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医药导报
医药导报, 2019, 38(1): 92-95
doi: 10.3870/j.issn.1004-0781.2019.01.021
顶空气相色谱-质谱法测定依度沙班原料药中遗传毒性物质
Simultaneous Determination of the Contents of Genotoxicity Impurities in Edoxaban by Head-space GC-MS
徐艳梅1,, 裴丽娟2, 杜高锋1, 宋更申1
1.河北省药品检验研究院,石家庄 050011
2.河北医科大学第一医院药剂科, 石家庄 050031
XU Yanmei1,, PEI Lijuan2, DU Gaofeng1, SONG Gengshen1
1.Institute for Drug Control of Hebei Province, Shijiazhuang 050011, China
2.Department of Pharmacy, the Frist Hospital of Hebei Medical University,Shijiazhuang 050031, China

作者简介 徐艳梅(1983-),女,河北廊坊人,主管药师,硕士,从事药物分析工作。ORCID:0000-0001-6636-5721。电话:0311-85212008,E-mail:13785197771@163.com。
中图分类号: R973.2;R927.2     文献标识码: B     文章编号: 1004-0781(2019)01-0092-04
摘要:

目的 建立内标法测定依度沙班原料药中遗传毒性杂质甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯的含量。方法 采用顶空气相色谱-质谱法,以DB-WAX毛细管柱(30 m×0.25 mm,0.25 μm)为色谱柱,程序升温,高纯氦气为载气,流速为0.6 mL·min-1,进样口温度为110 ℃,进样方式为分流进样,分流比为20:1;顶空进样,平衡温度为60 ℃,平衡时间为30 min,进样体积1 mL;检测器为质谱检测器,离子源为EI源,离子源温度为200 ℃,接口温度为150 ℃,扫描方式为选择离子检测,电子能量为70 eV。结果 甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯在0.05~3.0 μg·mL-1(r≥0.998 5)浓度范围内线性关系良好,加样回收率分别为96.4%,96.1%和96.5%,RSD分别为2.0%,1.9%,1.9%(n=6)。结论 该方法简便、快速、灵敏度高、专属性好,可为依度沙班原料药的质量控制提供参考依据。
关键词: 依度沙班 ; 杂质 ; 遗传毒性 ; 甲磺酸甲酯 ; 甲磺酸乙酯 ; 甲磺酸异丙酯 ; 顶空气相色谱-质谱法

Abstract:

Objective To establish a method for the determination of the genotoxic impurities (methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate) in edoxaban with internal standard method. Methods A head-space GC-MS method was used. The column was DB-WAX capillary column (30 m×0.25 mm,0.25 μm) by programmed temperature, carrier gas was high purity helium, column flow was 0.6 mL·min-1, the inlet temperature was 110 ℃, sample mode split injection with a split ratio of 20:1;Head-space samp-ling was conducted, equilibrium temperature was 60 ℃, equilibrium time was 30 min, injection volume was 1 mL; Detector was a mass spectrometer detector, the ion source was EI source and source temperature was 200 ℃, the interface temperature was 150 ℃, scanning method was selective ion monitoring, electron energy was 70 eV. Results The linear range of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate were 0.05-3.0 μg·mL-1(r≥0.998 5); Recoveries were 96.4%,96.1% and 96.5%, respectively, and RSD were 2.0%,1.9% and 1.9%(n=6). Conclusion This method is simple, rapid, sensitive, with good specificity, and it can provide a reference for the quality control of edoxaban raw material with internal standard.
Key words: Edoxaban ; Genotoxic impurities ; Methyl methanesulfonate ; Ethyl methanesulfonate ; Isopropyl methanesulfonate ; Head-space GC-MS method

