柱前衍生化RP-HPLC法鉴别及测定麻黄中的生物胺类成分

Identification and determination of biogenic amines in Ephedrae Herba by RP-HPLC with precolumn derivatization

目的：从中药麻黄所含阿魏酰组胺和麻黄根碱的结构以及可能的生源关系推测其尚有未被发现的组胺、腐胺、亚精胺和精胺等成分,建立柱前衍生化RP-HPLC法测定麻黄及麻黄根中11种生物胺。方法：药材样品经1 mol·L-1盐酸提取,正丁醇-二氯甲烷（1∶1）萃取富集,以丹磺酰氯为柱前衍生试剂进行衍生化,RP-HPLC法测定。采用Shimadzu C18色谱柱（200mm×4.6 mm,5μm）,以乙腈（A）-水（B）为流动相,梯度洗脱（0-7 min,45%A→50%A;7-25 min,50%A→90%A;25-35min,90%A;35-40 min,90%A→45%A）,流速为0.8 m L·min-1,检测波长为254 nm,柱温为30℃。结果：甲胺、尸胺、组胺、亚精胺与精胺的线性范围为0.10-10.00 mg·L-1（r〉0.999）,色胺、腐胺的线性范围为0.10-50.00 mg·L-1（r〉0.999）,2-苯乙胺、5-羟色胺、酪胺与多巴胺的线性范围为0.25-10.00 mg·L-1（r〉0.999）,平均回收率在75.6%-101.1%之间。麻黄中含有甲胺、色胺、腐胺、尸胺、组胺、亚精胺、多巴胺与精胺。麻黄根中含有甲胺、腐胺、尸胺、组胺、亚精胺与精胺。结论：经方法学验证,该方法可用于测定麻黄中甲胺、色胺、2-苯乙胺、腐胺、尸胺、组胺、5-羟色胺、酪胺、亚精胺、多巴胺与精胺11种生物胺类成分。这是首次发现在麻黄中含有生物胺类成分,同时也证实了麻黄中存在组胺、腐胺、精胺和亚精胺的推测,并为其生源关系提供了证据。该研究方法也可用于其他中药或天然药物化学成分的深入研究。

Objective： To develop a method for the determination of eleven biogenic amines in Ephedrae Herba and Ephedrae Radix et Rhizome by RP- HPLC with precolumn derivatization as it was speculated that histamine,putrescine,spermidine and spermine should exist according to the structures of feruloyl histamine and ephedradine A- D and their possible biogenetic relationship in Ephedrae Herba. Methods： Biogenic amines in Ephedrae Herba were extracted with 1 mol·L- 1hydrochloric acid,concentrated and purified by n- butyl alcohol- dichloromethane（ 1∶ 1） extraction,derivatized with dansyl chloride,and separated by RP- HPLC. The separation was performed on a Shimadzu C18column（ 200 mm × 4. 6 mm,5 μm） with gradient elution（ 0- 7 min,45% A →50% A; 7- 25 min,50% A→90% A; 25- 35 min,90% A; 35- 40 min,90% A→45% A） using acetonitrile（ A） and water（ B） as the mobile phase at a flow rate of 0. 8 m L·min- 1. The detection wavelength was 254 nm. The column temperature was 30℃. Results： The calibration curves were linear in the ranges of 0. 10- 10. 00 mg·L- 1（ r 0. 999） for methylamine,cadaverine,histamine,spermidine and spermine,0. 10- 50. 00 mg·L- 1（ r 0. 999） for tryptamine and putrescine,0. 25- 10. 00 mg·L- 1（ r 0. 999） for 2- phenylethylamine,serotonin,tyramine and dopamine,respectively. The average recoveries varied from 75. 6% to 101. 1%. Ephedrae Herba contained methylamine,tryptamine,putrescine,cadaverine,histamine,spermidine,dopamine and spermine. Ephedrae Radix et Rhizome contained methylamine,putrescine,cadaverine,histamine,spermidine and spermine. Conclusion： The method has acceptable sensitivity,precision,accuracy,selectivity and recovery,and can be used to determine eleven biogenic amines（ methylamine,tryptamine,2- phenylethylamine,putrescine,cadaverine,histamine,serotonin,tyramine,spermidine,dopamine and spermine） in Ephedrae Herba. This is the first report that biogenic amines are existed in Ephedrae Herba. The results confirmed the speculation of existence of histamine,putrescine,spermidine and spermine in Ephedrae Herba,and provided the experimental evidence of its biogenetic relationship. This methodology may be an effective way to study the chemical constituents in traditional Chinese medicine and natural medicine.