衍生气相色谱法测定甲磺酸伊马替尼中甲磺酸烷基酯类
甲磺酸脂类检测方法
Determination of Methyl Methanesulfonate, Ethyl Methanesulfonate and Isopropyl Methanesulfonate Impurities in Lopinavir API by GC/MS/MS using Electron IonizationVeenaeesh P, *Manikumar G, Manjusha. P, Padmasri ADepartment of Pharmaceutical Analysis and Quality Assurance, School Of Pharmaceutical Sciences and Technologies, Jawaharlal Nehru Technological University Kakinada, Andhra Pradesh, India.ABSTRACTA high sensitive rapid Gas Chromatography mass spectrometry method was developed and validated for the determination of three carcinogenic Alkyl methanesulfonates viz. methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate impurities in Lopinavir API (active pharmaceutical ingredient). Alkyl methanesulfonates have been highlighted as potential genotoxic impurities (PGIs). The optimum separation was achieved between methyl methanesulfonate, ethyl methanesulfonate, isopropyl methanesulfonate on DB–624 capillary column (30mt×0.32mm), 1.8m, with EI in Selective ion monitoring (SIM) mode under programming temperature was used. Methanol was used as diluents. This method was validated as per International Conference on Harmonization guidelines. The proposed method was specific, linear, accurate, rugged and precise. The calibration curves showed good linearity over the concentration range of 0.7 to 2.1 ppm. The correlation coefficient was >0.999 in each case. Method had very low limit of detection (LOD) as 0.12 ppm for Methyl methanesulfonate, 0.13 ppm for Ethyl methanesulfonate and 0.11 ppm for Isopropyl methanesulfonate and limit of quantification (LOQ) as 0.37 ppm for MMS, 0.38 ppm for EMS, 0.34 ppm for respectively for the analytes. Accuracy was observed within 70%–130% for the analytes. This method can be further extended a good quality control tool for low level quantitation of Alkyl methanesulfonate impurities in other API.Keywords: Ethyl methanesulfonate, GC/MS/MS, isopropyl methanesulfonate, lopinavir, methylmethanesulfonateReceived 17 Dec 2013 Received in revised form 03 Jan 2014 Accepted 05 Jan 2014*Address for correspondence:Manikumar G.Department of Pharmaceutical Analysis and Quality Assurance, School Of Pharmaceutical Sciences and Technologies, Jawaharlal Nehru Technological University Kakinada, Andhra Pradesh, India.E-mail: veenaeesh169@INTRODUCTIONLopinavir is an antiretroviral of theprotease inhibitor class. Lopinavirinhibiting the HIV viral protease enzyme. This prevents cleavage of the gag-polpolyprotein and, therefore, improper viral assembly results. This subsequently results in non-infectious, immature viral particles [1]. Its chemical name was (2S)-N-[(2S, 4S, 5S)-5-[2-(2, 6-dimethyl phenoxy) acetamido] -4-hydroxy -1,6-diphenyl hexan-2-yl] -3-methyl -2-(2-oxo-1,3 diazinan-1-yl)butanamide with molecular weight 628.810and its molecular formula was C37H48N4O5 [2].Impurities in pharmaceuticals are theunwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during formulation, or upon aging of both API and formulated APIs to medicines.Figure 1: Structure of the LopinavirThe presence of these unwanted chemicalseven in small amounts may influence the efficacy and safety of the pharmaceuticalproducts. The potential genotoxic impurities (PGIs) are known to induce genetic mutations or chromosomal aberrations and are reported as known carcinogens in rats and mice. The different pharmacopoeias such as the British Pharmacopoeia (BP) and the United States Pharmacopoeia (USP), are slowly incorporating limits to allowable levels of impurities present in the APIs or formulations. International Conference on Harmonization (ICH) has published guidelines on impurities in new drug substances, product and residual solvents, including the identification and monitoring of impurities found in drug products [3]. Experimental:MATERIALS AND METHODSMethyl methanesulfonate, ethylmethanesulfonate and isopropyl methanesulfonate (MMS, EMS, and IPMS respectively) were purchased from Sigma Aldrich, Fluka,Acros Organics. Methanol was procured from Rankem (HPLC grade). Pure sample of Lopinavir was obtained from well reputed research laboratory. Instrument:GC-MS analysis was carried out on GCMS-QP7000 system ( Agilent) having GCMS Mass hunter software. AMSs were separated on DB-624 capillary column (Agilent Technologies, 30 m×0.32 mm, i.d.