依度沙班(edoxyaban)是一种新型Xa因子抑制药[1,2],具有起效快、抗凝效果可逆、具有剂量依赖的效果等特点。2015年以来,美国食品药品管理局(FDA)、欧洲药品管理局(EMA)和瑞士药品管理局相继批准依度沙班用于非瓣膜性心房颤动患者脑卒中及系统性栓塞、急性静脉栓塞患者深静脉血栓和肺栓塞的预防及治疗[3]。依度沙班原料药在合成工艺中有一步甲磺酰化的过程,其中甲磺酰氯易与醇类反应产生甲磺酸酯[4]。依度沙班合成工艺中将甲醇和乙醇作为反应溶剂,因此依度沙班原料药中可能存在甲磺酸甲酯(methyl methanesulfonate)、甲磺酸乙酯(ethyl methanesulfonate)及甲磺酸异丙酯(isopropylmethanesulfonate)3种杂质。甲磺酸酯类物质是一种潜在性基因毒性杂质[5,6],这些杂质的DNA烷基化作用会产生诱变效应、致癌效应和致畸效应[7],严重威胁人类健康,因此需要严格控制药品中甲磺酸酯的限度。根据EMEA发布的《遗传毒性杂质限度指导原则》相关规定,按照毒理学担忧阈值(TTC)作为评价大部分遗传毒性杂质的阈值,则遗传毒性杂质摄入量最大限值为1.5 μg·d-1[8,9]。依度沙班作为抗凝药物使用时,每日通常使用剂量为30 mg,按此计算含甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯不得过50 μg·g-1。近年来,气相色谱(GC)法、气相色谱-质谱联用(GC-MS)法和衍生化-气相色谱-质谱联用(HS-GC-MS)法已被广泛应用于甲磺酸酯类基因毒性杂质检测[10,11]。笔者参考《欧洲药典》(EP)8.0 版中关于甲磺酸盐中甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯的测定方法,采用HS-GC-MS法对甲磺酸依度沙班原料药中上述3 种遗传毒性杂质进行测定。

1 仪器与试药
1.1 仪器

GC-MS 2010 Plus色谱仪(日本Shimadzu公司);DU305型电子天平(瑞士Mettler-Toledo公司,感量:0.01 mg)。

1.2 试药

依度沙班原料药(河北志恒医药科技有限公司,批号:160031,160032,160033);甲磺酸甲酯对照品(JSK SCIENTIFIC LTD,批号:LU80L27);甲磺酸乙酯对照品(Atta Aesar,批号:10169291);甲磺酸异丙酯对照品(ACROS,批号:A0314222);甲磺酸丁酯(南京强山化工有限公司,批号:20130027);乙腈和水均为色谱纯,无水碘化钠和硫代硫酸钠均为分析纯。

2 方法与结果
2.1 GC-MS条件

色谱柱为DB-WAX毛细管柱(30 m×0.25 mm,0.25 μm);检测器为MS检测器;进样口温度为110 ℃;采用程序升温,起始温度为40 ℃,维持3 min,以20 ℃·min-1的速率升温至150 ℃,维持2 min,然后以20 ℃·min-1的速率升温至230 ℃,维持10 min;进样方式为分流进样,分流比20:1;载气为高纯氦气;柱流量为0.6 mL·min-1;顶空进样,炉温60 ℃;平衡时间30 min;进样体积1 mL,进样时间1 min;离子源温度200 ℃;接口温度150 ℃;扫描方式为选择离子扫描(SIM);电子能量为70 eV。各待测成分的定性、定量信息见表1,质谱图见图1。

表1 待测成分的定性、定量信息
Tab.1 Qualitative and quantative data of the ingredient under test
成分 m/z
定量 定性
碘代甲烷 142 127
碘代乙烷 156 127
碘代异丙烷 170 127
碘代丁烷 184 127

表1 待测成分的定性、定量信息

Tab.1 Qualitative and quantative data of the ingredient under test


图1 碘代甲烷、碘代乙烷、碘代异丙烷和碘代丁烷的质谱图

Fig.1 Mass spetrum of methyl iodide, ethyl iodide, iso-propyl iodide and iodo-2-methylpropane

2.2 溶液配制

2.2.1 衍生化试剂 精密称取无水碘化钠60.0 g和硫代硫酸钠30 mg置50 mL量瓶中,加水溶解并稀释至刻度,摇匀,即得。

2.2.2 内标溶液 精密称取甲磺酸丁酯25 mg,置25 mL量瓶中,加稀释液(水-乙腈20:80)溶解并稀释至刻度,摇匀,精密量取该溶液50 μL,置于10 mL量瓶中,加稀释液稀释至刻度,摇匀,另精密量取该溶液2 mL,至50 mL量瓶中,加稀释液稀释并定容至刻度,摇匀,作为内标溶液。

