitros closed-system in the high value range were higher. The results of closed-system were slightly better than opened-system. Conclusions The revered-phase HPLC with 5-FU as an internal standard is recommended as a candidate reference method for serum UA determination. There were some biases among the results of uricase method in different systems.