人血浆中枸橼酸HPLC测定方法建立及其在连续性肾脏替代疗法患者行局部枸橼酸钠抗凝中的应用

Establishment of HPLC method for determination of citric acid in human plasma and its application in local sodium citric acid anticoagulation in patients with continuous renal replacement therapy

目的建立测定人血浆中枸橼酸浓度的HPLC方法,探讨连续性肾脏替代疗法(CRRT)患者行局部枸橼酸钠抗凝(RCA)中滤器前、滤器后和滤过液血浆中枸橼酸浓度相关性。方法血浆样品用甲醇沉淀蛋白,测定枸橼酸的条件:色谱柱为Eclipse Plus C18柱(250 mm×4.6 mm,5μm);流动相为0.1%磷酸水溶液-甲醇(98∶2);柱温:35℃;进样体积:20μL;体积流量:0.6 mL·min^(-1);检测波长:215 nm。采用建立的HPLC方法对40例使用RCA的CRRT患者的滤器前、滤器后和滤过液血浆中的枸橼酸浓度进行测定。采用Pearson相关性分析对滤器前、滤器后和滤过液血浆中枸橼酸浓度进行相关性分析,记录每位患者枸橼酸泵速,观察滤器的凝血程度,根据血液净化治疗后透析器和管路的凝血情况进行分级。结果建立的HPLC方法检测血浆中枸橼酸浓度的专属性良好,基质效应为90.25%~101.23%,回收率在89.90%~97.00%,日内精密度RSD在2.60%~8.94%,日间精密度RSD在2.48%~7.31%,样品在室温下放置0、12、24 h及4℃下放置1、2、3、4周,稳定性均良好。滤器前血浆枸橼酸浓度为(2.93±0.97)mmol·L^(-1),滤器后血浆枸橼酸浓度为(2.42±0.77)mmol·L^(-1),滤过液血浆中枸橼酸浓度为(2.89±0.81)mmol·L^(-1);滤过液血浆中枸橼酸浓度与滤器前、滤器后血浆枸橼酸浓度的Pearson相关系数分别为0.672(P=0.00)、0.734(P=0.00)。40例患者滤器凝血情况:0~1级32例,2级7例,3级1例。结论建立的HPLC分析方法符合生物样品含量测定要求,可用于血浆中枸橼酸的浓度检测。在RCA应用于CRRT患者时,滤过液血浆枸橼酸浓度可代替滤器后血浆枸橼酸浓度评价滤器抗凝效果,减少反复检测导致的诊断性失血。

Objective To establish a high performance liquid chromatography(HPLC)method for the determination of the concentration of citric acid in human plasma,and to investigate the correlation between the concentration of citric acid in the prefilter,post-filter and filtered plasma in patients undergoing local sodium citric acid anticoagulation(RCA)in continuous renal replacement therapy(CRRT).Methods Plasma samples were precipitated with methanol,and the conditions for determination of citric acid were as follows:The chromatographic column was Eclipse Plus C18 column(250 mm×4.6 mm,5μm),the mobile phase was 0.1%phosphoric acid solution-methanol(98∶2),column temperature was 35℃,injection volume was 20μL,volume flow was 0.6 mL·min^(-1),detection wavelength was 215 nm.The established HPLC method was used to determine the concentration of citric acid in the pre-filter,post-filter and filtered plasma of 40 patients with CRRT using RCA.Pearson correlation analysis was used to analyze the correlation between the citric acid concentration in the pre-filter,post-filter and filtered plasma,the citric acid pump speed of each patient was recorded,the coagulation degree of the filter was observed,and according to the coagulation condition of the dialyzer and the pipeline after the blood purification treatment was graded.Results The specificity of the established HPLC method for the determination of citric acid concentration in plasma was good.The matrix effect was 90.25%—101.23%,the recovery rate was 89.90%—97.00%,the intra day precision RSD was 2.60%—8.94%,and the inter day precision RSD was 2.48%—7.31%.The samples were kept at room temperature for 0,12,24 hours and 4℃for 1,2,3,and 4 weeks,with good stability.The citric acid concentration in pre-filter plasma was(2.93±0.97)mmol·L^(-1),the citric acid concentration in post-filter plasma was(2.42±0.77)mmol·L^(-1),and the citric acid concentration in filtered plasma was(2.89±0.81)mmol·L^(-1).The Pearson correlation coefficient between the concentration of citric acid in the filtered plasma and the concentration of citric acid in pre-filter,and post-filter was 0.672(P=0.00)and 0.734(P=0.00),respectively.Coagulation condition of filter in 40 patients:32 cases were grade 0—1,seven cases were grade 2,one case was grade 3.Conclusion The established HPLC method meets the requirements of biological sample content determination,and can be used to determine the concentration of citric acid in plasma.When RCA is applied to patients with CRRT,the citric acid concentration of the filtered plasma can replace the citric acid concentration of postfilter plasma to evaluate the anticoagulant effect of the filter and reduce the diagnostic blood loss caused by repeated testing.