高效液相色谱法测定人乳汁中羟氯喹浓度

Determination of Hydroxychloroquine in Human Breast milk by HPLC

目的:建立人乳汁中羟氯喹浓度的HPLC测定方法。方法:取乳汁样本200μL,以氯喹为内标,在0.45mmol·L^(-1) NaOH碱性条件下以甲基叔丁基醚液液提取,氮气吹干后用50μL流动相复溶,取20μL进样。Shimadzu Shim-pack CLC-ODS柱(150×6 mm,5μm)色谱柱分离,流动相为45 mmol·m L^(-1)磷酸二氢钾水溶液:乙腈=87:13(H3P04调节流动相p H至3.0),流速为1.0 mL·min^(-1),柱温40℃,荧光检测激发波长和发射波长分别为337 nm和371 nm。结果:标准曲线在200~2000 ng·m L^(-1)范围内线性良好(r=0.999 9),低、中、高浓度质控样本(400、800、1 500 ng·mL^(-1))的批内、批间变异(RSD)分别为1.07%~2.93%(n=5)和1.82%~3.91%(n=5),方法学和提取回收率分别为90.84%~91.90%(n=10)和67.04%~69.61%(n=3),内标的提取回收率为67.70%±3.94%(n=3)。结论:本方法灵敏度高,准确性好,简便快捷,适用于人乳汁中羟氯喹浓度的测定。

Objective： To develop a high-performance liquid chromatographic（HPLC） method for determination of hydroxychloroquine in human breast milk. Methods： Chloroquine was used as the internal standard. 200 mL breast milk sample were extracted with methyl-tert-butyl ether（MTBE） in an alkaline condition after adding 0.45mmol·L^-1 NaOH solution, followed by drying with nitrogen and re-dissolving with 50 μL mobile phase. The separation was carried out on a Shimadzu Shim-pack CLC-ODS column（150×6 mm, 5μm）at 40°C. The mobile phase consisted of acetonitril -45m mol·mL^-1 KH2PO4 buffer solution（13：87）（pH adjusted to 3.0 by H3PO4）. The flow rate was 1.0mL·min^-1 and the injection volume was 20 μL. Fluorescence detection was performed at excitation and emission wavelengths of 337 nm and 371 nm, respectively. Results： The calibration curve of hydroxychloroquine was linear in the range of 200-2 000ng·mL^-1（r =0.999 9）. The intra-run and inter-run RSDs were 1.07%-2.93%（n =5）and 1.82%-3.91%（n =5）for the three check samples（400, 800, 1500 ng.mL^（-1）） respectively. The accuracy was 90.84%-91.90%（n =10）. The extraction recoveries were 67.04%-69.61% and 67.70%±3.94% for hydroxychloroquine and IS, respectively（n=3）. Conclusion： The method is simple, sensitive, and accurate, and it can be used for the measurement of hydroxychloroquine concentration in human breast milk.