目的 建立測定人血漿中枸櫞酸濃度的 HPLC 方法,探討連續(xù)性腎臟替代療法(CRRT)患者行局部枸櫞酸鈉抗凝(RCA)中濾器前、濾器后和濾過液血漿中枸櫞酸濃度相關(guān)性。方法 血漿樣品用甲醇沉淀蛋白,測定枸櫞酸的條件:色譜柱為 Eclipse Plus C₁₈柱(250 mm×4.6 mm,5 μm);流動相為 0.1% 磷酸水溶液-甲醇(98:2);柱溫:35℃;進(jìn)樣體積:20 μL;體積流量:0.6 mL·min^-1;檢測波長:215 nm。采用建立的 HPLC 方法對 40 例使用 RCA 的 CRRT 患者的濾器前、濾器后和濾過液血漿中的枸櫞酸濃度進(jìn)行測定。采用 Pearson 相關(guān)性分析對濾器前、濾器后和濾過液血漿中枸櫞酸濃度進(jìn)行相關(guān)性分析,記錄每位患者枸櫞酸泵速,觀察濾器的凝血程度,根據(jù)血液凈化治療后透析器和管路的凝血情況進(jìn)行分級。結(jié)果 建立的 HPLC 方法檢測血漿中枸櫞酸濃度的專屬性良好,基質(zhì)效應(yīng)為 90.25%~101.23%,回收率在 89.90%~97.00%,日內(nèi)精密度RSD在2.60%~8.94%,日間精密度RSD在2.48%~7.31%,樣品在室溫下放置0、12、24 h及4℃下放置1、2、3、4周,穩(wěn)定性均良好。濾器前血漿枸櫞酸濃度為(2.93±0.97) mmol·L^-1,濾器后血漿枸櫞酸濃度為(2.42±0.77) mmol·L^-1,濾過液血漿中枸櫞酸濃度為(2.89±0.81) mmol·L^-1;濾過液血漿中枸櫞酸濃度與濾器前、濾器后血漿枸櫞酸濃度的 Pearson 相關(guān)系數(shù)分別為 0.672(P=0.00)、0.734(P=0.00)。40 例患者濾器凝血情況:0~1 級 32 例,2 級 7 例,3 級 1 例。結(jié)論 建立的HPLC 分析方法符合生物樣品含量測定要求,可用于血漿中枸櫞酸的濃度檢測。在 RCA 應(yīng)用于 CRRT 患者時,濾過液血漿枸櫞酸濃度可代替濾器后血漿枸櫞酸濃度評價濾器抗凝效果,減少反復(fù)檢測導(dǎo)致的診斷性失血。

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, 5 μm), the mobile phase was 0.1% phosphoric acid solution-methanol (98: 2), column temperature was 35℃, injection volume was 20 μL, 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.

R917

江蘇省藥學(xué)會-奧賽康基金科研項(xiàng)目(A201941);2019 泰州市科技支撐社會發(fā)展指導(dǎo)性項(xiàng)目(SSF20190065)