WO2023102741A1 - 一种药物中亚硝胺类杂质的检测方法 - Google Patents

一种药物中亚硝胺类杂质的检测方法 Download PDF

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WO2023102741A1
WO2023102741A1 PCT/CN2021/136130 CN2021136130W WO2023102741A1 WO 2023102741 A1 WO2023102741 A1 WO 2023102741A1 CN 2021136130 W CN2021136130 W CN 2021136130W WO 2023102741 A1 WO2023102741 A1 WO 2023102741A1
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mobile phase
solution
nitrosamine
liquid chromatography
performance liquid
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PCT/CN2021/136130
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English (en)
French (fr)
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殷敏敏
胡成松
范加红
张晖
吴毓渊
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南通联亚药业股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the invention relates to the field of chemical analysis, in particular to a method for detecting nitrosamine impurities in medicines.
  • the signal-to-noise ratio of the nitrosamine impurities is greater than 10;
  • the nitrosamine impurities include N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitroso-N-ethylisopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodi-n-propylamine (NDPA), N-nitroso-N-methylaniline (NMPA), N-nitrosodibutyl
  • NMBA N-nitroso-N-methyl-4-aminobutyric acid
  • NMBA N-nitroso-N-methyl-4-aminobutyric acid
  • the detection method meets the requirements of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitroso-N-methylaniline (NMPA) simultaneously.
  • NDMA N-nitrosodimethylamine
  • NDEA N-nitrosodiethylamine
  • NMPA N-nitroso-N-methylaniline
  • the signal-to-noise ratio is greater than 10.
  • the nitrosamine impurity is N-nitrosodiethylamine (NDEA), whose signal-to-noise ratio is greater than 14.
  • the nitrosamine impurity is N-nitroso-N-methylaniline (NMPA), and its signal-to-noise ratios are 18.6, 24.1 and 42.9.
  • NMPA N-nitroso-N-methylaniline
  • (1) Prepare respectively 40-120mg/mL drug test solution and 0.5ng/mL-6.5ng/mL nitrosamine impurity reference substance solution, the drug test solution and reference substance in the configuration process
  • the solution is prepared through multiple dilutions, and the diluent is independently selected from any one or more of methanol, ethanol, isopropanol or water, wherein the last diluent used is that the volume ratio of methanol and water is (0.5 -2): a mixed solvent of 1;
  • the test conditions of high-performance liquid chromatography include: mobile phase A is 0.05-1.0vol% formic acid aqueous solution, mobile phase B is 0.05-1.0vol% formic acid methanol solution, and the chromatographic column is Agilent Poroshell 120 SB-AQ, with an inner diameter of 4-5mm, column length 150mm, particle size 2.7-4 ⁇ m, flow rate 0.4-0.6mL/min, injection volume 15-25 ⁇ L, column temperature 35-45°C, sample chamber temperature 5-15°C, The running time is 32-40min;
  • the test conditions of triple quadrupole tandem mass spectrometry include: 0-5.4 minutes mobile phase waste, ion source is APCI, polarity is positive, drying gas temperature is 200-300°C, evaporation chamber temperature is 300-400°C, The drying gas flow rate is 3-5L/min, the atomizing gas pressure is 30-40psi, the capillary voltage is 1400-1600V, the corona needle current is 5-7 ⁇ A, the monitoring mode is multiple reaction monitoring, and the chromatogram type is MRM.
  • nitrosamine impurities NDIPA and NDPA are isomers of each other, and it is difficult to separate them by conventional methods.
  • the separation of nitrosamine impurities NDIPA and NDPA is realized by continuously optimizing the test conditions of high performance liquid chromatography, and the separation degree is >1.5, which can accurately detect the content of nitrosamine impurities NDIPA and NDPA in medicine.
  • the selection of the triple quadrupole tandem mass spectrometer ion source detector has a great influence on the detection effect.
  • the ESI electrospray ionization source
  • the expected detection cannot be achieved.
  • APCI atmospheric pressure chemical ionization source
  • the multi-reaction monitoring mode selects data in a targeted manner for mass spectrometry signal acquisition, performs signal recording on ions that conform to the rules, and removes the interference of ion signals that do not conform to the rules. Therefore, the present invention selects the multi-reaction monitoring mode to improve detection Sensitivity and quantitative accuracy.
