三重四极杆液质联用测定利多卡因中痕量基因毒性杂质

Determination of trace genotoxic impurity in lidocaine by HPLC- MS/MS method

关键词:  利多卡因  N-亚硝基-去乙基利多卡因  基因毒性  三重四极杆质谱

Key words:  lidocaine  N-(2,6-dimethylphenyl)-2-(ethyl(nitroso)amino)acetamide  genotoxic impurities  triple quadrupole mass spectrometry

摘要:

建立了一种测定利多卡因中基因毒性杂质N-亚硝基-去乙基利多卡因(NDLC)的液相色谱-三重四极杆质谱(HPLC-MS/MS)联用检测方法。方法 采用资生堂 C18(150 mm × 4.6 mm，5 μm)色谱柱，以乙酸铵溶液-乙腈为流动相，进行程序洗脱；进样器温度5 ℃，流速1.0 mL·min-1；柱温30 ℃；进样体积10 μL。质谱采用HSEI源，正离子多反应监测模式(MRM)，监测离子对为m/z 236.2→105.1(定量离子对)和m/z 236.2→58.1(定性离子对)，标准曲线法定量。结果 NDLC在1.058 ~ 21.160 ng·mL-1浓度范围内线性关系良好，相关系数(r)为0.999 7；检出限0.349 ng·mL-1，定量限1.058 ng·mL-1，平均回收率99.1%，RSD(n=9)为1.5%。应用该方法对3批利多卡因原料药中的NDLC进行测定，检出量在9~12 ng·g-1范围内，未超过可接受限度。结论 该方法灵敏准确，经验证方法专属、精密、准确，满足痕量检测需求，可为工艺监控和质量控制提供支持。可将监控数据溯源至工艺设计源头，在合成阶段规避高风险物料与反应，从本质上减少其生成。

Abstract:

OBJECTIVE A sensitive and accurate liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS) method was developed for the determination of genotoxic impurity N-nitroso-deethyllidocaine (NDLC) in lidocaine. METHODS Chromatographic separation was achieved on a Shiseido C18 column (150 mm × 4.6 mm， 5 μm) using a gradient elution with ammonium acetate solution and acetonitrile as the mobile phase. The injection temperature was maintained at 5 ℃ with a flow rate of 1.0 mL·min-1. The column temperature was set at 30 ℃， and the injection volume was 10 μL. Mass spectrometric detection was performed using an HESI source in positive ion multiple reaction monitoring (MRM) mode. The monitored ion transitions were m/z 236.2 → 105.1 (quantitative ion pair) and m/z 236.2 → 58.1 (qualitative ion pair). Quantification was carried out using the standard curve method. RESULTS The method demonstrated a linear range of 1.058-21.160 ng·mL-1 for NDLC with a correlation coefficient (r) of 0.999 7. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.349 ng·mL-1 and 1.058 ng·mL-1， respectively. The average recovery was 99.1% with an RSD of 1.5% (n = 9). The validated method was successfully applied to the determination of NDLC in three batches of lidocaine bulk drug， with detected levels ranging from 9-12 ng·g-1， well below the acceptable limit. CONCLUSION This method is sensitive and accurate. Validation has demonstrated its specificity, precision, and accuracy, meeting the requirements for trace-level analysis. It can therefore provide support for process monitoring and quality control. The monitoring data generated can be traced back to the process design stage, enabling the mitigation of risks associated with high-risk materials and reactions during synthesis. This approach fundamentally reduces the formation of the target analyte.