×1.8 μm). Chromatographic Conditions:The GC oven temperature program utilizedan initial temperature of 110°C and aninitial holding time of 15min, and thenincreased at 25°C/min to 225°C. The final temperature was held for 15min. The injection temperature, GC-MS interface and ion source temperature were 200°C, 270°C and 230°C, respectively. Helium was used as the carrier gas with a flow rate of 2ml/min. All the three alkyl methanesulfonates were identified using the National Institute of Standard Technology (NIST) mass spectral library. Validation was done in Selective Ion Monitoring (SIM) mode. Preparation of solutions:Standard solution:MMS, EMS, IPMS, and NBMS stock solutions were prepared by dissolving 10mg individually in 100ml of diluent. Methanol was used as diluent. MMS, EMS, IPMS, and NBMS mixture solution 10µg/ml was prepared by diluting the appropriate volume of above stock solution with diluent. Sample solution:Weigh and transfer accurately 500mg of sample into 10ml volumetric flask and make upto the mark with diluent. Further diluted the solution to get the concentration of 0.00009mg/mL of MMS, EMS & IPMS. Results and Discussion:All the three alkyl methanesulfonates are liquids, hence it was planned to separate them by gas chromatography, identify and confirm them by mass spectrometry. Initially the experiments were carried out by using DB-1 column for the separation of AMSs, but the resolutions were found to be very poor. Then, this column was replaced by DB-5 capillary column and same result was found. Hence, DB-624 column was used and good resolutions were observed. An optimum injection volume of 1μl was chosen. The split ratio was fixed as 1:1 depending on the detector response. An initial column temperature of 110° was found to be optimum. The present method is validated as per ICH guidelines.MMS, EMS, and IPMS mixture solution (10 ppm) was injected and the limit of detection (LOD) and the limit of quantification (LOQ) values were determined at the lowest concentrations at which signal-to-noise ratio is 3 and 10, respectively. LOD values for all the AMSs were found to be 0.12 ppm for MMS, 0.13 ppm for EMS and 0.11 ppm for IMS and limit of quantification (LOQ) as 0.37 ppm for MMS, 0.38 ppm for EMS, 0.34 ppm for respectively.Linearity of the method was checked by plotting calibration curves between the peak areas versus the concentration of AMSs over the range 0.7-2.1 ppm. The slope, intercept and correlation coefficient values were derived from liner least-square regression treatment. The correlation coefficient values reported in (Table 1) indicate the best linearity of the method.Table 1: Linear Regression Data for Calibration CurvesComponent Slope Intercept Correlation coefficient (R) R2 Methyl methanesulfonate 18265.9329 822.6058 0.9979 0.9958 Ethyl methanesulfonate 35282.7514 843.7394 0.9981 0.9962 Isopropyl methanesulfonate 59399.9033 -77.8838 0.9980 0.9961The precision of the method was evaluated in terms of repeatability and intermediateprecision. The repeatability is determined by calculating the relative standarddeviation (% RSD) of six replicatedeterminations by injecting freshly prepared 10 ppm mixture solution separately on the same day. For intermediate precision, 10 ppm mixturesolution was injected on six different days. The low % RSD values via peak areasconfirm the good precision of the developedmethod (Table 2).Table 2: Data for Method Precision Preparation No. MMS EMS IPMS1 1.8 1.9 1.62 1.8 2.0 1.73 1.8 2.0 1.74 1.8 2.0 1.65 1.7 1.9 1.66 1.8 2.0 1.7 Average ppm 1.8 2.0 1.7 % RSD 2.9 0.9 1.2AMSs were not detected when three pure Rand D samples of Lopinavir were analyzed in the present method. Hence, the accuracyof the method was determined by spiking AMSs mixture at three concentration levels (0.7, 1 and 2.1 ppm) to 500 mg of Lopinavir and making the volume to 10 ml withdiluent. Each