  • the diluent is independently selected from any one or more of methanol, ethanol, isopropanol or water, wherein the diluent used for the last time is that the volume ratio of methanol and water is (0.5-2 ): 1 mixed solvent;
  • step (2) The drug test solution obtained in step (1) and the nitrosamine impurity reference solution are respectively injected into a high-performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and the drug test solution is calculated by the external standard method.
  • the testing conditions of high performance liquid chromatography include: mobile phase A is 0.1vol% formic acid aqueous solution, mobile phase B is 0.1vol% formic acid methanol solution, chromatographic column is Agilent Poroshell 120 SB-AQ, internal diameter is 4-5mm, The column length is 150mm, the particle size is 2.7-4 ⁇ m, the flow rate is 0.5mL/min, the injection volume is 20 ⁇ L, the column temperature is 40°C, the sample chamber temperature is 10°C, and the running time is 35min;
  • the detection method includes the following steps:
  • the diluent is independently selected from any one or more of methanol, ethanol, isopropanol or water, wherein the diluent used for the last time is that the volume ratio of methanol and water is (0.5-2 ): 1 mixed solvent;
  • step (2) The drug test solution obtained in step (1) and the nitrosamine impurity reference solution are respectively injected into a high-performance liquid chromatography-triple quadrupole tandem mass spectrometer for detection, and the drug test solution is calculated by the external standard method.
  • Adopt gradient program to carry out elution in the detection of described high performance liquid chromatography, described gradient program is:
  • the test conditions of triple quadrupole tandem mass spectrometry include: 0-5.4 minutes mobile phase waste, ion source is APCI, polarity is positive, drying gas temperature is 250°C, evaporation chamber temperature is 350°C, drying gas flow rate is 4L/min, the nebulizer gas pressure is 35psi, the capillary voltage is 1500V, the corona needle current is 6 ⁇ A, the monitoring mode is multiple reaction monitoring, and the chromatogram type is MRM.
  • Metformin hydrochloride need testing solution of 80mg/mL and 0.5ng/mL-6.5ng/mL nitrosamine impurity reference substance solution are prepared respectively;
  • Said medicine need test solution and reference substance solution in configuration process Prepared through multiple dilutions, the diluent is independently selected from any one or more of methanol, ethanol, isopropanol or water, wherein the last diluent used is that the volume ratio of methanol and water is (0.5- 2): a mixed solvent of 1;
  • step (1) obtains and the nitrosamine impurity reference substance solution inject high performance liquid chromatography-triple quadrupole tandem mass spectrometer respectively and detect, and adopt external standard method to calculate metformin hydrochloride The content of nitrosamine impurities in the test sample;
  • the testing conditions of high performance liquid chromatography include: mobile phase A is 0.1vol% formic acid aqueous solution, mobile phase B is 0.1vol% formic acid methanol solution, chromatographic column is Agilent Poroshell 120 SB-AQ, internal diameter is 4-5mm, The column length is 150mm, the particle size is 2.7-4 ⁇ m, the flow rate is 0.5mL/min, the injection volume is 20 ⁇ L, the column temperature is 40°C, the sample chamber temperature is 10°C, and the running time is 35min;
  • the test conditions of triple quadrupole tandem mass spectrometry include: 0-5.4 minutes mobile phase waste, ion source is APCI, polarity is positive, drying gas temperature is 300°C, evaporation chamber temperature is 350°C, drying gas flow rate is 4L/min, the nebulizer gas pressure is 35psi, the capillary voltage is 1500V, the corona needle current is 6 ⁇ A, the monitoring mode is multiple reaction monitoring, and the chromatogram type is MRM.
  • the drug is a pharmaceutically acceptable salt that is easily soluble in water, and the retention time under chromatographic conditions is less than or equal to 5.4 minutes.
  • the drug is a pharmaceutically acceptable salt of metformin and its intermediate or preparation.
  • the drug is metformin hydrochloride and its intermediates or preparations.
