Kamaris Georgios, Tsami Maria, Lotca Georgiana-Roxana, Almpani Sofia, Markopoulou Catherine K
Laboratory of Pharmaceutical Analysis, Department of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.
Molecules. 2024 Nov 23;29(23):5535. doi: 10.3390/molecules29235535.
In recent years, the detection of nitrosamine precursors has become an important issue for regulatory authorities such as the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). The present study provides a pre-column derivatization method for the analysis of dimethylamine (DMA) and diethylamine (DEA) in pharmaceutical products using HPLC and a fluorescence detector. Appropriate chromatographic parameters, including mobile phase composition (organic solvent, buffer, pH), elution type, flow rate, temperature, and λexcitation/emission, were investigated. Analysis was performed at λ = 450 nm and λ = 540 nm on a C column (at 40 °C) using gradient elution as a mobile phase with Eluent A: Phosphoric Acid Buffer (20 mM, pH = 2.8) and Eluent B: methanol, with a flow of 0.8 mL/min. The method was validated according to ICH specifications in terms of linearity (0.5-10 ng/mL for DMA and 5-100 ng/mL for DEA), specificity, and robustness, as well as repeatability, intermediate precision (%RSD < 2.9), and accuracy (% recovery 98.2-102.0%). The derivatization process was optimized using the "Crossed D-Optimal" experimental design procedure, where one mixture component was cross-correlated with two factors. The stability of the samples was studied over a period of one month. To process the samples (pharmaceuticals), various purification techniques were tried using solid/liquid or liquid/liquid extraction with dichloromethane. Finally, a straightforward solid-phase extraction (SPE, C) method was chosen prior to derivatization. The method was successfully applied, since the extraction recoveries were >81.6% for DMA (0.5 ppm) and >81.1% for DEA (5 ppm). Based on the results obtained and the available literature, the scientific community seeks, by proposing flexible analytical methods, to delimit the problem of nitrosamines.
近年来，亚硝胺前体的检测已成为欧洲药品管理局（EMA）和美国食品药品监督管理局（FDA）等监管机构的重要议题。本研究提供了一种柱前衍生化方法，用于使用高效液相色谱（HPLC）和荧光检测器分析药品中的二甲胺（DMA）和二乙胺（DEA）。研究了合适的色谱参数，包括流动相组成（有机溶剂、缓冲液、pH值）、洗脱类型、流速、温度以及激发/发射波长。在C柱（40℃）上，以磷酸缓冲液（20 mM，pH = 2.8）作为流动相A，甲醇作为流动相B，流速为0.8 mL/min，采用梯度洗脱，在λ = 450 nm和λ = 540 nm处进行分析。该方法根据国际人用药品注册技术协调会（ICH）规范，在线性（DMA为0.5 - 10 ng/mL，DEA为5 - 100 ng/mL）、特异性、稳健性以及重复性、中间精密度（%RSD < 2.9）和准确度（%回收率98.2 - 102.0%）方面进行了验证。使用“交叉D - 最优”实验设计程序对衍生化过程进行了优化，其中一种混合组分与两个因素进行交叉关联。对样品的稳定性进行了为期一个月的研究。为了处理样品（药品），尝试了各种纯化技术，采用二氯甲烷进行固/液或液/液萃取。最后，在衍生化之前选择了一种简单的固相萃取（SPE，C）方法。该方法成功应用，因为DMA（0.5 ppm）的萃取回收率>81.6%，DEA（5 ppm）的萃取回收率>81.1%。基于所获得的结果和现有文献，科学界通过提出灵活的分析方法来界定亚硝胺问题。