determination was carried out for three times. The recovery datapresented in (Table 3) indicates the accuracy of the method.Table 3: Data for Accuracy of MMS, EMS, IPMS% Accuracy% RecoveryMMS EMS IPMS40% Level 100.0 100.0 95.7 112.3 105.3 97.1 95.9 102.7 92.860% Level 94.5 95.6 92.2 97.3 95.6 91.3 107.3 98.2 92.2100% Level 100.5 102.7 95.3 100.5 104.8 97.7 97.8 103.7 96.5120% Level 93.6 96.9 91.395.0 96.2 90.396.3 96.2 90.3Table 4: Ruggedness Data of MMS, EMS, IPMSSample MMS EMS IPMSDay-1 Day-2 Day-1 Day-2 Day-1 Day-2Sample-1 1.8 1.8 1.9 1.8 1.6 1.7 Sample-2 1.8 1.9 2.0 1.8 1.7 1.7 Sample-3 1.8 1.8 1.9 1.8 1.7 1.7 Sample-4 1.8 1.8 2.0 1.8 1.6 1.7 Sample-5 1.7 1.8 1.9 1.8 1.6 1.7 Sample-6 1.8 1.8 2.0 1.8 1.7 1.8 Average ppm 1.8 1.8 1.9 1.8 1.7 1.7 % RSD 2.9 2.4 0.8 0.7 1.2 1.4The mass spectra of EMS, IPMS, and MMS, shows Parent peaks in (fig. 3) at m/z109 (C3H8O3S), 123 (C4H10O3S) and 80 (C2H6O3S), respectively corresponding to their molecular weights. In the varied gas chromatographic conditions of ±1° on the carrier gas flow, ±2° on the initial oven temperature, the retention times and peak areas of AMSs were found to be same indicating the robustness of the method. CONCLUSIONThe aim of this study is to develop a GC-MSmethod that can quantify three alkylmethanesulfonates in Lopinavir. The developed GC-MS method was optimizedbased on the resolutions of AMSs peaks andvalidated as per ICH guidelines. The method well suits for the intended purpose.Table 5: Data for RobustnessParameter conditionRetention time MMS EMS IPMSActual 5.2 7.4 8.7 Flow : 1.9 mL/min 5.3 7.6 8.9 Flow : 2.1 mL/min 5.1 7.3 8.5Column oven temperature- 108°C 5.5 7.9 9.2 Column oven temperature 112°C 4.9 7.0 8.2Figure 5: Chromatograms of Optimized method for MMS, EMS & IPMSFigure 6: Mass Spectra of MMS, EMS, and IPMSREFERENCES1.www.drugbank.ca/drugs/DB016012./wiki/Lopinavir3./july_sep_2010/301-310.pdf4.P.R.Kakadiya, B.P.Reddy, V.Singh, S.Ganguly,T.G.ChandraSekhar. Low level determinations of methyl methanesulfonate and ethyl methanesulfonate impurities in Lopinavir and Ritonavir Active pharmaceutical ingredients by LC/MS/MS using electrospray ionization. Journal of Pharmaceutical and Biomedical Analysis (2011);55 (2):379–384.5.Chitturi Sr, Sreenivas Rao Ch, RaghavaReddy A, V.Himabindu, Ramesh D. Impurity Profile Study Of Lopinavir And Validation Of HPLC Method For The Determination Of Related Substances In Lopinavir Drug Substance. J Pharm Biomed Anal (2008);48(5):1430-40.6. A. Suneetha, S.Kathirvel, G.Ramachandrika.A Validated RP HPLC Method ForSimultaneous Estimation Of Lopinavir And Ritonavir In Combined Dosage Form.International Journal of Pharmacy and Pharmaceutical Sciences (2011);3(1):49-51.7.Yoshiko Usami etal. SimultaneousDetermination Of Lopinavir, Ritonavir And Efavirenz By HPLC Method. Chemical and Pharmaceutical Bulletin (2003);51(6):715-718.8.Madhukar A. Jagadeeshwar K*, Naresh K.,Armstrong Vinod Raj, B. Jayapaul. Simple and sensitive analytical method development and validation of lopinavir bulk drug by RP-HPLC. Der Pharma Chemica (2011);3(6):494-499.9.R.Vijaya Lakshmi etal. Development andvalidation of a RP-HPLC method for determination of lopinavir in bulk and pharmaceutical dosage form. International Journal Of Research In Pharmacy And Chemistry (2011);2(2):413-417.10.Chandrasekhar K, Seshachelam K, Hari BabuB. A novel validated LC method forquantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products. Biomed Chromatogr (2007);21(7):716-23.11.Li W. Trace Analysis Of Residual MethylMethanesulfonate, Ethyl Methanesulfonate And Isopropyl Methanesulfonate In Pharmaceuticals By Capillary Gas Chromatography With Flame Ionization Detection. J Chromatogr A (2000);1046(2):297-301. 12.Colon I etal. Determination of methyl andethyl esters of methanesulfonic, benzenesulfonic and p-toluenesulfonic acids in active pharmaceutical ingredients by solid-phase microextraction (SPME) coupled to GC/SIM-MS. J Pharm Biomed Anal (2005);39(3-4): 477-85.13.Jagadeeshwar K etal. Simple and sensitiveanalytical method development and validation of lopinavir bulk drug by RP-HPLC. Der Pharma Chemica (2011);3 (6): 494-499.14.Gohlke, R.S. Time-of-Flight MassSpectrometry and Gas-Liquid Partition Chromatography. Analytical Chemistry (1959);31(4): 535.。