  • the term "greater than” includes the prime number. For example, if the signal-to-noise ratio is greater than 10, it means that the number 10 is included in addition to the value greater than 10.
  • the method for analyzing and measuring nitrosamine impurities in medicine provided by the present invention, by optimizing the conditions of high performance liquid chromatography and triple quadrupole tandem mass spectrometry, all eight kinds of nitrosamine impurities are effectively separated in the chromatogram, Moreover, the signal-to-noise ratio of nitrosamine impurities is high, and has the beneficial effects of high sensitivity, strong specificity, rapid analysis and strong anti-interference.
  • the separation of nitrosamine impurities NDIPA and NDPA is realized, and the separation degree is >1.5, and the content of nitrosamine impurities NDIPA and NDPA in the drug can be accurately detected.
  • the present invention plays an important role in formulating and improving the quality standards of drugs containing nitrosamine impurities, and also provides a reference for research on nitrosamine impurities.
  • Fig. 1 the MRM collection of illustrative plates of impurity NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, NDPA and NDBA reference substance solution in embodiment 1;
  • Fig. 2 the MRM collection of illustrative plates of impurity NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, NDPA and NDBA reference substance solution in embodiment 2;
  • Fig. 3 the MRM collection of illustrative plates of impurity NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, NDPA and NDBA reference substance solution in embodiment 3;
  • Fig. 4 The MRM spectrum of impurity NDMA, NMBA, NDEA, NEIPA, NDIPA, NMPA, NDPA and NDBA reference substance solution in comparative example 1;
  • Fig. 5 the MRM spectrum of impurity NDMA reference substance solution in comparative example 2;
  • Fig. 6 MRM spectra of impurities NMBA, NDEA, NEIPA, NDIPA, NMPA, NDPA and NDBA reference substance solution in Comparative Example 2.
  • a method for detecting nitrosamine impurities in medicine comprising the following steps:
  • Metformin hydrochloride test solution of 80mg/mL and 0.5ng/mL-6.5ng/mL of nitrosamine impurities reference substance solution were prepared respectively;
  • metformin hydrochloride sustained-release tablet need testing solution: get metformin hydrochloride sustained-release tablet appropriate amount, grind into powder, accurately weigh powder appropriate amount, put in the centrifuge tube, accurately measure methanol appropriate amount, put in the centrifuge tube, Vortex, shake well, centrifuge, then take the supernatant to dilute and filter to make a test solution with a concentration of 80mg/mL;
  • step (1) obtains and the nitrosamine impurity reference substance solution inject high performance liquid chromatography-triple quadrupole tandem mass spectrometer respectively and detect, and adopt external standard method to calculate metformin hydrochloride The content of nitrosamine impurities in the test sample;
  • the test conditions of high-performance liquid chromatography include: mobile phase A is 0.1vol% formic acid aqueous solution, mobile phase B is 0.1vol% formic acid methanol solution, and the chromatographic column is Agilent Poroshell 120 SB-AQ, with an inner diameter of 4-5mm and a column length of The particle size is 150mm, the particle size is 2.7-4 ⁇ m, the flow rate is 0.5mL/min, the injection volume is 20 ⁇ L, the column temperature is 40°C, the sample chamber temperature is 10°C, and the running time is 35min;
  • Adopt gradient program to carry out elution in the detection of described high performance liquid chromatography, described gradient program is:
  • the test conditions of triple quadrupole tandem mass spectrometry include: 0-5.4 minutes mobile phase waste, ion source is APCI, polarity is positive, drying gas temperature is 350°C, drying gas flow rate is 11L/min, atomizing gas pressure is 50psi, the capillary voltage is 6000V, the monitoring mode is multiple reaction monitoring, and the chromatogram type is MRM.
  • a method for detecting nitrosamine impurities in medicines The specific steps are the same as in Comparative Example 1, except that the mobile phase A is 1 vol% formic acid aqueous solution, and the mobile phase B is 1 vol% formic acid methanol solution.
  • a method for detecting nitrosamine impurities in medicines the specific steps are the same as those in Comparative Example 1, the difference is: triple quadrupole tandem mass spectrometry: the drying gas temperature is 300°C.
