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

Preliminary exploration and structure identification of related substances of midazolam injection by 2D-LC-IT-TOF/MS

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 C_(18)(4.6 mm×250 mm,5μm)色谱柱,以甲醇-磷酸盐缓冲液(取同体积的0.1 mol·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p H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 )C_(18)(2.0 mm×50 mm,2.1μm)色谱柱,以甲酸溶液(甲酸调节p H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 orhophosphorie 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 InertsurstainTM 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.