GC-MS法测定尿液中麻黄汤代谢产物麻黄类生物碱的浓度

Simultaneous Determination of Ephedrine Alkaloids by GC-MS in Human Urine after Oral Administration of Mahuang Decoction

目的:建立 GC-MS 测定麻黄汤代谢产物麻黄类生物碱浓度的方法。方法:尿样经5 mol·L^(-1)氢氧化钾碱化后,用乙酸乙酯萃取。以二苯胺为内标,采用 GC-MS 分离并同时测定了人体口服麻黄汤后尿中5种麻黄碱的浓度,检测方式为选择离子(m/z 44,m/z 58,m/z 72,m/z 169)。色谱条件:色谱柱为 DB-5MS 毛细管柱(30 m×250μm,0.25μm),载气为 He(1 mL·min^(-1)),无分流进样,进样量1μL。起始温度70℃(1 min)125℃(5min)270℃(5 min)。用该方法测定6名健康志愿者单次口服麻黄汤后尿液中麻黄碱类代谢产物的浓度。结果:在24 min 内去甲伪麻黄碱(NMP)、去甲麻黄碱(NME)、麻黄碱(E)、伪麻黄碱(PE)和甲基麻黄碱(ME)达到良好分离。尿液中 NMP、NME、E、PE 和 ME 的线性范围分别为2.0～20μg·L^(-1)(r=0.997)、4.0～40μg·L^(-1)(r=0.998)、20～200μg·L^(-1)(r=0.999)、10～100μg·L^(-1)(r=0.999)和2.0～2μg·L^(-1)(r=0.997)。5种麻黄生物碱的回收率均在88.74%～102.70%之间。日内和日间精密度均≤10%。结论:方法简单、灵敏、准确,重复性好,可用于含麻黄中药复方代谢产物麻黄类生物碱的测定。

Objective ：To establish a gas chromatography- mass spectrometry（ GC -MS）method for the simultaneous determination of ephedrine alkaloids in human urine after oral administration of Mahuang decoction. Methods： The urine was alkalized with 5 mol·L^-1 KOH and extracted with ethyl acetate. The extracts were analyzed by GC - MS under selected - ion monitoring（SIM） mode（ m/z 44, m/z 58, m/z 72, m/z 169）, using diphenylamine as internal standard. Capillary column DB -5MS（30m×250μm,0.25μm）was used. Helium was used as carrier gas （ 1 mL·min^-1 ）. A1 μL volume was injected （splitless）. The GC oven temperature program was as follows ：70 ℃ （1 min） （5 ℃ ·min^-1）→125 ℃ （5 min） （10℃·min^-1） ;270℃（5 min）. The concentrations of norpseudoephedrine （ NMP）, norephedrine （ NME ）, ephedrine （ E ）, pseudoephedrine （ PE ） and methylephedrine （ ME ） in human urine in healthy volunteers were determined. Results： NMP, NME, E, PE and ME were well separated in 24 minutes. Five calibration curves were established in the range of 2.0 - 20μg·L^-1 for NMP（ r = 0. 997 ） and ME（ r = 0. 997 ）, 4.0 - 40μg·L^-1 for NME （ r = 0. 998 ）, 20 - 200μg·L^-1 for E （ r = 0. 999 ） and 10 - 100μg·L^-1 for PE （ r = 0. 999）. The recoveries of NMP, NME, E, PE and ME were in the range of 88.74% - 102.70% respectively. Precision（ RSD）within day and between days were less than ≤ 10% for all ephedrines. Conclusion：The method shows excellent separation, linearity, specificity, accuracy, precision and is suitable for separation and quantitation of ephedrines in the metabolites of traditional Chinese medicine remedy.