  • Example 3 See Table 3 for the signal-to-noise ratio of each impurity chromatographic peak in Example 3, and see Figure 3 for the MRM spectrum.
  • Example 3 except that the drying gas temperature is different from that of Example 1, other test conditions are the same as those of Example 1. It can be seen from Figure 3 and Table 3 that the signal-to-noise ratio range of each impurity chromatographic peak is 14.9-252.4, which is not as good as in Example 1, but the expected detection effect can be achieved.
  • a method for detecting nitrosamine impurities in medicines The specific steps are the same as in Example 1, except that the detector of the triple quadrupole tandem mass spectrometer is an ESI ion source.
  • Comparative Example 1 can only detect NMBA, and the remaining seven impurities have no response; in Comparative Example 2, the signal-to-noise ratio of NDMA, NDEA and NMPA chromatographic peaks is low, the response is poor, and the requirement of quantitative limit cannot be reached; Example 1 In -3, the signal-to-noise ratio of NDMA, NDEA and NMPA chromatographic peaks is high, the response is good, the sensitivity is high, and the specificity is strong, which can meet the requirements of detection; the signal-to-noise ratio of the chromatographic peaks of embodiment 1 is the best, and the response is the best. The comparison of detection methods is shown in Table 6 and Table 7.
  • Table 8 The separation degree of NDIPA and NDPA in embodiment 1, embodiment 2, embodiment 3, comparative example 1, comparative example 2
  • Each nitrosamine impurity is within the corresponding concentration range, and the concentration to be measured has a good linear relationship with the peak area, and r is greater than or equal to 0.99. See Table 10 for the linearity and range results of each impurity.
  • test solution of related substances was placed at room temperature for one day and then sampled for detection.
  • the detected amount of each impurity had no significant change, and no new impurities were detected, indicating that the product was placed at room temperature for one day and the solution had good stability.
  • the detected amount of impurities and the number of detected impurities have no obvious changes.
  • Example 4 the impurity reference substance was used for complete methodological verification, and nitrosamine impurities could be accurately and effectively detected.
  • the detection sensitivity of the invention is high, which matches the limit of nitrosamine impurities; the linear relationship is good; the precision and repeatability are good; the recovery rate and accuracy are good; the solution stability is good; and the durability is good.
