HPLC-DAD法同时检测抗炎类中药制剂中非法掺入的6种四环素类抗生素

HPLC-DAD simultaneous determination of six tetracycline antibiotics mixed into anti-inflammatory preparations of traditional Chinese medicine

目的建立同时检测抗炎类中成药中非法掺入四环素类抗生素的专属性方法。方法采用HPLC-DAD法。色谱柱为AgilentC18柱（250mm×4．6mm，5μm），醋酸盐缓冲液[取醋酸铵17．36g，乙二胺四醋酸二钠3．35g，加水适量溶解后，加三乙胺9mL，加水至1000mL，用醋酸调节pH至8．8]-乙腈（88：12），流速1．2mL／min；柱温35℃。检测波长280nm。通过液相色谱保留时间和紫外光谱信息，对消炎类中成药的提取液进行定性鉴别和定量测定。结果土霉素、美他环素、β-多西环素、多西环素、盐酸四环素、盐酸金霉素的线性范围分别为11．6-232．4pg／mL（r=0．9998），12．7-190．9μg／mL（r=0．9995），105．0-862．0μg／mL（r=O．9997），20．50-410．0pg／mL（r=0．9987），23．28-598．4μg／mL（r=0．9990），48．60-971．6μg／mL（r=0．9989）。平均加样回收率（n=6）分别为98．3％，97．5％，96．2％，98．4％，101．1％，96．1％；RSD分别为1．2％，0．5％，0．9％，1．5％，0．8％和1．9％。结论该方法操作简单，重复性好，可作为分析中药抗炎制剂中非法掺入土霉素、美他环素、β-多西环素、多西环素、盐酸四环素、盐酸金霉素的有效检测方法。

Objective To develop a specific method for simultaneous identification of six tetracycline antibiotics illegally added into anti-inflammatory preparations of traditional Chinese medicine. Methods Analysis was performed on an Agilent C18 （4.6mm×250mm, 5μm） column. The mobile phase was acetate buffer solution （ammonium acetate 17.36g and EDTA 3.35g were dissolved in sufficient water. Then added trietlylamine 9mL, acetic acid was added to adjust the pH to 8.8. The solution was diluted with water to 1000mL）-acetonitrile（88：12） The flow rate was 1.2 mL/min, the detection wavelength was 280nm and the column temperature was 35℃. After separated by HPLC, the organic extraction of Chinese medicine preparations was analyzed. The ultraviolet spectra information and retention time were used to identify and determinate the six drugs by comparison with those of reference substances. Results The calibration curves were linear in the ranges of 11.6-232.4μg/mL for oxytertracycline （r=0.9998）, 12.7-190.9μg/mL for metacycline （r=0.9995）, 105.0-862.0μg/mL for 13-doxycycline（r=0.9997）, 20.50~410.0μg/mL for doxycyclin（r=0.9987）, 23.28-598.4μg/mL for tetracycline （r=0.9990）, 48.60-971.6μg/mL for chlortetracycline（r=0.9989）.The average recoveries（n=6） were 98.3%, 97.5%, 96.2%, 98.4%, 101.1% and 96.1%; RSDs were 1.2%, 0.5%, 0.9%, 1.5%, 0.8% and 1.9%, respectively. Conclusion The method is specific, sensitive and accurate, thus it can be used to detect the illegal Chinese traditional medicine anti-inflammatory preparations.