目的: 建立抗脂肪肝治疗药4,4’-二甲氧基-5,6,5’,6’-双(亚甲二氧基)-2’-吗啉亚甲基联苯-2-甲酸甲酯甲磺酸盐(简称IMH)中氯代烷烃类遗传毒性杂质4,4’-二甲氧基-5,6,5’,6’-双(亚甲二氧基)-2’-氯甲基-2-甲酸甲酯(简称氯代物)的分析方法。方法: 采用正相高效液相色谱(NP-HPLC)法,选用Phenomenex Luna® Silica色谱柱(250 mm×4.6 mm, 5 μm),以正己烷-异丙醇(95∶5)为流动相,流速0.8 mL·min-1,检测波长226 nm,柱温35 ℃,进样量50 μL;采用外标法对氯代物进行定量。结果: 在水相中易分解的氯代物在该正相色谱方法条件下能够达到有效分离,专属性良好;氯代物质量浓度在0.03~3.0 μg·mL-1的范围内呈现良好的线性关系,检测限和定量限分别为0.15 ng和0.31 ng;平均回收率处于99%~103%,精密度及溶液稳定性良好。3批实测样品中均未检出氯代物,表明后续工艺能有效清除本品中的氯代物。结论: 该色谱方法快速、准确、灵敏,能够对IMH中的氯代物进行准确的定性定量分析,有效控制药物安全。同时,可为含有卤代烷烃类遗传毒性杂质的其他药物的质量控制提供参考方法。
Objective: To establish an analytical method for 4, 4’-dimethoxy-5, 6, 5’, 6’-bis(methylenedioxy)-2’-chloromethyl-2-methyl formate(chloride) that was genotoxic impurity of chloroalkanes in 4, 4’-dimethoxy-5, 6, 5’, 6’-bis(methylenedioxy)-2’-morpholine methylbiphenyl-2-methyl formate methanesulfonate(IMH), a new drug for the treatment of fatty liver disease. Methods: The NP-HPLC method was carried out using a Phenomenex Luna® Silica column(250 mm×4.6 mm, 5 μm) maintained at 35 ℃. The mobile phase was a mixture of n-hexane and isopropanol in a constant proportion of 95∶5 at a flow rate of 0.8 mL·min-1. The detection wavelength was 226 nm, and the injection volume was set at 50 μL. Chloride was quantified by external standard method. Results: Chloride that was easy to decompose in aqueous phase could be effectively separated under the condition of NP-HPLC, and the specificity was good. The linear relationship of chloride was good in the concentration ranges of 0.03-3.0 μg·mL-1, and the detection limit and quantitative limit were 0.15 ng and 0.31 ng, respectively. The average recovery was 99%-103%, and the precision and solution stability were good. The chlorides in 3 batches of IMH samples were below the detection limit, thereby indicating that the subsequent process could effectively remove chloride in IMH. Conclusion: The chromatographic method is rapid, accurate and sensitive, which can accurately analyze chloride in IMH, and effectively control the drug safety.At the same time, it can provide a reference method for the quality control of other drugs containing haloalkane genotoxic impurities.