2.2.3 空白溶液 精密量取水-乙腈(20:80)0.5 mL与内标溶液0.5 mL,置20 mL顶空瓶中,立即密封,摇匀,作为空白溶液。

2.2.4 对照品溶液 精密称取甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯各约50 mg,分别加甲苯溶解并稀释至10 mL,摇匀;精密量取50 μL,置同一25 mL量瓶中,加内标溶液稀释并定容,摇匀,作为对照品贮备液。精密量取对照品贮备液150 μL,置10 mL量瓶中,加内标溶液稀释定容,即得。分别精密量取该溶液0.5 mL及衍生化试剂0.5 mL,置20 mL顶空瓶中,立即密封,摇匀,作为对照品溶液。

2.2.5 供试品溶液 精密称取样品30 mg,置20 mL顶空瓶中,加0.5 mL内标溶液及0.5 mL衍生化试剂,立即压盖密封,超声溶解,作为供试品溶液。

2.3 专属性考察

取空白溶液、甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯对照品溶液和供试品溶液各1 mL,按“2.1”项色谱条件,记录色谱图。内标衍生物碘代丁烷保留时间为5.52 min,甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的衍生物碘代甲烷、碘代乙烷和碘代异丙烷的保留时间分别为2.87,3.52,3.81 min,各峰之间的分离度均>2.0,符合检测要求。

2.4 检测限和定量限考察

取对照品溶液,加内标液逐级稀释成不同质量浓度,分别精密量取该溶液0.5 mL及衍生化试剂0.5 mL,置20 mL顶空瓶中,摇匀,立即密封。按“2.1”项色谱条件分别进样,测定各杂质的检测限(S/N=3)和定量限(S/N=10)。结果,甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯检测限分别为5,5和10 ng·mL-1,定量限为15,15和30 ng·mL-1。

2.5 线性关系考察

精密量取对照品贮备溶液0.05,0.1,0.5,1.0,3.0 mL,置10 mL量瓶中,加内标溶液稀释定容,制得3种成分浓度分别约0.05,0.1,0.5,1.0,3.0 μg·mL-1,分别精密量取该溶液0.5 mL及衍生化试剂0.5 mL,置20 mL顶空瓶中,立即密封,摇匀,作为线性工作溶液。按“2.1”项下色谱条件分别进样,记录色谱图。以对照品溶液的质量分数为横坐标,主成分与内标峰面积比值为纵坐标,绘制标准曲线,3种杂质的线性结果见表2。实验结果表明,甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯均在0.05~3.0 μg·mL-1浓度范围内线性关系良好。

表2 3种成分的线性结果
Tab.2 Linearity of three kinds of components
成分 线性方程 r 线性范围/
(μg·mL-1)
甲磺酸甲酯 Y=18 763.82X-156.58 0.998 5 0.05~3.0
甲磺酸乙酯 Y=15 074.22X-112.76 0.999 0 0.05~3.0
甲磺酸异丙酯 Y=10 859.26X-48.63 0.998 9 0.05~3.0

表2 3种成分的线性结果

Tab.2 Linearity of three kinds of components

2.6 精密度实验

取“2.5”项下线性工作溶液(3),按“2.1”项色谱条件连续进样6次,记录色谱图。经计算得A甲磺酸甲酯/A内标、A甲磺酸乙酯/A内标和A甲磺酸异丙酯/A内标的RSD分别为2.4%,3.0%,2.9%(n=6),表明仪器精密度良好。

2.7 重复性实验

精密称取样品(批号:160031)30 mg,置20 mL顶空瓶中,加入0.15 μg·mL-1标准溶液0.5 mL及衍生化试剂0.5 mL,立即压盖密封,超声溶解,按“2.1”项色谱条件分别进样,记录色谱图。测定加标样品中甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的含量,平行测定6次。结果表明该方法重复性良好,满足测定要求。

2.8 加样回收率实验

精密称取样品(批号:160031)30 mg,共9份,分别置于20 mL顶空瓶中,精密加入对照品贮备溶液(10 μg·mL-1)2.25, 1.50和0.75 mL,每种浓度平行制备3份进行测定,分别加入衍生化试剂0.5 mL,立即压盖密封,超声溶解,按“2.1”项下色谱条件分别进样,记录色谱图。根据内标法计算甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的平均回收率(n=9)分别为96.4%,96.1%和96.5%。结果见表3。

