盐酸二甲双胍制剂中N-亚硝胺基因毒性杂质的GC-MS/MS测定及N-亚硝基二甲胺的产生机制解析

Determination of N-nitrosamine genotoxic impurities in metformin preparations by GC-MS/MS and investigation on generation mechanism of N-nitrosodimethylamine

目的:建立气相色谱-质谱联用法同时测定盐酸二甲双胍制剂中6种N-亚硝胺类基因毒性杂质:N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)、N-亚硝基乙基异丙胺(NEiPA)、N-亚硝基二异丙胺(NDiPA)、N-亚硝基二丙胺(NDPA)、N-亚硝基二丁胺(NDBA),并对缓释制剂中NDMA的产生机制进行探索。方法:采用聚乙二醇为固定相的毛细管柱(TG-WAX,30 m×0.25 mm×0.25μm);起始温度40℃,维持0.5 min,20℃·min^(-1)升温至200℃,60℃·min^(-1)升温至240℃,维持5 min;进样口温度为250℃;载气为氦气;碰撞气为氩气;流速为1 mL·min^(-1);进样体积为2μL;质谱采用电子轰击离子化离子源(EI),选择反应监测(SRM)模式,实现了6种N-亚硝胺杂质的色谱分离及定量检测。结果:6种N-亚硝胺杂质在0.25~50 ng·mL^(-1)浓度范围内线性关系良好(r>0.999);检测限为0.01~0.07 ng·mL^(-1),定量限为0.04~0.2 ng·mL^(-1);平均加样回收率为97.4%~116.0%(n=9);精密度、重复性、稳定性试验的RSD均<10%。139批盐酸二甲双胍制剂中仅检出NDMA,且超限样品全部来自缓释制剂,研究发现,原料药中残留的合成起始物二甲胺与缓释骨架材料羟丙甲纤维素中的亚硝酸盐是产生NDMA的主要因素;同时,辅料被氧化后产生的活性氧物质也能够氧化原料药中的二甲胺进而生成NDMA。结论:本方法灵敏度高,专属性强,结果准确且重现性好,适用于盐酸二甲双胍制剂中N-亚硝胺杂质的定量分析;此外,在盐酸二甲双胍缓释片制剂工艺过程中,须严格控制原料药以及辅料的质量,从而避免或减少杂质NDMA的产生。

Objective:To establish a gas chromatography-tandem mass spectrometry(GC-MS/MS)method forsimultaneous determination of six potential N-nitrosamine genotoxic impurities(NGIs)including N-nitrosodimethylamine(NDMA),N-nitrosodiethylamine(NDEA),N-nitrosoethylisopropylamine(NEiPA),N-nitrosodiisopropylamine(NDiPA),N-nitrosodiphenylamine(NDPA),and N-nitrosodibutylamine(NDBA)in different metformin preparations.The generation mechanism of NDMA in metformin sustained preparations was also investigated.Methods:Chromatographic separation was achieved on capillary column using polyethylene glycol as stationary mobile phase(30 m×0.25 mm×0.25μm);the column temperature was initially set at 40℃(0.5 min hold),and rose to 200℃at a rate of 20℃·min^(-1),then ramped at a rate of 60℃·min^(-1)to 240℃(5 min hold);the injection temperature was set at 250℃,helium and argon was used as carrier gas and collisional gas respectively;the flow rate was 1 mL·min^(-1)and the injection volume was 2μL.Detection was performed on tandem mass spectrometry equipped with electron impact ion source.Quantification was performed on the selected reaction monitoring(SRM)mode.Results:The six NGIs showed good linear relationships within the range of 0.25-50 ng·mL^(-1)(r>0.999);the detection limits were 0.01-0.07 ng·mL^(-1)and the quantification limits were 0.04-0.2 ng·mL^(-1).The average recovery fall between 97.4%and 116.0%(n=9),and the precision,repeatability,and stability were fine with RSD value less than 10%.Only NDMA was detected in 139 batches of metformin preparations,and all samples with NDMA content exceeding the limit were metformin sustained release tablets.Results indicated that the residual dimethylamine in active pharmaceutical ingredients(API)and nitrile in excipient are the main factors to generate NDMA.Furthermore,reactive oxygen species produced by oxidation of excipients can oxidize dimethylamine in API to form NDMA.Conclusions:This method showes satisfactory sensitivity,specificity,accuracy and repeatability,and is suitable for quantification of NGIs in metformin preparations.In addition,during the preparation process of metformin sustained release tablets,the quality of API and excipients should be strictly controlled to avoid or reduce the generation of NDMA.