Abstract As a potential DNA damaging substance, genotoxic impurities have been concerned by regulatory authorities in various countries. Two genotoxic impurities were found in imatinib mesylate which was a classical small molecule inhibitor of tyrosine kinase, and the analysis method has never been reported. A LC–MS/MS method was developed for the analysis of two genotoxic impurity materials: N -(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine (IMA) and N -(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine (IMN). The HPLC method utilized an ACQUITY UPLC HSS T3 C18 (150 × 2.1 mm, 1.7 μm) maintained at 40 °C. The mobile phase consisted of 0.02 M ammonium formate buffer (pH 3.4) and acetonitrile (containing 0.05% formic acid) in gradient elution mode. Detection was performed by triple quadrupole mass spectrometry fitted with ESI probe functioning in the positive ion mode and the following m/z 278/106 and 308/262 were used as qualifier and quantifier transitions. Flow rate was 0.4 mL min −1 . The validation data demonstrated that the method had high sensitivity and selectivity. A linear calibration curve (correlation coefficient, r > 0.998) was obtained at the concentration range of 0.02–35.27 and 0.02–28.86 ng mL −1 , respectively. The LOD of IMA in drug substances, tablets, and capsules were 0.0039, 0.0043, and 0.0044 ng mL −1 , and LOD of IMN were 0.0034, 0.0035, and 0.0036 ng mL −1 , respectively. Precision and accuracy were within the acceptable limit. The drug substances, tablets, and capsules from three manufacturers were used for inspection and all samples met the requirements. The developed LC–MS/MS method is robust and can be applied to detect the genotoxic impurities in imatinib mesylate. Graphic Abstract Mixed solution of Imatinib, IMA and IMN. A new LC-MS/MS method was developed for the analysis of two genotoxic impurities materials: N -(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine and N -(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine (IMN)

中文翻译：

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LC-MS/MS 检测原料药中的两种基因毒性杂质及甲磺酸伊马替尼的制备

摘要 作为一种潜在的DNA损伤物质，基因毒性杂质已受到各国监管部门的关注。在酪氨酸激酶经典小分子抑制剂甲磺酸伊马替尼中发现了两种基因毒性杂质，分析方法未见报道。开发了一种 LC-MS/MS 方法来分析两种遗传毒性杂质材料：N -(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine (IMA) 和 N -(2-甲基-5-硝基苯基）-4-（吡啶-3-基）嘧啶-2-胺（IMN）。HPLC 方法使用保持在 40 °C 的 ACQUITY UPLC HSS T3 C18（150 × 2.1 mm，1.7 μm）。流动相由梯度洗脱模式下的 0.02 M 甲酸铵缓冲液（pH 3.4）和乙腈（含 0.05% 甲酸）组成。检测通过配备 ESI 探针的三重四极杆质谱仪在正离子模式下运行，以下 m/z 278/106 和 308/262 用作定性离子对和定量离子对。流速为0.4mL min -1 。验证数据表明该方法具有较高的灵敏度和选择性。线性校准曲线（相关系数，r > 0.998）分别在 0.02–35.27 和 0.02–28.86 ng mL -1 的浓度范围内获得。原料药、片剂和胶囊中 IMA 的 LOD 分别为 0.0039、0.0043 和 0.0044 ng mL -1 ，IMN 的 LOD 分别为 0.0034、0.0035 和 0.0036 ng mL -1 。精密度和准确度在可接受的范围内。对3家生产厂家的原料药、片剂、胶囊进行检验，样品均符合要求。开发的 LC-MS/MS 方法稳定可靠，可用于检测甲磺酸伊马替尼中的基因毒性杂质。图解摘要伊马替尼、IMA 和 IMN 的混合溶液。开发了一种新的 LC-MS/MS 方法来分析两种遗传毒性杂质材料：N -(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine 和 N -(2-methyl- 5-硝基苯基)-4-(pyridin-3-yl) pyrimidin-2-amine (IMN)