表3 3种成分的回收率结果
Tab.3 Recovery results of three kinds of components
成分 取样量/
mg		 原有量 加入量 测得量 加样回收
率/%
μg
甲磺酸甲酯 30.00 0.004 5 2.25 2.21 98.02
30.00 0.004 5 2.25 2.20 97.58
30.00 0.004 5 2.25 2.21 98.02
30.00 0.004 5 1.50 1.47 97.70
30.00 0.004 5 1.50 1.46 97.03
30.00 0.004 5 1.50 1.46 97.03
30.00 0.004 5 0.75 0.72 95.40
30.00 0.004 5 0.75 0.70 92.73
30.00 0.004 5 0.75 0.71 94.07
甲磺酸乙酯 30.00 0.005 7 2.25 2.20 97.52
30.00 0.005 7 2.25 2.19 97.08
30.00 0.005 7 2.25 2.21 97.97
30.00 0.005 7 1.50 1.45 96.29
30.00 0.005 7 1.50 1.44 95.62
30.00 0.005 7 1.50 1.46 96.95
30.00 0.005 7 0.75 0.73 96.57
30.00 0.005 7 0.75 0.70 92.57
30.00 0.005 7 0.75 0.71 93.91
甲磺酸异丙酯 30.00 0.000 0 2.25 2.19 97.33
30.00 0.000 0 2.25 2.22 98.67
30.00 0.000 0 2.25 2.21 98.22
30.00 0.000 0 1.50 1.46 97.33
30.00 0.000 0 1.50 1.45 96.67
30.00 0.000 0 1.50 1.43 95.33
30.00 0.000 0 0.75 0.73 97.33
30.00 0.000 0 0.75 0.70 93.33
30.00 0.000 0 0.75 0.71 94.67

表3 3种成分的回收率结果

Tab.3 Recovery results of three kinds of components

2.9 样品含量测定

按“2.1”项色谱条件和“2.2”项对照品溶液和供试品溶液的制备方法操作,测定3批原料药中3种遗传毒性物质,以内标法计算甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的含量,测定结果见表4。

表4 样品中3种遗传毒性物质含量测定结果
Tab.4 Content determination on three kinds of genotoxic impurities in samples μg·g-1
批号 甲磺酸甲酯 甲磺酸乙酯 甲磺酸异丙酯
160031 0.15 0.19 0.00
160032 0.11 0.17 0.00
160033 0.09 0.16 0.00

表4 样品中3种遗传毒性物质含量测定结果

Tab.4 Content determination on three kinds of genotoxic impurities in samples μg·g-1

3 讨论

本研究采用内标法进行定量分析,选取待测物的同系物甲磺酸丁酯作为内标物,其衍生化产物为碘代丁烷,与待测物质完全分离,且灵敏度较高。因此,甲磺酸丁酯可作为这3种遗传毒性杂质含量测定的内标物。

因本品理化性质原因,溶解性较差,在溶剂筛选过程中分别考察了甲醇、异丙醇、二氯甲烷和乙腈等溶剂。实验结果表明,选用混合溶剂水-乙腈(20:80)效果最佳。

实验中考察了不同极性的色谱柱,结果表明,3种待测组分和内标物在DB-WAX毛细管柱(30 m×0.25 mm,0.25 μm)上的峰形和分离度最佳。

实验采用DB-WAX毛细管柱内径为0.25 mm,采用常用流速1.0 mL·min-1不能使3种待测组分和内标物达到分离度的要求,故考察了1.0,0.8和0.6 mL·min-13种不同流速对分离度的影响,结果表明,流速为0.6 mL·min-1时,分离效果最好。

色谱分离时,不仅载气流速影响分离度,而且升温程序的不同也会导致色谱峰分离度的变化。由于3种待测组分和内标物的沸点接近,为了使之达到很好的分离,需采用较低的起始温度进行实验,最终选择40 ℃作为起始温度,程序升温进行实验。

顶空条件的优化主要考察了两个方面,包括炉温和平衡时间的选择。实验考察了不同炉温对实验结果的影响,结果表明,炉温为60 ℃时,能够使3种待测组分和内标物充分气化。不仅炉温会影响气化程度,平衡时间也会影响气化程度,不同的平衡时间直接影响顶空进样的浓度,故考察了平衡时间的影响,最终选择平衡时间为30 min。