衍生气相色谱法测定甲磺酸伊马替尼中甲磺酸烷基酯类
衍生气相色谱法测定甲磺酸伊马替尼中甲磺酸烷基酯类作者：连小刚李孝壁程红艳来源：《中国医药科学》2013年第17期[摘要] 目的建立衍生气相色谱法同时检测甲磺酸伊马替尼中的甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯三种甲磺酸烷基酯。
方法采用NaI-H2O碘化衍生，利用ZB-WAX石英毛细管柱（30m×0.53mm，1.0μm）分离，氢火焰离子化（FID）检测器。
结果方法在0.070～13.0 μg/mL具有良好的线性（r>0.999），检出限0.2ppm，回收率94.1%～111.3%。
结论该方法可用于甲磺酸伊马替尼中甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯三种甲磺酸烷基酯成分的同时碘化衍生分离分析，方法简便，结果可靠、重现性好。
[关键词] 甲磺酸伊马替尼；衍生化；气相色谱；甲磺酸甲酯；甲磺酸乙酯；甲磺酸异丙酯[中图分类号] R927.2 [文献标识码] B [文章编号] 2095-0616（2013）17-128-03甲磺酸伊马替尼（imatinib mesylate）化学名称为4-[（4-甲基-1-哌嗪）甲基]-N-[4-甲基-3-[4-（3-吡啶）-2-嘧啶]氨基]苯基]-苯胺甲磺酸盐，是一种高效特异的酪氨酸激酶抑制剂[1]。
在合成工艺中用到甲醇、异丙醇，片剂生产中用到乙醇溶剂，在生产过程中，有可能与甲磺酸产生具有基因毒性的甲磺酸烷基酯类：甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯[2-3]。
近几年基因毒性杂质成为人们关注的焦点[4]，甲磺酸、苯甲磺酸等磺酸类物质与微量的低级醇在合成反应中生成烷基磺酸酯如甲磺酸甲酯（MMS）、甲磺酸乙酯（EMS）、异丙基甲磺酸酯（IMS）、正丁基甲磺酸酯（NBMS）[5]，以及芳基磺酸酯如苯磺酸甲酯（MBS）、苯磺酸乙酯（EBS）、对甲苯磺酸酯（MP-TS），这些物质可与DNA发生烷基化反应[6-7]，从而可能成为引发癌症的诱因，因此控制药物中该类杂质的毒理学关注阈值（TTC）水平非常重要[8-9]，欧洲医药评价署（EMEA），美国食品和药物管理局（FDA）及国际药品注册协调会议（ICH）先后对基因毒性杂质做出限度规定。
顶空-气相色谱-质谱法同时测定7种甲磺酸酯类杂质
第51卷第3期2019年9月郑州大学学报(理学版)J.Zhengzhou Univ.(Nat.Sci.Ed.)Vol.51No.3Sep.2019顶空•气相色谱•质谱法同时测定7种甲磺酸酯类杂质王少敏,牛颖1,代敏1,魏一1,刘宏民2(1.郑州大学化学与分子工程学院河南郑州450001； 2.新药创制与药物安全性评价河南省协同创新中心河南郑州450001)摘要：建立了一种同时测定药物中甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯、甲磺酸正丙酯、甲磺酸仲丁酯、甲磺酸异丁酯、甲磺酸正丁酯等7种甲磺酸酯类基因毒性物质的原位衍生-顶空-气相色谱-质谱法，改变衍生反应溶剂和用量，优化了反应时间、温度等反应条件以及孵化温度、平衡时间等顶空萃取条件.经方法学验证,7种甲磺酸酯类化合物在各自的线性范围内线性关系良好(K〉0.9990)，方法检出限(S/E=3)为0.21-2.64-g/L,定量限(S/S二10)为0.70-8.81-//L,日内、日间RSD分别小于10.3%和5.07%,加标回收率为88.2%-115,1%.对替比培南酯中间体中甲磺酸酯类杂质进行测定，均检出甲磺酸甲酯、甲磺酸乙酯和甲磺酸正丙酯残留•该方法可用于活性物质中甲磺酸酯类杂质的同时测定•关键词：烷基甲磺酸酯；衍生；顶空-气相色谱-质谱；检测中图分类号：R917文献标志码：A文章编号：1671-6841(2019)03-0104-05DOI：10.13705/j.issn.1671-6841.20182400引言基因毒性物质是一类能对DNA结构造成破坏而产生致癌性的物质，因此对药物中此类物质的检测引起人们广泛关注*_3+.在制药过程中，烷基磺酸常用作原料药的成盐试剂来提高药物的稳定性和溶解性,磺酸酯的前体物质(如磺酰氯)也常用作反应试剂或催化剂,这些应用都可能生成磺酸酯类基因毒性物质*4+.替比培南酯是第1个可口服的碳青霉烯类抗生素，对大多数临床分离的菌株均具有比青霉素及头抱类抗生素更强的抗菌性，但由于在其侧链的生产中使用了甲磺酰氯*5+,会与残留的溶剂醇生成甲磺酸酯，因此需要对替比培南酯侧链中的甲磺酸酯类杂质进行检测.目前检测甲磺酸酯的方法主要有GC-MS法*6"、LC-MS法*和气相色谱-微池电子捕获法*14].微池电子捕获检测器灵敏度高，但线性范围窄；质谱(MS)检测器因具有选择性强、灵敏度高、线性范围宽等优势而成为重要的检测手段*15].由于直接进样测定难以避免原料药本身对仪器的污染及离子化效率低等问题,在GC-MS方法中，使用低挥发性的衍生试剂，通过顶空萃取挥发性衍生产物可有效降低基质干扰.目前采用较多的方法是以乙腈/水(体积比80:20)混合溶剂为衍生基质的碘化钠衍生方法*_0],本文改进了碘化钠衍生方法，将甲磺酸酯与碘化钠在2-己酮/水(体积比3:97)溶剂中反应得到相应的碘代烷烃产物,在优化样品的顶空萃取条件和色谱条件的基础上，实现了同时对7种甲磺酸酯类杂质的定性、定量检测,可用于活性物质中甲磺酸酯类杂质的检测.1实验部分1.1仪器与试剂Trace ISQ™气质联用仪，配套有Triplus顶空自动进样器、顶空进样瓶(20mL)和Xcclibur数据处理系统收稿日期：2018-08-27基金项目：国家自然科学基金项目(21772180).作者简介：王少敏(1973—)，女，河南新乡人，教授，主要从事药物分析研究,E-mail%wshcomin@.第3期王少敏，等：顶空-气相色谱-质谱法同时测定7种甲磺酸酯类杂质105（EI离子源，美国Thermo Scientific公司）；FiveEasy PlusTM pH计（瑞士Mettler Toledo公司）；Milli-Q Reference超纯水系统（美国Millipore公司）.甲磺酸甲酯（MMS,98.0%,吴睿化学有限公司）；甲磺酸正丙酯（，-PMS）和甲磺酸异丁酯（z-BMS）（>98.0%,梯希爱化成工业发展有限公司）;甲磺酸仲丁酯（s-BMS）和甲磺酸正丁酯（，-BMS）（>98.0%,苏州麦凯生物医药科技有限公司）;甲磺酸乙酯（EMS,98.5%）、甲磺酸异丙酯（z-PMS,97.0%）、碘化钠（99.0%）、2-己酮（99.0%）（百灵威科技有限公司）;内标CHCi$（99.8%,J.T.Bake-公司）；硫代硫酸钠（99.0%,Sigma Ald-ich公司）;其他试剂均为色谱纯或分析纯.1.2溶液的配制衍生试剂：称取60g碘化钠和30mg硫代硫酸钠，加水溶解并稀释至50mL,摇匀备用.内标溶液：量取CHCi$适量，用2-己酮溶解并稀释，配制质量浓度为14.84g/L的内标储备液，再用超纯水稀释至质量浓度为7.42m//L的内标溶液，摇匀备用.甲磺酸酯类混合标准溶液：精密量取MMS、EMS、z-PMS、,-BMS、s-BMS、z-BMS、,-BMS标准品适量，用2-己酮溶解并稀释，配制质量浓度约为5g/L的储备液，摇匀，于4T冷藏备用.精密量取标准品储备液适量,用超纯水分别稀释至质量浓度约为1、1、3、2、2、2、2mg/L的混合标准溶液，现制现用.1.3衍生反应混合标准溶液的衍生：精密量取混合标准溶液200-L置于20mL顶空瓶中，加入超纯水320-L、2-己酮30-L、内标溶液50-L和衍生试剂400-L,立即加盖密封，涡旋混匀，于60°C下反应20min.样品溶液的衍生：精密称取原料药25mg置于20mL顶空瓶中，加入超纯水520-L和2-己酮30-L,涡旋使其溶解，再加入内标溶液50-L和衍生试剂400-L,立即加盖密封，涡旋混匀，于60C下反应20me.1.4色谱-质谱条件色谱柱为TR-wax MS柱（30mX0.25mm,0.25-m）;程序升温：40C保持6min，然后以20C/min升至240C，保持5min&载气为高纯氦气；流速为0.8mL/min,恒流模式;进样口温度200C;进样量100-L;进样方式为分流进样，分流比50:1；孵化室温度80C，平衡时间为30min.质谱条件：离子源温度250C&传输线温度250C；EI离子源能量70eV；检测模式为选择离子扫描（SIM）.定量监测离子:m/z142（碘甲烷）,m/z 156（碘乙烷）,m/z170（碘代正丙/异丙烷）,m/z184（碘代正丁/异丁/仲丁烷）；m/z83（CHC13）.定性监测离子:m/z127（碘代烷烃）,m/z118（CHCi3）.2结果分析与讨论2.1衍生反应条件2.1.1反应溶剂和用量目前的碘化钠衍生方法均是以乙腈/水（体积比80:20）混合溶剂为衍生基质,衍生结束后经顶空萃取，大量的强极性乙腈进入仪器中会产生很强的溶剂峰,不仅降低离子源灯丝的寿命,还容易引起色谱柱的填料流失.因此，对一些出峰位置不干扰检测物目标组分的有机相进行了考察.