  • the detection method provided by the present invention has high sensitivity, strong specificity, rapid analysis, strong anti-interference ability, and can accurately determine potential genotoxic impurities in drugs, which is an improvement in the process of drug raw materials and preparations.

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Abstract

本发明提供一种药物中亚硝胺类杂质的检测方法，使用高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量。本发明能够同时检测8种亚硝胺类杂质，8种亚硝胺类杂质的信噪比均大于10，并且N-亚硝基二异丙基胺和其同分异构体N-亚硝基二正丙基胺的分离度大于1.5，该方法具有灵敏度高，专属性强，分析快速，抗干扰性强的优点，尤其适用于盐酸二甲双胍亚硝胺类杂质的检测。

Description

一种药物中亚硝胺类杂质的检测方法 技术领域
本发明涉及化学分析领域，特别涉及一种药物中亚硝胺类杂质的检测方法。
背景技术
亚硝胺类化合物被国际癌症研究组织判定为2A类致癌物。亚硝胺类杂质属于ICH M7(R1)(《评估和控制药物中DNA反应性(致突变)杂质以限制潜在致癌风险》)指南中提及的“关注队列”物质。N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)、N-亚硝基-N-乙基异丙基胺(NEIPA)、N-亚硝基二异丙基胺(NDIPA)、N-亚硝基二正丙基胺(NDPA)、N-亚硝基-N-甲基苯胺(NMPA)、N-亚硝基二丁基胺(NDBA)和N-亚硝基-N-甲基-4-氨基丁酸(NMBA)是药物中较为常见的亚硝胺类杂质，一定程度上会影响药物的药效和用药安全性。自2018年7月在缬沙坦原料药中检出了亚硝胺类杂质以来，国内外多家药企都曾因在其他沙坦类的药物和非沙坦类的药物(如雷尼替丁、二甲双胍等)中检出超限亚硝胺类杂质而启动召回。
2020年1月6日，国家药品监督管理局组织起草了《化学药物中亚硝胺类杂质研究技术指导原则(征求意见稿)》，要求对于明确有亚硝胺类杂质残留风险的品种应建立合适的分析方法，确保成品中亚硝胺类杂质低于限度要求。亚硝胺类杂质有多种产生原因，如工艺产生、降解途径和污染引入等。由于亚硝胺类杂质在人体中可接受限度较小，微量杂质的检测和控制难度大。因此，为了更好地检测药物中亚硝胺类杂质 的质量，有必要提供一种检测方法，以准确检测亚硝酸胺类杂质的含量，从而保证用药的安全性。
发明内容
为了解决上述技术问题，本发明提供了一种药物中亚硝胺类杂质的检测方法，具体技术方案如下：
(1)分别配制药物供试品溶液和亚硝胺类杂质对照品溶液；
(2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
所述亚硝胺类杂质的信噪比大于10；
所述亚硝胺类杂质包括N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)、N-亚硝基-N-乙基异丙基胺(NEIPA)、N-亚硝基二异丙基胺(NDIPA)、N-亚硝基二正丙基胺(NDPA)、N-亚硝基-N-甲基苯胺(NMPA)、N-亚硝基二丁基胺(NDBA)和N-亚硝基-N-甲基-4-氨基丁酸(NMBA)中的一种或数种。
在本发明中，所述药物供试品包括但不限于药物原料药、药物中间体和药物制剂。
需要说明的是，所述外标法是所属领域的一种公知的定量方法，本发明在此不进行限定。
作为本发明的优选方案，该检测方法同时满足N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)和N-亚硝基-N-甲基苯胺(NMPA)的信噪比大于10。
作为本发明的优选方案，所述亚硝胺类杂质为N-亚硝基二甲胺(NDMA)，其信噪比大于13。
作为本发明的优选方案，所述亚硝胺类杂质为N-亚硝基二甲胺(NDMA)，其信噪比为13.7、14.9和32.7。
作为本发明的优选方案，，所述亚硝胺类杂质为N-亚硝基二乙胺(NDEA)，其信噪比大于14。
作为本发明的优选方案，所述亚硝胺类杂质为N-亚硝基二乙胺(NDEA)，其信噪比为14.3、15.2和20.7。
作为本发明的优选方案，所述亚硝胺类杂质为N-亚硝基-N-甲基苯胺(NMPA)，其信噪比大于18。
作为本发明的优选方案，所述亚硝胺类杂质为N-亚硝基-N-甲基苯胺(NMPA)，其信噪比为18.6、24.1和42.9。
需要说明的是，所述信噪比(Signal to Noise Ratio，SNR)是特定参数(信号)值与非特异性参数(噪声)的比值。本发明中，选用高效液相色谱-三重四级杆串联质谱仪进行检测，在亚硝胺类杂质对照品溶液浓度相同和进样体积相同的条件下，信噪比越高，说明检测方法灵敏度越高。
作为本发明的优选方案，所述检测方法包括以下步骤：
(1)分别配制40-120mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液，在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
(2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
其中，高效液相色谱的测试条件包括：流动相A为0.05-1.0vol％的甲酸水溶液，流动相B为0.05-1.0vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为 0.4-0.6mL/min，进样体积为15-25μL，柱温为35-45℃，样品室温度为5-15℃，运行时间为32-40min；