本研究采用HS-GC-MS方法对依度沙班合成过程中可能产生的基因毒性物质甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯进行含量测定,对于确保临床用药的安全性和有效性具有重要的意义。

The authors have declared that no competing interests exist.
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依度沙班为新型口服抗凝药,于2015年1月8日经美国FDA批准用于降低非瓣膜性心房颤动患者的卒中和系统性栓塞风险。本文对其作用机制、药理学、药动学、临床评价、用法用量、安全性、不良反应等方面作了综述。
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[3] 张清,罗素新,唐炯.新型Xa 因子抑制剂——依度沙班在心房颤动患者抗凝治疗中的研究进展[J].心血管病学进展,2016,37(2):151-155.
心房颤动是最常见的心律失常,也是缺血性卒中的主要危险因素之一。一直以来华法林都是心房颤动患者预防脑卒中和系统性血栓的一线抗凝药,但由于其治疗窗窄、药物及食物之间相互作用,需频繁监测国际标准化比值等不足,限制了其在临床上的应用。新型口服抗凝药如达比加群酯、利伐沙班和阿哌沙班目前均已上市,且广泛用于非瓣膜性心房颤动患者预防脑卒中和系统性血栓的治疗中。依度沙班是最新的用于抗凝治疗的Xa因子抑制剂,实验结果显示,依度沙班在非瓣膜性心房颤动患者预防脑卒中及系统性血栓事件上效果不亚于华法林,总的出血事件及心血管死亡事件发生率更低。现对依度沙班的临床研究及临床应用进展做一综述。
DOI:10.16806/j.cnki.issn.1004-3934.2016.02.000      URL    
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[4] 潘林玉,梁斌,张福利.依度沙班合成路线图解[J].中国医药工业杂志,2013,44(11):1170-1173.
依度沙班（edoxaban，1），化学名为N-（5-氯吡啶．2．基）-N＇-[（1s，2R，4s）-4-（N，N-二甲基氨甲酰基）]．2-[（5-甲基-4，5，6，7-四氢-1，3-噻唑并[5,4-C]-吡啶-2．甲酰胺基）环己基]草酰胺，是日本第-三共株式会社开发的凝血因子Xa抑制剂，
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[5] 范达,涂家生.顶空气相色谱法测定注射用甲磺酸吉米沙星中基因毒性杂质[J].药学进展,2014, 38(3):220-223.
目的：建立测定注射用甲磺酸吉米沙星中甲磺酸烷基酯类基因毒性杂质的顶空气相色谱法。方法：以Supelco 毛细管柱（30m×250μm，0．25μm）为色谱柱，程序升温：氮气为载气，流速为0．5mL·min-1：采用微池电子捕获检测器，检测器温度为250℃：顶空进样，平衡温度为60℃，平衡时间为30min，进样口温度为110℃，进样量为1mL;分流比为1：10。结果：基因毒性杂质甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯在限度为20％～120％的范围内线性关系良好，定量限分别为0．0100、0．2500和1．2500μg·L-1．检测限分别为0．0020、0．0100和0．2500μg·L-1，平均回收率分别为99．20％、99．45％和99．98％（n=9）。结论：该方法准确、简便，适用于甲磺酸吉米沙星中基因毒性杂质的检测及限度控制。
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[6] European Medicines Agency.Guideline on the Limits of Genotoxic Impurities[S].2006:4-7.
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[7] LI W.Trace analysis of residual methyl methanesulfonate,ethyl methanesulfonate and isopropyl methanesulfonate in pharmaceuticals by capillary gas chromatography with flame ionization detection[J].J Chromatogr A,2004,1046(1/2):297-301.
A capillary gas chromatographic method using flame ionization detection was developed and validated for the trace analysis (ppm level) of methyl methanesulfonate, ethyl methanesulfonate, and isopropyl methanesulfonate in pharmaceutical drug substance. The method utilizes a megabore capillary column with bonded and crosslinked polyethylene glycol stationary phase. A dissolve-and-injection approach was adopted for sample introduction in a splitless mode. The investigated sample solvents include acetonitrile, ethyl acetate, methylene chloride, 1,2-dichloromethane, and toluene. Aqueous mixtures of acetonitrile and water can also be used as sample solvent. A limit of detection of about 1 mug/g (1 ppm) and limit of quarnitation of 5 mug/g (5 ppm) were achieved for the mesylate esters in drug substance samples. The method optimization and validation are also discussed in this paper. Copyright 2004 Elsevier B.V. All rights reserved.
DOI:10.1016/j.chroma.2004.06.095      PMID:15387202      URL    
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[8] WOLLEIN U,SCHRAMEK N.Simultaneous determination of alkyl mesilates and alkyl besilates in finished drug products by direct injection GC/MS[J].Eur J Pharm Sci,2012,45(1/2):201-204.
