二维液相-离子肼-飞行时间质谱法对咪达唑仑注射液中有关物质的初探与结构解析

目的：应用在线脱盐-高效液相色谱-离子肼-飞行时间质谱（2D-LC-IT-TOF/MS）对咪达唑仑注射液在药典条件下检出的杂质进行了解析鉴定，并推断了咪达唑仑的降解途径。方法：分离液相色谱条件：采用ZORBAX Eclipse Plus C₁₈（4.6 mm×250 mm，5 μm）色谱柱，以甲醇-磷酸盐缓冲液（取同体积的0.1mol·L^-1磷酸溶液与0.03 mol·L^-1三乙胺溶液混合，用0.1 mol·L^-1氢氧化钠溶液调节pH至3.5）（65∶35）为流动相等度洗脱；捕集成分脱盐液相色谱条件：采用Inertsurstain_TM C₁₈（2.0 mm×50 mm，2.1 μm）色谱柱，以甲酸溶液（甲酸调节pH为3.5）为流动相A，甲醇为流动相B，进行梯度洗脱。质谱条件：采用ESI正离子，雾化气为氮气（1.5 L·min^-1），干燥气为氮气（10 L·min^-1），脱溶剂管温度200℃。根据多级质谱信息来推定检出的有关物质结构。结果：在咪达唑仑注射液中共检出3个有关物质，推定分别为咪达唑仑同分异构体、英国药典杂质D和杂质G，其中咪达唑仑同分异构体目前未见国内外文献报道，实验证明其为咪达唑仑在酸性条件下产生的降解产物。结论：建立的方法可用于咪达唑仑注射液有关物质检查，并为咪达唑仑注射液进一步的质量控制与工艺优化研究提供可靠分析手段。

Objective: To establish a 2-dimensional HPLC/ion trap/time-of-flight mass spectrometric method(2D-LC-IT-TOF/MS) for the detection and identification of the related substances described in the Pharmacopoeias, and to discuss the possible degration pathways of midazolam. Methods: The separation was carried out on ZORBAX Eclipse Plus C₁₈(4.6 mm×250 mm, 5 μm) column using a mixture of 65 volumes of methanol and 35 volumes of phosphate buffer solution(equal volumes of 0.1 mol·L^-1 orhophosphoric acid and 0.03 mol·L^-1 triethylamine. pH was adjusted to 3. 5 with 0. 1 mol·L^-1 NaOH) as the mobile phase with a flow rate of 1. 0 mL·min^-1. The related substances of interest were trapped on an Inertsurstain_TM C₁₈(2. 0 mm×50 mm, 2. 1 μm) column separately and then desalted by using formic acid solution(adjust the pH with formic acid solution to 3. 5) as mobile A and methanol as mobile B in a gradient elution. IT-TOF/MS was conducted by electrospray positive ionization with the nebulized gas of 1. 5 L·min^-1,dry gas N2 of 10. 0 L·min^-1,desolation tube temperature at 200℃. The related substances detected were then characterized according to their multi-level product MS behaviors. Results: Three related substances were detected in midazolam injection and their structures were identified to be midazolam's isomer,and the impurity D and G described in BP. The isomer,not reported before,was the degradation product of midazolam under acidic stress. Conclusion: The established method is suitable for the identification of impurities in midazolam injection and useful for quality control and process optimization of midazolam injection.

[1] GHONEIM MM, BLOCK RI. The interaction of midazolam and flumazenil on human memory and cognition[J]. Anesthesiology, 1993, 79(6):1183
[2] NAGUIB M, SAMARKANDI AH. The comparative dose-response effects of melatonin and madazolam for premedication of adults[J]. Anesth Analg,2000, 91(2):473
[3] 孙丽华,杨桐伟.咪达唑仑的临床应用进展[J].吉林医学, 2006, 27(12):1469 SUN LH, YANG TW. Progress in clinical application of midazolam[J]. Jilin Med J,2006, 27(12):1469
[4] MINORU W, TOMONORI T, MASAKO A, et al. Effects of glucocorticoide on pharmacokinetics and pharmacodynamics of midazolam in rats[J]. Life Sci,1998, 63(19):1685
[5] 原皓,邹亮,孙莉.全麻醉术中应用咪达唑仑镇静的安全性及有效性临床分析[J].医学综述, 2015, 21(7):1337 YUAN H, ZOU L, SUN L. Analysis on the safety and efficacy of midazolam sedation in general anesthesica operation[J]. Med Recapit,2015, 21(7):1337
[6] 许涛,杨涛,曹好好.咪达唑仑或丙泊酚联用右美托咪定对颅脑创伤患者镇静作用的比较[J].中国医院药学杂志,2015, 35(4):326 XU T,YANG T,CAO HH. Comparative analysis of sedative effects mediated by dexmedetomidine in combination with propofol or midazolam in traumatic brain injury patients[J]. Chin J Hosp Pharm,2015, 35(4):326
[7] 胡春伟.高效液相色谱法同时测定肝微粒体中咪达唑仑及代谢产物含量[J].中国医院用药评价与分析,2016, 16(1):42 HU CW. Determination of content of midazolam and its metabolite in live microsome by HPLC[J]. Eval Anal Drug Use Hosp China, 2016, 16(1):42
[8] 郑琴,郝伟伟,胡双.液相色谱-串联质谱法同时测定大鼠血浆中咪达唑仑和非那西丁及其动力学研究[J].中国新药与临床杂志, 2016, 16(1):42 ZHENG Q, HAO WW, HU S. Simultaneous determination of pharmacokinetics of midazolam and phenacetion in rat plasma by LC-MS/MS[J]. Chin J New Drugs Clin Rem,2016, 16(1):42
[9] 刘雅,路红梅.老年病人麻醉前肌肉注射咪达唑仑的安全剂量[J].中华麻醉学杂志, 2005, 25(8):628 LIU Y,LU HM. Safe dosage of midazolam in elderly patients before anesthesia[J]. Chin J Anesthesiol,2005, 25(8):628
[10] 中国药典2015年版.二部[S]. 2015:747 ChP 2015. Vol Ⅱ[S]. 2015:747
[11] BP 2015 Vol Ⅲ[S]. 2015:843
[12] USP 38-NF 33[S]. 2015:4397
[13] 符义刚,李莉维.咪达唑仑共轭体系研究[J].化学与生物工程, 2010, 27(10):34 FU YG,LI LW. Study on conjuated system of midazolam[J]. Chem Bioeng, 2010, 27(10):34
[14] PDR Staff. Medical Economics Staff. Physicians'Desk Reference. 66 Edition[M]. London:PDR Network,2011:2794
[15] 高辉,温学森,马小军.二维液相色谱技术在药物分析中的应用[J].药物分析杂志, 2007, 27(4):616 GAO H, WEN XS, MA XJ. The technique of dimensional liquid chromatography and its application in drug[J]. Chin J Pharm Anal, 2007, 27(4):616