2010年版中国药典品种盐酸多西环素及其制剂含量和有关物质测定用HPLC法的建立

2010年版中国药典品种盐酸多西环素及其制剂

含量和有关物质测定用HPLC法的建立

袁耀佐1*；张玫1；钱文1；杨志明2；赵恂1

（1 江苏省食品药品检验所，南京 210008 ；2 常州制药厂有限公司，常州 213018）

摘要目的：建立先进的盐酸多西环素及其制剂含量和有关物质测定方法，用于2010年版中国药典。方法：用十八烷基硅烷键合硅胶为填充剂（pH值适用范围应大于9）；以醋酸盐缓冲液[0.25mol/L醋酸铵－0.1mol/L乙二胺四醋酸二钠－三乙胺（100：10：1），用冰醋酸或氨水调节pH值至8.8]－乙腈（85：15）为流动相；柱温35℃；检测波长为280nm，流速：1.0ml/min；进样体积：20µl。结果：新色谱条件中用醋酸铵代替草酸铵，解决了《中国药典》2005年版流动相中容易结晶析出堵塞系统管路的问题；通过调整流动相pH值提高了柱效，通过添加EDTA钠和三乙胺改善色谱峰的对称性，使部分原来色谱条件无法有效分离杂质C和杂质F在新系统中能与相邻峰完全分离。代表性样品色谱图中检出6个主要杂质峰，用杂质对照品对杂质A(ß-多西环素)、B(美他环素)、E(土霉素)进行了定位；用文献方法制备含已知杂质的溶液对杂质C(4-表多西环素)、D(4-表-6-表多西环素)进行了定位；用制备薄层色谱制得杂质F(2-乙酰-2-脱碳酰胺多西环素)溶液(纯度大于99%)，用LC-ESI-IP-MS2对主成分的结构进行了鉴定，用该溶液对杂质F 进行了定位，土霉素、美他环素、ß-多西环素相对于多西环素的相对响应因子（relative response factor，RRF）分别为0.95、1.29、0.77。建立的方法在0.07～122.556µg/ml范围内线性良好（r=1.0），检测限和定量限分别约为0.016、0.063µg/ml，多西环素及已知杂质的检测精密度良好，片剂和胶囊的回收率分别为99.2%(n=9)、99.4%(n=9)（RSD%均为0.2%），主成分在0.01mol/L的溶剂中可以稳定24小时。微调该色谱条件，可用于土霉素、四环素、金霉素、米诺环素和美他环素的含量和有关物质的测定。结论：建立的方法优于CP2005、USP31/NF26、BP2008、Ph.Eur 6.0、JPⅩⅤ中同品种方法，被2010年版中国药典采用。

关键词盐酸多西环素；高效液相色谱法；含量测定；有关物质；中国药典2010版

The Establishment of Chromatographic Analysis Method of Doxycycline Hyclate and Its Preparations for Pharmacopoeia of the People’s Republic of China 2010

Yuan Yao-zuo1, Zhang Mei1，Qian Wen1，Yang Zhi-ming2 and Zhao Xun1

(1.Jiangsu Institute for drug control, Nanjing 210008; 2.Changzhou Pharmaceutical factory Changzhou 213018) Abstract Objectives To establish a better chromatographic analysis method of doxycycline hyclate and its preparations for Pharmacopoeia of the People’s Republic of China 2010 . Methods Carry out the method for high performance liquid chromatogram, using a column packed with octadecysilane bonded silica gel and a mixture of a buffer solution which consist

of 100 volumes of 0.25 mol/L ammonium acetate solution, 10 volumes of 0.1 mol/L ethyldiaminetetraacetic acid disodium

salt solution and 1 volume of triethyamine adjusting pH to 8.8 with glacial acetic acid –acetonitrile (85:15) as the mobile phase, maintain the column temerature at 35℃, detection wavelength is 280nm, flow rate is 1 mL·min-1, injection volume is

20µl.Results The chromaotograhic mobile phase of doxycycline hyclate and its preparations used in CP2005 was changed completely, substituting ammonium acetate for ammonium oxalate as buffer salt to avoid the tunnel block of chromatographic system, substituting acetonitrile for dimethylformamide and tetrahydrofuran, the adjustment of pH value of mobile phase improves the separation ability, the addition of triethyamine and ethyldiaminetetraacetic acid disodium improves the symmetry of peaks. Impurity C and impurity F which can’t be separated effectively with their neighboring components in

CP2005 chromatographic system can be separated completely under the new chromatographic system. Impurity A

(6-epidoxycycline), B(metacycline), E(oxytetracycline) were located in the chromatogram by impurity standards. Impurity C (4-epidoxycycline), D(4-epi-6-epi-doxycycline) were located in the chromatogram by impurity solutions prepared according as literature methods, impurity F was located by the solution of impurity F which prepared by a preparative TLC method, and identified by LC-ESI-IP-MS2, the relative response factors of oxytetracycline, metacycline and 6-epidoxycycline were 0.95, 1.29 and 0.77 respectively. The linear range was 0.07～122.556µg/ml (r=1.0). The precision of method is good.The average recovery of the tablet method was 99.2%(n=9) and RSD% 0.2%, the average recovery of the capsule method was 99.4%(n=9) and RSD% 0.2%. Doxycycline can be stable in a 0.01 mol/L hydrochloric acid solution for 24hours. This chromatographic system can be applied to the determination of oxytetracycline, tetracycline, chlorotetracycline, minocycline and metacycline

by adjusting the mobile phase ratio of acetate buffer and acetonitrile. Conclusions the established method was better than the corresponding methods of CP2005,USP31/NF26,BP2008,Ph.Eur 6.0 and JPⅩⅤ and was used in CP2010.

Key words Doxycycline hyclate High performance liquid chromatography Assay Related substances Pharmacopoeia of the People’s Republic of China 2010

*袁耀佐，男，1968.03.10，博士，副主任药师，主要从事抗生素药物质量分析。