目的 应用在线脱盐-高效液相色谱-离子阱-飞行时间质谱(2D-LC-IT-TOF/MS)法鉴定盐酸多西环素片在药典条件下检出的有关物质结构,并推断盐酸多西环素在溶液状态下的降解途径。方法 LC 1stD色谱条件:InertSustain^TM C₁₈(4.6 mm×150 mm,5 μm)色谱柱,醋酸盐缓冲液[0.25 mol·L^-1醋酸铵-0.1 mol·L^-1 EDTA钠盐-三乙胺(100∶10∶1),用醋酸或氨水调节pH值至8.8]-乙腈(85∶15)为流动相进行等度洗脱;LC 2ndD 色谱条件:InertSustain^TM C₁₈(2.1 mm×50 mm,2 μm)色谱柱,0.1%甲酸-水溶液为流动相A,0.1%甲酸-乙腈为流动相B,进行梯度洗脱;质谱条件:ESI正负离子同时检测,雾化气流速1.5 L· min^-1,干燥气流速10 L· min^-1,脱溶剂管温度200 ℃。根据多级质谱信息来推定药典条件下检出的有关物质结构和强制降解溶液中产生的杂质结构。结果 在盐酸多西环素片中共检出8个有关物质,推定出其中4个含量大于0.1%的有关物质的结构,分别为4-表多西环素、美他环素、β-多西环素、2-乙酰-6-脱碳酰胺多西环素。盐酸多西环素溶液在碱性条件下易降解,生成脱酮基开环物;在高温条件下,容易产生4-表多西环素,并伴有少量4-表-6-表多西环素的生成;在酸性条件和光照条件下相对稳定。结论 建立的方法可用于盐酸多西环素中杂质的鉴定,为盐酸多西环素的质量控制与工艺优化研究提供可靠的分析手段。

OBJECTIVE To rapidly identify the related substances in doxycycline hyclate tablets and investigate the possible degradation pathways of doxycycline hyclate solution by 2D-LC-IT-TOF/MS. METHODS Firstly,the chromatography was carried out using the 1stD InertSustain^TM C₁₈(4.6 mm×150 mm,5 μm) column and a mobile phase containing a mixture of buffer solution(0.25 mol·L^-1 ammonium acetate solution-0.1 mol·L^-1 ethyldiaminetetraacetic acid disodium: triethyiamine=100∶10∶1)-acetonitrile(85∶15). Solution pH was adjusted to 8.8 with glacial acetic acid. Then a InertSustain^TM C₁₈(2.1 mm×50 mm,2 μm) column was used with 0.1% formic acid-water as mobile A and 0.1% formic acid-acetonitrile as mobile B in a gradient elution mode in 2ndD. Electrospray ionization (ESI) source was tested in both positive and negative ion modes. Nebulized gas flow was 1.5 L· min^-1. Dry gas flow was 10 L· min^-1. The desolvation tube temperature was kept at 200 ℃. Related substances were characterized according to multi-level MS behaviors. The 2D-LC-IT-TOF/MS method was employed to identify the structures of impurities in forced degradation solutions and illuminate the degradation pathways of doxycycline hyclate solution. RESULTS A total of eight related substances were detected in doxycycline hyclate tablets. Four of them had a content of more than 0.1%, and their structures were identified to be 4-epidoxycycline, metacycline, β-epidoxycycline and 2-acetyl-2-decarbamoyldoxycycline. The solution of doxycycline hyclate easily degraded under alkaline condition and generated an open loop compound taken off the keto group. The solution was sensitive to heat, and generated 4-epidoxycycline with a little amount of 4-epi-6-epidoxycycline; but the solution was stable under illumination and acidic condition. CONCLUSION The established method is suitable for rapid identification of impurities in doxycycline hyclate, which can be applied as a useful analytical tool for quality control and drug process optimization of doxycycline hyclate.