[1] REDDY AVB, JAAFAR J, UMAR K, et al. Identification, control strategies, and analytical approaches for the determination of potential genotoxic impurities in pharmaceuticals:a comprehensive review[J].J Sep Sci, 2015, 38(5):764
[2] TEASDALE A, ELDER DP. Analytical control strategies for mutagenic impurities:current challenges and future opportunities?[J].Trends Analyt Chem, 2018, 101(9):66
[3] 王萍, 徐彩虹, 陈仙,等. 原料药中基因毒性杂质控制的研究进展[J].中国现代应用药学, 2015, 32(1):119
WANG P, XU CH, CHEN X, et al. Development of genotoxic impurities control in active pharmaceutical ingredient[J].Chin J Mod Appl Pharm, 2015, 32(1):119
[4] ELDER DP, LIPCZYNSKI AM, TEASDALE A. Control and analysis of alkyl and benzyl halides and other related reactive organohalides as potential genotoxic impurities in active pharmaceutical ingredients(APIs)[J].J Pharm Biomed Anal, 2008, 48(3):497
[5] POZNIAK A, MULLER L, SALGO M, et al. Elevated ethyl methanesulfonate(EMS) in nelfinavir mesylate(Viracept?, Roche):overview[J].AIDS Res Ther, 2009, 6(1):18
[6] 文海若, 闫明, 王亚楠,等. 药物杂质遗传毒性评价策略与监管研究[J].中国药事, 2020, 34(2):131
WEN HR, YAN M, WANG YN, et al. Strategy and regulatory research for genotoxicity evaluation of impurities[J].Chin Pharm Aff, 2020, 34(2):131
[7] 程方洁, 付晓婷, 郜淑晓,等. 阿哌沙班中基因毒性杂质的LC-MS/MS检查[J].中国新药杂志, 2020, 29(7):816
CHENG FJ, FU XT, GAO SX, et al. Determination of genotoxic impurities in apixaban by LC-MS/MS[J].Chin New Drugs J, 2020, 29(7):816
[8] European Medicines Agency. Guidelines on the Limits of Genotoxic Impurities[EB/OL].(2006-06-28)[2022-2-24].https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-limits-genotoxic-impurities_en.pdf
[9] The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Guideline M7(R1):Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk[EB/OL].2017[2022-2-24].https://database.ich.org/sites/default/files/M7_R1_Guideline.pdf
[10] 中华人民共和国药典2020年版. 四部[S].2020:527
ChP 2020. Vol Ⅳ[S].2020:527
[11] 吴松, 孙华, 张金兰,等. 一类左旋双环吗啉,其制备方法、药物组合物和应用:中国, CN112851626A[P].2021-05-28
WU S, SUN H, ZHANG JL, et al. The Invention Relates to a Class of L-Bicyclic Morpholine, a Preparation Method, a Pharmaceutical Composition and Application Thereof:China, CN112851626A[P].2021-05-28
[12] 吴松, 孙华, 张文轩,等. 一类双环醇类衍生物及其制备和应用:中国, CN107488162A[P].2017-12-19
WU S, SUN H, ZHANG WX, et al. A Class of Bicyclic Alcohol Derivatives and Their Preparation and Application: China, CN107488162A[P].2017-12-19
[13] MATVEEVA OA, KOVALEVA EL. Modern approaches to estimating the content of genotoxic impurities in drugs (a review)[J].Pharm Chem, 2016, 49(11):765
[14] 马磊, 马玉楠, 陈震,等. 遗传毒性杂质的警示结构[J].中国新药杂志, 2014, 23(18):2106
MA L, MA YN, CHEN Z, et al. Structural alerts of genotoxic impurities[J].Chin New Drugs J, 2014, 23(18):2106
[15] 王昕, 王卫, 唐素芳. HPLC法测定苯丙氨酯原料药及片剂的含量和有关物质[J].中国药品标准, 2021, 22(2):105
WANG X, WANG W, TANG SF. Determination of the related substances and assay of phenprobamate and phenprobamate tablets by HPLC[J].Drug Stand China, 2021, 22(2):105
[16] 程智, 王孝艳, 雍子宜,等. 高效液相色谱法测定富马酸比索洛尔原料药及其制剂中的有关物质[J].中南药学, 2021, 19(7):1405
CHENG Z, WANG XY, YONG ZY, et al. Determination of related substances in bisoprolol fumarate raw materials and its preparations by HPLC[J].Cent South Pharm, 2021, 19(7):1405
[17] 张媛媛, 张文轩, 吴松,等. HPLC法测定阿法骨化醇中有关物质[J].药物分析杂志, 2021, 41(1):160
ZHANG YY, ZHANG WX, WU S, et al. Determination of related substances in alfacalcidol by HPLC[J].Chin J Pharm Anal, 2021, 41(1):160