82020.9998 has been established for concentrations between 0.01 and 1.3302μg/ml. Validation of the method was carried out on a sample matrix containing MMS, EMS, IMS, MBS and EBS at relevant levels and was further confirmed on finished products containing APIs as mesilate salts (Bromocriptine mesilate, Doxazosin mesilate).
DOI:10.1016/j.ejps.2011.11.008      PMID:22115865      URL    
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[9] RAMAKRISHNA K,RAMAN NV,RAO KM, et al.Deve-lopment and validation of GC-MS method for the determination of methyl methanesulfonate and ethyl methanesulfonate in imatinib mesylate[J].J Pharm Biomed Anal,2008,46(4):780-783.
A gas chromatography–mass spectrometry (GC–MS) method has been developed for the identification and determination of two carcinogenic and genotoxic mesylate esters viz. methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) in imatinib mesylate (INM). The method was optimized based on the peak shapes and resolution of MMS and EMS. The method was validated as per International Conference of Harmonization (ICH) guidelines in terms of limits of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy, specificity and robustness. The LOD and LOQ values were found to be 0.3 and 1.002μg/ml, respectively. The method is linear within the range of 1–1502μg/ml for both the compounds. These mesylate esters were not found in three different batches of pure and pharmaceutical formulations of INM.
DOI:10.1016/j.jpba.2007.11.013      PMID:18178357      URL    
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[10] RAMAN N V,PRASAD A V,REDDY K R,et al.Determi-nation of genotoxic alkyl methane sulfonates and alkyl paratoluene sulfonates in lamivudine using hyphenated techniques[J].J Pharm Anal,2012,2(4):314-318.
Two highly sensitive methods for the determination of genotoxic alkyl methane sulfonates (AMSs) and alkyl paratoluene sulfonates (APTSs) in lamivudine using hyphenated techniques have been presented. AMSs were determined by GC–MS method using GSBP-INOWAX (3002m×0.2502mm×0.2502μm) column. Temperature program was set by maintaining at 10002°C initially for 302min, then rised to 22002°C at the rate of 1502°C/min and maintained at 22002°C for 1602min. N,N-dimethyl formamide was used as diluent. APTSs were determined by LC-MS using Zorbax, Rx C8, 25002mm×4.602mm, 502μm column as stationary phase. 0.0102M ammonium acetate is used as buffer. The mixture of buffer and methanol in 75:25 (v/v) ratio was used as mobile phase A and mixture of buffer and methanol in 5:95 (v/v) ratio was used as mobile phase B. The gradient program (T/%B) was set as 0/28, 16/50, 17/100, 23/100, 27/28 and 40/28. Both the methods were validated as per International Conference on Harmonization guidelines. Limit of quantitation was found 1.5 μg/mL for AMSs and was in the range of 1.0–1.502μg/mL for APTSs.
DOI:10.1016/j.jpha.2012.03.003      URL    
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[11] European Directorate for Quality and Medicines&Health-Care(EDQM).Methyl,ethyl and isopropyl methanesulfonate inactive substances[S]//European Pharmacopoeia 8.0.Strasbourg:Council of Europe,2012.
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关键词（key words）
依度沙班
杂质
遗传毒性
甲磺酸甲酯
甲磺酸乙酯
甲磺酸异丙酯
顶空气相色谱-质谱法
Edoxaban
Genotoxic impurities
Methyl methanesulfonate
Ethyl methanesulfonate
Isopropyl methanesulfonat...
Head-space GC-MS method
作者
徐艳梅
裴丽娟
杜高锋
宋更申
XU Yanmei
PEI Lijuan
DU Gaofeng
SONG Gengshen