目的: 采用柱前衍生化-高效液相色谱-串联质谱联用技术建立骨肽注射液中生物胺类物质定量测定方法,并通过影响因素试验和加速试验考察其生物胺类物质的稳定性。方法: 采用丹磺酰氯衍生化法对样品进行前处理,经ZORBAX SB-C18色谱柱梯度洗脱分离,电喷雾离子源正离子模式下多反应监测,对骨肽注射液中10种生物胺进行定量测定,并于高温条件、强光照条件和加速试验条件下考察生物胺类物质的稳定性。结果: 方法学考察结果显示10种生物胺线性关系良好,相关系数均大于0.990,方法检测限为0.01~0.10 ng·mL-1,定量限为0.05~0.30 ng·mL-1,准确性、重复性和耐用性较好。稳定性研究结果显示不同条件下骨肽注射液中生物胺类物质变化存在差异,高温条件下腐胺增多,精胺和亚精胺减少,强光照条件及加速试验条件中生物胺类物质含量均存在变化。结论: 方法学验证表明该方法可用于同时测定骨肽注射液中10种生物胺,为骨肽及其它药品质量标准中生物胺类物质检测方法的建立和完善提供了参考,同时生物胺类物质稳定性研究结果表明高温及强光条件可对其稳定性产生影响,提示药品生产及贮存过程中应加强质控和监管,从而保障药品质量的稳定性。
Objective: To establish a pre-column derivatization high performance liquid chromatography-mass spectrometry to determine biogenic amines in osteopeptide injections and determine the changes of biogenic amines after the stability influence factor test and accelerated test. Methods: The osteopeptide injections were separated by a ZORBAX SB-C18 chromatographic column and gradient elution after derivatization by dansyl chloride. Ten kinds of biogenic amines was determined by mass spectrometry with electrospray ion source and multiple reaction monitoring in positive mode. The osteopeptide injections were placed under high temperature, strong light and accelerated experimental conditions to inspect the stability of biogenic amines. Results: Method validation showed that the linear relationship of 10 biological amines was good, and the correlation coefficients were higher than 0.990. The detection limits were 0.01-0.10 ng·mL-1, the quantitation limits were 0.05-0.30 ng·mL-1. Good accuracy, repeatability and durability were obtained. Under different conditions, the changes of biogenic amines in osteopeptide injections were significant. The accumulation of putrescine increased under high temperature, while spermine and spermidine decreased. The biogenic amines were unstable under strong light and accelerated test. Conclusion: Method validation shows that the method can be applicable to simultaneous determine 10 biogenic amines in osteopeptide injections. It provided a reference to establish and improve the quality standards of biogenic amines in osteopeptide and other drugs. In addition, the research of the stability of biological amines shows that high temperature and strong light will affect the stability of biogenic amines. Therefore, quality control and supervision should be strengthened during drug production and storage to ensure the stability of drug.
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