一种采用分数析因设计的超高效液相色谱-串联质谱法，用于同时测定新冠病毒抑制剂莫努匹韦原料药及其制剂剂型中的痕量遗传毒性杂质，并对其进行定量分析。
A sensitive UPLC-MS/MS method for the simultaneous assay and trace level genotoxic impurities quantification of SARS-CoV-2 inhibitor-Molnupiravir in its pure and formulation dosage forms using fractional factorial design.
作者信息
Nakka Srinivas, Muchakayala Siva Krishna, Manabolu Surya Surendra Babu
机构信息
Department of Chemistry, School of Science, GITAM Deemed to be University, Hyderabad 502329, India.
Analytical Research and Development, Catalent Pharma Solutions, 1100 Enterprise Drive, Winchester, KY, 40391, USA.
出版信息
Results Chem. 2023 Dec;6:101019. doi: 10.1016/j.rechem.2023.101019. Epub 2023 Jun 27.
Two potential genotoxic impurities were identified (PGTIs)-viz. 4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (PGTI-1), and 1-(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-one (PGTI-II) in the Molnupiravir (MOPR) synthetic routes. COVID-19 disease was treated with MOPR when mild to moderate symptoms occurred. Two (Q)-SAR methods were used to assess the genotoxicity, and projected results were positive and categorized into Class-3 for both PGTIs. A simple, accurate and highly sensitive ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was optimized for the simultaneous quantification of the assay, and these impurities in MOPR drug substance and formulation dosage form. The multiple reaction monitoring (MRM) technique was utilized for the quantification. Prior to the validation study, the UPLC-MS method conditions were optimised using fractional factorial design (FrFD). The optimized Critical Method Parameters (CMPs) include the percentage of Acetonitrile in MP B, Concentration of Formic acid in MP A, Cone Voltage, Capillary Voltage, Collision gas flow and Desolvation temperature were determined from the numerical optimization to be 12.50 %, 0.13 %, 13.6 V, 2.6 kV, 850 L/hr and 375 °C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 °C and flow rate at 0.5 mL/min. The method was successfully validated as per ICH guidelines, and demonstrated excellent linearity over the concentration range of 0.5-10 ppm for both PGTIs. The Pearson correlation coefficient of each impurity and MOPR was found to be higher than 0.999, and the recoveries were in between the range of 94.62 to 104.05% for both PGTIs and 99.10 to 100.25% for MOPR. It is also feasible to utilise this rapid method to quantify MOPR accurately in biological samples.
在莫努匹拉韦（MOPR）的合成路线中鉴定出了两种潜在的基因毒性杂质（PGTIs），即4-氨基-1-((2R,3R,4S,5R)-3,4-二羟基-5-(羟甲基)四氢呋喃-2-基)嘧啶-2(1H)-酮（PGTI-1）和1-(2R,3R,4S,5R)-3,4-二羟基-5-(羟甲基)四氢呋喃-2-基)嘧啶-2,4(1H,3H)-酮（PGTI-II）。当出现轻至中度症状时，使用MOPR治疗新冠肺炎。采用两种（Q）-SAR方法评估基因毒性，预测结果为阳性，两种PGTIs均归类为3类。优化了一种简单、准确且高灵敏度的超高效液相色谱-质谱联用（UPLC-MS/MS）方法，用于同时定量分析MOPR原料药和制剂剂型中的该分析物及这些杂质。采用多反应监测（MRM）技术进行定量。在验证研究之前，使用析因设计（FrFD）优化UPLC-MS方法条件。通过数值优化确定的优化关键方法参数（CMPs）包括MP B中乙腈的百分比、MP A中甲酸的浓度、锥孔电压、毛细管电压、碰撞气流和脱溶剂温度，分别为12.50%、0.13%、13.6 V、2.6 kV、850 L/hr和375 °C。在Waters Acquity HSS T3 C18柱（100 mm×2.1 mm，1.8 µm）上，以0.13%甲酸水溶液和乙腈为流动相，采用梯度洗脱模式，柱温保持在35 °C，流速为0.5 mL/min，实现了优化的色谱分离。该方法按照ICH指南成功验证，两种PGTIs在0.5-10 ppm浓度范围内均表现出良好的线性。发现每种杂质与MOPR的皮尔逊相关系数均高于0.999，两种PGTIs的回收率在94.62%至104.05%之间，MOPR的回收率在99.10%至100.25%之间。利用这种快速方法准确量化生物样品中的MOPR也是可行的。
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