按1.3所述过程操作,其他条件不变,分别加入30-L的有机相苯甲醚、氯苯、正己烷、甲基叔丁基醚、乙酸正丁酯、2-庚酮、正丁醇、2-己酮，与未加入有机相的各组分对比分析，发现2-己酮效果最好.同时对2-己酮的用量也进行了优化，确定2-己酮/水（体积比3:97）的最佳用量为30-L.2.1.2衍生试剂用量分别加入0.2、0.3、0.4、0.5mL衍生试剂，按1.3所述过程操作，通过调整超纯水用量使顶空瓶内的液体总量保持在1mL.结果表明,当衍生试剂加入0.4mL时可达到最佳衍生效果,略低于文献* 9-10+中0.5mL的用量.2.1.3衍生温度和时间按1.3所述过程操作，分别在40、50、60、70C下衍生反应20min后进样测定,发现60C下衍生效果最佳.在此基础上,考察了反应时间10、20、30、40min对衍生反应的影响，确定最佳反应时间为20min.2.2顶空萃取条件将衍生后的样品分别在50、60、70、80C下平衡30min后顶空进样分析.结果发现,随着温度的升高,所检测到的组分含量逐渐增大，其中相对分子质量高的化合物受温度的影响程度较大，考虑到顶空瓶内压力和106郑州大学学报（理学版）第51卷气密性等问题，选择在80T下平衡.在此基础上，考察了在80T下平衡时间对分析结果的影响，结果表明，平衡30min时萃取效果最好，因此将顶空萃取条件设为80T下平衡30min.2.3混合标准溶液和衍生产物的稳定性将新制的甲磺酸酯类混合标准溶液于室温下放置，依次取放置0、20、40、60'80min的样品进行分析，发现在20min内各个组分可保持稳定，超出20min后，s-BMS最容易分解，其次是z-PMS和z-BMS.对衍生产物的稳定性进行考察，4个由混合标准溶液配制的样品衍生后于室温下放置，每隔24h检测1个样品，发现碘代烷烃产物在3d内都能保持稳定.2.4方法学验证2.4.1线性范围、精密度和回收率将甲磺酸酯类混合标准溶液稀释至7个质量浓度，以目标物和内标的峰面积比对样品质量浓度进行线性回归，得到线性回归方程及相关系数.通过混合标准溶液逐级稀释，由信号响应信噪比（S/E）来确定检出限（S/E=3）和定量限（S/E=10），结果如表1所示.分析物标准曲线的线性相关系数均大于0.9990，检出限为0.21-2.64-g/L，定量限为0.70-8.81-g/L，对应的实际样品的检出限为&4-106ng/g，定量限为28-352ng/g.取混合标准溶液（MMS、EMS、i-PMS、n-PMS、s-BMS、i-BMS、,-PMS 的质量浓度分别为10、9、27、18、22、22、18-g/L）进行日内、日间平行分析，经过精密度计算，相对标准偏差（RSD，n=3）为0.95%-10.3%,满足方法学的要求.称取替比培南酯中间体样品25mg（含微量MMS、EMS和n-PMS杂质），分别加入混合标准溶液（MMS、EMS、z-PMS、n-PMS、s-BMS、z-BMS、n-PMS的质量浓度分别为10、9、27、18、22、22、18-g/L）进行回收率试验，各目标物的加标回收率为88.2%-115.1%（n=3"，满足方法学的要求.表1甲磺酸酯类化合物的线性范围、回归方程、相关系数、精密度和回收率Tab.1The linear ran/e,regression equation#correlation coefficient#precision and recovery of methanesulfonate compounds化合物线性范围/（-/匸1）线性回归方程相关系数检出限/（-/L）定量限/(-/LT)日内RSD/%日间RSD S%平均回收率/%MMS 2.50-400Y=0.6102&X-1.31200.99960.53 1.7810.3 5.07101.6 EMS 2.25-360Y=1.1878&X-2.22850.99960.210.70 2.90 4.13112.3i-PMS 6.75-1080Y=2.1168&X-5.90910.9998 1.32 4.390.95 1.64115.1n-PMS 4.50-720Y=4.5657&X-3.46430.99990.65 2.15 1.45 2.8588.2s-PMS11.0-880Y=2.2816&X+8.37120.9991 2.648.81 1.43 3.1896.4i-PMS 5.50-880Y二0.3489&P+1.20500.9990 1.15 3.84 1.25 2.5499.5n PWMS 4.50-720Y二0.3250&P+1.46310.99940.85 2.85 2.56 4.45108.22.4.2低级醇的干扰性实验文献* 9-10+中提出，空气中的甲醇会与碘化钠衍生试剂发生亲核取代反应，生成碘甲烷，进而对检测准确性造成一定的影响.而在本方法中，未见空白样品中有甲醇带来的干扰.作为补充,按照混合标准溶液的配制方法配制了甲醇和乙醇的混合溶液，于顶空瓶中的质量浓度均为127-//L,按同样的方法进行衍生分析，也未观察到碘甲烷或碘乙烷谱峰的出现，表明空气或样品中残留的甲醇或乙醇对该方法的检测结果没有影响.文献* 9-10+中的干扰可能是由于使用了大量有机相溶剂乙腈，促进了干扰产物的生成.2.4.3样品分析按上述方法对某药企生产的5批替比培南酯中间体进行分析，其SIM色谱图如图1所示.由图1（a）可见，7min内所有目标化合物均已出峰，内标CHCf（IS）的保留时间为5.47min,对应MMS、EMS、i-PMS、n-PMS、s-BMS、i-BMS、n-PMS的衍生物的保留时间分别为2.25、2.77、3.06、4.05、4.91、5.17、6.43min.图1（b）中保留时间为3.68、3.80min的峰来源于溶剂本底.图1（c）为实际样品的SIM色谱图（图1（C）中的小图分别为实际样品中rn/z142、m/z156和rn/z170的萃取离子流图），均检出MMS、EMS和n-PMS，来源于生产中甲醇、乙醇、丙醇等溶剂与甲磺酰氯的副反应，而生产中未用到的异丙醇和正、异、叔丁醇没有检测到，5批样品（S1-S5）的测定结果见表2.第3期王少敏，等：顶空-气相色谱-质谱法同时测定7种甲磺酸酯类杂质107s wcq,s图1混合标准品溶液(a)、空白溶液(V)和实际样品(c)的SIM色谱图Fig.l Selected-ion monitoring chromatograms of mixed standard solution(a),blank solution(V)and the sample(c)表25批样品的测定结果Tab.2Deieominaiion oesuaisoooivebaichesoosampaesnggg 样品S1S2S3S4S5MMS0.1810.1990.1760.2130.188EMS0.2780.4380.4110.3740.414,-PMS0.0860.1430.0870.1400.1533结论文献[8,12+中报道的色谱-质谱法对药物中甲磺酸酯类杂质同时检测的数量最多有4种，本文方法可以同时对7种甲磺酸酯类杂质进行定性、定量分析,具有更广泛的适用性.与基于碘化钠衍生的GC-MS法相比，该方法避免了有机相乙腈的大量使用,仅使用少量2-己酮就可达到分析要求.由于衍生反应溶剂中有机相用量少,避免了空气或样品中残留的甲醇或乙醇对检测结果的影响•该方法具有适用范围广、灵敏度高、基质干扰小等特点,可用于药物中间体、原料药及成品药中甲磺酸酯类杂质的检测•参考文献：*1+NIKOLOVA T,MARINI F,KAINA B.Genotoxicity testing:ccmparison of the yH2AX focus assay with the alkaline and neutral ccmet assays*J+.Mutation reseerch/genetic toxicclogy and environmentai mutagenesis,2017,822:10-18.*2:钱建钦,张云峰,王建,等.UHPLC-MS法测定2种硫酸氢氯毗格雷晶型中的基因毒性杂质对甲苯磺酸甲酯*J]•药物分析杂志,2017,37(11)：1994-1999.*3+CAO X F,MITTELSTAEDT R A,PEARCE M G,et al.Quantitativv dose-response analysis of ethyS methanesulfonate genotoxicity in aduit gpt-delta transaenic micc*J].Unvironmentai and moleculac mutaaenesis,2014,55(5)：385-399.*4]SZEKELY G,AMORES S M C,GIT M,et al.Genotoxic impurities in pharmaccuticai manufacturing:sourccs,reeulations,and mitigation*J].Uhemical reviews,2015,115(16):8182-8229.*5:翟雪，隋强,时惠麟•替比培南酯合成路线图解*J]•中国医药工业杂志,2012,43(6)：503-506.*6+ZHANG C Z,HUANG L,WU Z G,et al.Determination ot sulfonato ester genotoxic impurities in imatinib mesylato by gas chroma-tooraphy with mass spectrometry*J].Journat of aeparation sciencc,2016,39(18):3558-3563.