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目的建立一种高效液相色谱法测定人全血中羟氯喹的浓度。方法取全血样本300μL,用3倍量的乙酸乙酯液液提取,以甲硝唑为内标,氮气吹干后用流动相60μL复溶,进样20μL。用Venusil ASB C18(4.6 mm×250 mm,5μm)色谱柱分离,流动相为20 mmol·L~(-1)甲酸铵水溶液-乙腈=85∶15(甲酸调节流动相p H至3.0),流速为0.8 m L·min~(-1),柱温为35℃,检测波长为254 nm。考察该方法的专属性,标准曲线和定量下限,精密度和准确度,稳定性和提取回收率。结果羟氯喹标准曲线方程为f=0.56×10~(-1)C+0.39×10~(-1)(r=0.998 2),在100~3000 ng·m L~(-1)内线性关系良好,日内、日间精密度RSD均小于5.5%,全血样品的提取回收率为(76.08±6.21)%(n=5)。结论本方法操作简洁,灵敏度较高,准确性好,适用于人全血中羟氯喹浓度的监测。
Objective To establish a method for the determination of hydroxychloroquine in human whole blood by high performance liquid chromatography. Methods Take whole blood sample 300μL, with 3 times the amount of ethyl acetate extract, with metronidazole as internal standard, dry with nitrogen after the mobile phase 60μL reconstituted, injection 20μL. The sample was separated on a Venusil ASB C18 column (4.6 mm × 250 mm, 5 μm) using a mobile phase of 20 mmol·L -1 ammonium formate-acetonitrile = 85:15 (formic acid adjusted mobile phase p H to 3.0) 0.8 m L · min -1, the column temperature was 35 ℃ and the detection wavelength was 254 nm. The specificity, standard curve and quantitation limits, precision and accuracy, stability and extraction recovery of this method were examined. Results The calibration curve of hydroxychloroquine was f = 0.56 × 10 ~ (-1) C + 0.39 × 10 ~ (-1) (r = 0.998 2) and linear in the range of 100 ~ 3000 ng · m L ~ (-1) RSD of intra-day and inter-day precision was less than 5.5%, and the recovery rate of whole blood samples was (76.08 ± 6.21)% (n = 5). Conclusion The method is simple, sensitive, accurate and suitable for the monitoring of hydroxychloroquine concentration in human whole blood.