*7]ALZAGA R,RYAN R W,TAYLOR-WORTH K,et at.A generic approach for the determination ot residues of alkylating agents in activv pharmaccuticat ingredients by in situ derivvtization-heedspacc-gas chromatoxraphy-mass spectrometTy*J+.Journat ot pharmaccutical and biomedical analysis,2007,45(3):472-479.*8+SITARAM C,RUPAKULA R B,REDDY B N,et al.Determination ot alky】methaneulfonates in doxezosin mesylate by gas chromatoxraphy-mass spectrometer*J].Indian journat of pharmaccuticat sciences,2011,73(1):107-110.*9:张萌萌,姚晓华，蒋元森,等•不同检测器-顶空气相色谱法测定甲磺酸达比加群酯中的甲磺酸乙酯和甲磺酸异丙酯*J].中国医药工业杂志,2015,46(10) :1108-1112.*10:易大为,邹宇,赵晓东,等.GC-MS法测定甲磺酸培氟沙星中甲磺酸酯杂质的研究*J]•中国抗生素杂志,2017,42(6):108郑州大学学报（理学版）第51卷521-525.[11]冯慧敏，杭太俊，高新桃，等-LC-MS法同时检测甲磺酸伊马替尼中3个磺酸酯基因毒杂质[J]•药物分析杂志$2014,34(12)%2202-2206.[12+G60T,SHI Y Y,ZHENG L,et al.Rapid and simultaneous determination of sulfonate ester genotoxic impurities in drug substance by liquid chromatography ccupled to tandem mass spectrometry：ccmparison of different ionization modes[J+.Journal of chromatography A,2014,1355%73-79.[13+ZHOU J,XU J,ZHENG X Y,et al.Determination of methyl methanesulfonato and ethyl methanesulfonato in methanesulfonic acid by derivatization followed by high-performance liquid chromatooraphy with ultraviolet detection[J+.Journal of separation sciencc, 2017,40(17)%3414-3421.[14]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.[15]陈俊苗，朱登勇，王少敏，等.HPLC-MSMS法快速鉴定Z/E马来酸桂哌奇特异构体[J]•郑州大学学报(理学版),2012,44(3)：94-96.Simultaneous Determination of Seven MethanesulfonateImpurities by Hea=pace-GC-MSWANG Shaomin1-2,NIU Ying1,DAI Min1,WEI Yi1,LIU Hongmin2；(1.College of Chemist—and Molecular Engineering,Zhengzhou University,Zhengzhou450001,China2.School of Collaborative Innovation Center rf New Drug Research and Safety Evaluation ofHenan Province,Zheeghou450001,China)AbstracC:A proccdura for the simultaneous determination of seven methanesulfonate genotoxio impurities,such as MMS,EMS,i-PMS,n-PMS,s-BMS,i-BMS,n-BMS,employed in drug synthesiz was developed and validated by derivatization-heedspace-GC-MS.The eeects of type and amouni of oraanio phase,aeaciion iempeaaiuaeand iimeaelaied io ihe de aiia iiaa iion we ae s iudied and iheopiimum condiB tions wera determined.The conditions including heating temperatura and equilibration time to the head-spacc method wera discussed and optimized,too.The mcthodoloxicol validation showed the good lincarita for all the analytes with the ccrrelation ccefficientt greeter than0.9990,the detection limitt(S/N=3) varying from0.21to2.64—g/L,and the110x1:0of quantitation(S/N=10)from0.70to8.81—g/L.The standard deviations of intra-day and inter-day wera less than10.3%and5.07%respectivaly,and the re-covvries of standard addition ranged from88.2%to115.1%.The method was succcssfully used to test te-bipenem intermediates and a was found that all t he samples tested ccntained MMS,EMS and n-PMS.The method was suitable for the simultaneeus analysis of the methanesulfonato impurities in activa substances.Key words:alkyl methanesulfonato；derivatization；headspacc-GC-MS&determination（责任编辑:孔薇）。
一种气相色谱-串联质谱检测甲磺酸甲酯的方法
一种气相色谱-串联质谱检测甲磺酸甲酯的方法
气相色谱-串联质谱（GC-MS/MS）是一种常用的分析技术，可以用于甲磺酸甲酯的检测。
下面是一种可能的方法：
1. 样品准备：取一定量的甲磺酸甲酯样品，加入适量的溶剂，进行溶解和稀释。
可以选择有机溶剂如甲醇或乙醚。
2. 样品注射：将样品溶液注射到气相色谱仪的进样口中。
3. 气相色谱分离：使用一个适当的毛细管柱，将甲磺酸甲酯及其他杂质物质进行分离。
常用的柱材料是聚硅氧烷（PDMS）或聚酯。
4. 串联质谱检测：色谱柱的出口连接到一个串联质谱仪。
在质谱仪中，通过碰撞电离或化学电离等方式将甲磺酸甲酯和其他化合物进行离子化。
然后，以母离子或特定的离子对进行选择性监测。
5. 数据分析：通过与已知标准溶液对比，确定甲磺酸甲酯的浓度。
可以利用质谱仪的软件进行数据分析和峰面积比较。
6. 质量控制：在分析过程中，使用标准物质对仪器进行校准和验证。
需要注意的是，在GC-MS/MS分析中，使用合适的柱和仪器条件，以确保甲磺酸甲酯能够得到良好的分离和检测。
另外，
在这个方法中，还可以使用内标物质进行质量校正，提高准确性和精确性。
LC—MS／MS测定甲磺酸伊马替尼中的伊马胺
LC—MS／MS测定甲磺酸伊马替尼中的伊马胺目的建立高效液相色谱-质谱联用测定甲磺酸伊马替尼中基因毒性杂质伊马胺的方法。
方法选用C18色谱柱（3.5μm，150mm×3.0mm），以0.05%甲酸的乙腈-20mmol/L甲酸铵为流动相，采用梯度洗脱进行分离，流速：0.5mL/min；柱温为40℃；进样量10μL。
对照品溶液选用TSQ Quantum Ultra质谱仪的选择离子检测（SIM）扫描方式进行检测。
结果该方法在2.5～100.0ng/mL范围内线性良好（0.998），定量限和检出限分别为2.5ng/mL和1.0ng/mL，方法回收率97.1%，稳定性较好。
结论本研究所建立的方法快速、灵敏、专属性强，适用于甲磺酸伊马替尼中基因毒性杂质伊马胺的测定。
标签：甲磺酸伊马替尼；LC-MS/MS；基因毒性杂质；伊马胺甲磺酸伊马替尼（imatinib mesylate）是瑞士Novartis公司研发的Bcr-Abl酪氨酸激酶受体抑制剂，2001年首次在美国上市[1]，商品名Gleevec。
临床主要用于治疗慢性髓性白血病（chronic myelogenous leukemia，CML）急变期[2-3]、加速期或α-干扰素治疗失败后的慢性期患者和治疗不能切除和发生转移的恶性胃肠道间质肿瘤（GIST）的成人患者[4]，治疗效果得到了广泛的认可。
伊马胺化学名N-（5-氨基-2-甲基苯基）-4-（3-吡啶基）-2-嘧啶胺是合成甲磺酸伊马替尼合成的中间体[5-7]，属于潜在的基因毒性杂质[8-9]。
根据美国食品和药物管理局（FDA）和国际药品注册协调会议（ICH）关于基因毒性杂质的指导原则要求[10-11]，本资料拟建立采用LC-MS/MS测定本品合成中间体伊马胺的含量方法。
1 仪器与试剂仪器：XS105电子分析天平（瑞士METTLER公司）；UltiMate 3000高效液相色谱仪（美国戴安公司）；TSQ Quantum Ultra三重四级杆串联质谱仪，配有电喷雾离子源（美国Thermo Scientific公司）。
衍生化GC-MS法测定甲磺酸萘莫司他原料药中甲磺酸甲酯与甲磺酸乙酯
衍生化GC-MS法测定甲磺酸萘莫司他原料药中甲磺酸甲酯与甲磺酸乙酯刘晓强;丁建【摘要】目的建立衍生化GC-M S法测定甲磺酸萘莫司他原料药中甲磺酸甲酯与甲磺酸乙酯.方法采用柱前衍生技术、程序升温法,以氦气为载气,以甲磺酸丁酯为内标,测定甲磺酸萘莫司他中甲磺酸甲酯和甲磺酸乙酯的含量.结果甲磺酸甲酯和甲磺酸乙酯的线性范围均为3.0～200.0 ng·mL-1,相关系数分别为0.9975和0.9999;检测限分别为0.9643和1.0078 ng·mL-1;定量限分别为2.8928和3.0223 ng·mL-1;进样精密度的RSD值分别为0.9％和1.3％;平均加样回收率分别为102％(RSD=2.9％)和106％(RSD=1.2％).结论该方法简单方便、快速易行,可作为控制甲磺酸萘莫司他中甲磺酸甲酯与甲磺酸乙酯含量的有效方法.%Objective To determinate mthylmesylate (MMS) and ethylmesylate (EMS) in nafamostat mesilate raw material by deriv-ative GC-MS .Methods The MMS and EMS were determined by pre-column derivation by temperature programming method .Heli-um was used as the carrier gas .The content of MMS and EMS were quantified with butylmesylate as internal standard .Results The linear calibration curves of MMS and EMS were both obtained over the range of 3 .0-200 .0 ng · mL -1 ,the correlation coeffi-cients were 0 .9975 and 0 .9999 .The limits of detection were 0 .9643 and 1 .0078 ng · mL -1 ,the limits of quantitation were 2.8928 and 3 .0223 ng · mL -1 .The RSDsof the injection precision were 0 .9％ and 1 .3％ .The extraction rates were 102％ and 106％ with RSD of 2 .9％ and 1 .2％ .Conclusion The method issimple and convenient ,quick and easy ,and can be used for the quality control of MMS and EMS in nafamostat mesilate .【期刊名称】《西北药学杂志》【年(卷),期】2018(033)001【总页数】3页(P40-42)【关键词】甲磺酸萘莫司他;甲磺酸甲酯;甲磺酸乙酯;GC-MS【作者】刘晓强;丁建【作者单位】常州市食品药品监督检验中心,常州 213101;常州市食品药品监督检验中心,常州 213101【正文语种】中文【中图分类】R927.2甲磺酸萘莫司他(nafamostat mesilate)化学名为6-脒基-2-萘基-4-胍基苯甲酸酯二甲磺酸盐,是Japan Tobacco公司研发的非肽类蛋白酶抑制剂[1],首次上市是在1986年的日本,主治临床急性胰腺炎[2-3]。
顶空气相色谱法测定甲磺酸伊马替尼中有机溶剂残留量
顶空气相色谱法测定甲磺酸伊马替尼中有机溶剂残留量摘要：目的：建立顶空气相色谱法测定甲磺酸伊马替尼中有机溶剂残留量。
方法：采用Agilent DB-624毛细管柱（30m×0.53mm，3.0？m）；氮气为载气；进样口温度为250℃；检测器温度为300℃；柱温为50℃。
顶空进样，平衡温度为90℃，平衡时间为30min；以二甲亚砜为溶剂。
结果：在本文色谱条件下，分离度良好，检测浓度在所考察的范围内与峰面积具有良好的线性，r=0.9990～0.9999；平均回收率为95.0%～104.6%，精密度试验及准确度试验的RSD均小于5%。
结论：本试验建立的色谱方法简便、准确，灵敏度高，适合甲磺酸伊马替尼有机溶剂残留量的检测。
关键词：甲磺酸伊马替尼残留溶剂顶空气相色谱法甲磺酸伊马替尼用于治疗慢性骨髓性白血病及恶性胃肠道间质肿瘤，治疗效果得到了广泛的认可。
甲磺酸伊马替尼的制备使用了甲醇、乙腈、四氢呋喃、氯仿和丁酮5种溶剂。
现有的研究工作中未见报道甲磺酸伊马替尼中5种残留溶剂的检查方法。
本文按中国药典2010年版二部[1]与人用药品注册技术规范国际协调会（ICH）[2]相关要求，并参考有关资料[3]建立了顶空气相色谱法检测甲磺酸伊马替尼中5种残留溶剂，该方法灵敏度高，结果准确。
一、仪器与试药仪器：Agilent 7890；顶空进样器：Agilent 7697。
其它试剂均为色谱纯。
甲磺酸伊马替尼样品（山东方明药业集团股份有限公司提供，批号为：201101001，201101002，201101003）。
二、方法与结果1.溶液的制备对照品溶液分别精密称取甲醇312.32mg，乙腈42.51mg，四氢呋喃73.22mg，氯仿60.85mg，丁酮520.32mg，同置一个100ml容量瓶中，加二甲亚砜稀释至刻度，振摇使溶解，即得对照品储备液。
精密量取储备液10ml，置100ml量瓶中，加DMF稀释至刻度，摇匀，即为对照品溶液。
毛细管气相色谱法测定甲磺酸伊马替尼原料药中4种有机溶剂残留量
毛细管气相色谱法测定甲磺酸伊马替尼原料药中4种有机溶剂残留量王善春;张喜全;李洋;李慧【摘要】目的建立可同时测定甲磺酸伊马替尼原料药中石油醚(60~90 ℃)、乙酸乙酯、乙醇和吡啶4种残留有机溶剂的分析方法.方法采用毛细管气相色谱法.色谱柱为DB-WAX毛细管柱,氢离子火焰检测器(FID),温度为200 ℃,进样口温度为180 ℃,程序升温,载气为氮气,流速为5 mL·min-1,进样量为1 μL.以 N,N-二甲基甲酰胺(DMF)为溶剂,采用外标法测定3批甲磺酸伊马替尼原料药中有机溶剂石油醚(60~90 ℃)、乙酸乙酯、乙醇和吡啶的残留量.结果 4种有机溶剂在建立的色谱条件下均能完全分离,在所考察的质量浓度范围内线性关系良好(r=0.9998~0.9999);进样精密度(RSD)均小于2.0%(n=5);乙酸乙酯、乙醇、吡啶的回收率均在97%~101%之间,石油醚的回收率为88.7%,RSD均小于2.0%(n=5);石油醚(60~90 ℃)、乙酸乙酯、乙醇和吡啶的检测限分别为0.58,0.42,0.18,0.61 ng,定量限分别为1.8,2.1,0.5,2.5 ng.结论该实验建立的方法简便、灵敏,结果准确、可靠.%Objective To establish a method for simultaneous determination of petroleum ether (60-90 ℃),ethyl acetate,ethanol and pyridineas residual organic solvents in imatinib mesylate API. Methods Capillary gas chromatography was adopted.The determination was performed on DB-WAX capillary column with FID detector.The injector temperature was 180 ℃and the temperature of FID was 200 ℃ by temperature programming with nitrogen serving as carrier gas at a flow rate of 5 mL·min-1.The injection volume was 1 μL.With N,N-dimethylformamide(DMF) serving as solvent,four kinds of organic solventresidues in the samples were calculated by external standard method. Results The four kinds of organic solvent were completely separated under the established chromatographic condition.A good linearity was obtained in the concentration ranges of them (r=0.999 8-0.999 9).The sampling precisions (RSDs) were less than 2.0%(n=5).The average recovery rates of ethyl acetate,ethanol and pyridine were 97%-101%and that of petroleum ether was 88.7%(RSD<2.0%,n=5).The limits of detection for them were 0.58,0.42,0.18 and 0.61 ng respectively(S/N=3:1).The limits of quantification for them were 1.8,2.1,0.5 and 2.5 ng,respectively (S/N=10:1). Conclusion The method is simple,sensitive,accurate and reliable.【期刊名称】《医药导报》【年(卷),期】2017(036)012【总页数】4页(P1390-1393)【关键词】伊马替尼,甲磺酸;色谱法,气相,毛细管;残留,有机溶剂【作者】王善春;张喜全;李洋;李慧【作者单位】正大天晴药业集团股份有限公司研究院,南京 210042;正大天晴药业集团股份有限公司研究院,南京 210042;正大天晴药业集团股份有限公司研究院,南京 210042;正大天晴药业集团股份有限公司研究院,南京 210042【正文语种】中文【中图分类】R979.1;R927.2甲磺酸伊马替尼(imatinib mesylate，商品名：Glive，格列卫)是由瑞士Novartis(诺华)公司研发的酪氨酸激酶抑制药，于2001年5月首次在美国上市[1]。
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衍生气相色谱法测定甲磺酸伊马替尼中甲磺酸烷基酯类目的建立衍生气相色谱法同时检测甲磺酸伊马替尼中的甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯三种甲磺酸烷基酯。
方法采用NaI-H2O碘化衍生，利用ZB-WAX石英毛细管柱（30m×0.53mm，1.0μm）分离，氢火焰离子化（FID）检测器。
结果方法在0.070～13.0 μg/mL具有良好的线性（r>0.999），检出限0.2ppm，回收率94.1%～111.3%。
结论该方法可用于甲磺酸伊马替尼中甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯三种甲磺酸烷基酯成分的同时碘化衍生分离分析，方法简便，结果可靠、重现性好。
标签：甲磺酸伊马替尼；衍生化；气相色谱；甲磺酸甲酯；甲磺酸乙酯；甲磺酸异丙酯甲磺酸伊马替尼（imatinib mesylate）化学名称为4-[（4-甲基-1-哌嗪）甲基]-N-[4-甲基-3-[4-（3-吡啶）-2-嘧啶]氨基]苯基]-苯胺甲磺酸盐，是一种高效特异的酪氨酸激酶抑制剂[1]。
在合成工艺中用到甲醇、异丙醇，片剂生产中用到乙醇溶剂，在生产过程中，有可能与甲磺酸产生具有基因毒性的甲磺酸烷基酯类：甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯[2-3]。
近几年基因毒性杂质成为人们关注的焦点[4]，甲磺酸、苯甲磺酸等磺酸类物质与微量的低级醇在合成反应中生成烷基磺酸酯如甲磺酸甲酯（MMS）、甲磺酸乙酯（EMS）、异丙基甲磺酸酯（IMS）、正丁基甲磺酸酯（NBMS）[5]，以及芳基磺酸酯如苯磺酸甲酯（MBS）、苯磺酸乙酯（EBS）、对甲苯磺酸酯（MP-TS），这些物质可与DNA发生烷基化反应[6-7]，从而可能成为引发癌症的诱因，因此控制药物中该类杂质的毒理学关注阈值（TTC）水平非常重要[8-9]，欧洲医药评价署（EMEA），美国食品和药物管理局（FDA）及国际药品注册协调会议（ICH）先后对基因毒性杂质做出限度规定。
由于本品中甲磺酸烷基酯类的限度在中国药典没有规定，有必要对样品中的甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的残留量进行检测，本方法参考欧洲药典，采用衍生化的方法[10]，通过检测碘甲烷、碘乙烷和2-碘丙烷来确定甲磺酸甲酯、甲磺酸乙酯和甲磺酸异丙酯的量。
1 仪器与试剂仪器：电子分析天平，梅特勒XS105；Agilent 7890 GC气相色谱仪，顶空进样器G1888；安捷伦色谱工作站；色谱柱：ZB-WAX（30m×0.53mm×1.0μm）。
试剂：甲醇、丙酮、乙酸乙酯、甲苯、异丙醇均为分析纯是国药集团化学试剂股份有限公司。
2 试验过程与结果2.1 色谱条件按照气相色谱法（中国药典2010年版二部附录ⅤE），用聚乙二醇20M交联聚合物为固定相（或极性相似）的毛细管柱（ZB-WAX 30m×0.53mm×1.0μL），氢火焰离子化（FID）检测器，进样口温度110℃；检测器温度260℃；柱温40℃维持10min，以每分钟20℃的升温速率升至160℃维持3min；顶空瓶平衡温度为80℃，平衡时间为30min。
取对照品溶液与供试品溶液分别顶空进样，进样体积3mL，记录色谱图，按碘甲烷、碘乙烷、2-碘丙烷的出峰顺序；按外标法以峰面积计算。
2.2 试验溶液配制供试品溶液配制：取本品适量，精密称定，按供试品与乙腈100︰1（mg︰mL）的比例配置供试品溶液，溶液经超声、薄膜过滤处理后，精密移取2mL加入顶空瓶中，加入3mL水、6g碘化钠后，扎盖密闭，作为供试品溶液。
对照品溶液配制：取甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯适量，精密称定，用乙腈制成每毫升含甲磺酸甲酯10μg、甲磺酸乙酯10μg、甲磺酸异丙酯10μg 的溶液，精密移取2mL加入顶空瓶中，加入3mL水、6g碘化钠后，扎盖密闭，作为对照品溶液。
2.3 系统专属性试验精密称取甲磺酸甲酯99.2mg、甲磺酸乙酯102.8mg、甲磺酸异丙酯104.8mg 分别置100mL量瓶中，用乙腈稀释至刻度，标记为1#、2#、3#溶液。
分别移取1#、2#、3#溶液各1mL分别置100mL量瓶中，用乙腈稀释至刻度，标记为4#、5#、6#液。
分别移取1#、2#、3#溶液各1mL置100mL量瓶中，用乙腈稀释至刻度，作为对照品溶液，标记为7#液。
分别精密移取4#、5#、6#、7#液各2mL分别置入顶空瓶中，并分别加入3mL 的水、6g的碘化钠，扎盖密闭，分别标记为A甲磺酸甲酯衍生化测试液、B甲磺酸乙酯衍生化测试液、C甲磺酸异丙酯衍生化测试液、D对照品溶液，分别进样A、B、C、D溶液，记录色谱图。
从表1可见，被测溶剂各峰的保留时间与对照品中各峰保留时间接近，最小分离度为3.1，符合实验要求，表明各溶剂的专属性强，专属性色谱图见图1。
2.4 定量限与检测限精密称取甲磺酸甲酯、甲磺酸乙酯、甲磺酸异丙酯适量，置100mL量瓶中，用乙腈溶解制成溶液，作为储备液。
逐步稀释储备液，进行样品衍生化，并分别进样至信噪比（峰高/基线噪声）为3左右为检测限，至信噪比为10左右为定量限，结果见表2。
由表2可见，该检测方法最小可准确检测0.6ppm的量，表明本方法对碘甲烷、碘乙烷、2-碘丙烷可以准确定量检测。
2.5 线性关系试验精密称取甲磺酸甲酯100.1mg、甲磺酸乙酯103.3mg、甲磺酸异丙酯107.8mg，分别置100mL量瓶中，用乙腈稀释定容。
各移取上述溶液1mL置5mL量瓶中，用乙腈稀释至刻表度作为贮备液；分别精密移取贮备液0.3、0.6、0.9、1.2mL分别置于20mL量瓶中，用乙腈稀释至刻度，摇匀，作为2#、3#、4#、5#液。
移取上述溶液各2mL分别置入顶空瓶中，并分别加入3mL 水、6g碘化钠，扎盖密闭，平行制备2份。
分别进样上述溶液，平行2次进样，取峰面积的平均值与相应浓度回归，实验数据记录及线性回归方程见表3。
表3可见，甲磺酸甲酯在0.0721～12.0μg/mL、甲磺酸乙酯在0.0604～12.4μg/mL、甲磺酸异丙酯0.0679～12.9μg/mL的范围内，线性相关系数皆不小于0.999，说明线性良好。
线性方程及相关系数见表3。
2.6 回收率精密称取甲磺酸甲酯101.3mg、甲磺酸乙酯100.8mg、甲磺酸异丙酯102.3mg 置100mL量瓶，乙腈稀释至刻度，作为储备液。
取供试品约300mg，置顶空瓶中精密称定，共称9份，用储备液进行配置成限量2、30、100ppm的测试液，置入顶空进样器中，定量测定的6个溶剂的平均回收率介于94.1%～111.3%之间，各溶剂回收率的最大相对标准偏差RSD为5.79%，表明本方法的准确性较好。
3 讨论在前期预实验回收率测试时发现，在衍生化过程中，样品与碘化钠也发生衍生化反应，干扰甲磺酸酯的检测，影响碘代烷的收率，因此，采用与样品不溶而与甲磺酸烷基酯易溶的乙腈溶剂，对样品中的甲磺酸烷基酯进行超声、萃取。
本研究建立了衍生气相色谱法测定甲磺酸伊马替尼中的基因毒性杂质甲磺酸烷基酯类的检测方法，该方法专属性好。
对检测限、定量限的测定，说明此方法具有较高的灵敏度，此外，该方法线性与范围良好、准确度好，能对样品中的基因毒性杂质甲磺酸烷基酯类化合物进行很好的控制。
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