所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为200-300℃，蒸发室温度为300-400℃，干燥气流量为3-5L/min，雾化气压力为30-40psi，毛细管电压为1400-1600V，电晕针电流为5-7μA，监测模式为多反应监测，色谱图类型为MRM。
在本发明中，色谱柱的选用对检测效果的影响很大，极性很高的药物，在常规反向色谱系统中无保留。为提高色谱系统的适用性和耐用性，本发明选择亲水型反相色谱柱Agilent Poroshell 120 SB-AQ，起始采用98％水相洗脱，实现了极性很高的药物与八种亚硝胺类杂质的分离效果。
亚硝胺类杂质NDIPA和NDPA互为同分异构体，常规方法难以实现分离。在本发明通过不断优化高效液相色谱的测试条件，实现了亚硝胺类杂质NDIPA和NDPA的分离，其分离度>1.5，可以准确检测药物中亚硝胺类杂质NDIPA和NDPA的含量。
在本发明中，采用梯度洗脱方式，提高了杂质在相同浓度和相同进样体积条件下的信噪比，进而提高了检测灵敏度和定量准确性。
在本发明中，三重四级杆串联质谱离子源检测器的选用对检测效果的影响很大，本发明在检测时，使用ESI(电喷雾电离源)离子源作为检测器，无法达到预期的检测效果；使用APCI(大气压化学电离源)离子源作为检测器，可以达到预期的检测效果。
在本发明中，多反应监测模式有针对性地选择数据进行质谱信号采集，对符合规则的离子进行信号记录，去除不符合规则离子信号的干扰，因此，本发明选择多反应监测模式，提高检测灵敏度和定量准确性。
作为本发明的优选方案，所述检测方法包括以下步骤：
(1)分别配制80mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
(2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
其中，高效液相色谱的测试条件包括：流动相A为0.1vol％的甲酸水溶液，流动相B为0.1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为250℃，蒸发室温度为350℃，干燥气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
作为本发明的优选方案，所述检测方法包括以下步骤：
(1)分别配制80mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一 种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
(2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
其中，高效液相色谱的测试条件包括：流动相A为1vol％的甲酸水溶液，流动相B为1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为250℃，蒸发室温度为350℃，干燥气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
作为本发明的优选方案，所述检测方法包括以下步骤：
(1)分别配制80mg/mL的盐酸二甲双胍供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
(2)将步骤(1)得到的盐酸二甲双胍供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算盐酸二甲双胍供试品中亚硝胺类杂质的含量；
其中，高效液相色谱的测试条件包括：流动相A为0.1vol％的甲酸水溶液，流动相B为0.1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为300℃，蒸发室温度为350℃，干燥气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
在优选的方案中，步骤(1)中前两次使用的稀释溶剂优选甲醇，最后一次使用的稀释剂优选甲醇和水的体积比为1:1的混合溶剂；
作为本发明的优选方案，所述药物为易溶于水的药学上可接受的盐，在色谱条件下的保留时间小于等于5.4分钟。
作为本发明的优选方案，所述药物为二甲双胍可药用盐及其中间体或制剂。
作为本发明的优选方案，所述药物为盐酸二甲双胍及其中间体或制剂。
在本发明中，术语“大于”包括本数。例如信噪比大于10，表示除了大于10的数值之外还包括本数10。
有益效果
1.本发明提供的分析测定药物中亚硝胺类杂质的方法，通过优化高效液相色谱和三重四级杆串联质谱的条件，在色谱图中将八种亚硝胺类杂质全部有效分离，且亚硝胺类杂质的信噪比高，具有灵敏度高、专属性强，分析快速，抗干扰性强的有益效果。
2.在本发明通过不断优化高效液相色谱的测试条件，实现亚硝胺类杂质NDIPA和NDPA的分离，分离度>1.5，可以准确检测药物中亚硝胺类杂质NDIPA和NDPA的含量。
3.本发明对于含有亚硝胺类杂质的药物质量标准的制定和提升具有重要的作用，也提供了亚硝胺类杂质研究的参考依据。
附图说明
图1：实施例1中杂质NDMA、NMBA、NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品溶液的MRM图谱；
图2：实施例2中杂质NDMA、NMBA、NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品溶液的MRM图谱；
图3：实施例3中杂质NDMA、NMBA、NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品溶液的MRM图谱；
图4：对比例1中杂质NDMA、NMBA、NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品溶液的MRM图谱；
图5：对比例2中杂质NDMA对照品溶液的MRM图谱；
图6：对比例2中杂质NMBA、NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品溶液的MRM图谱。
具体实施方式
下面通过具体实施方式进一步说明本发明。应当理解为，本发明的实施例仅仅是用于说明本发明，而不是对本发明的限制。在本发明技术 方案的基础上对本发明的简单改进或者采用惯用手段或成分进行等同替换所得得到的技术方案均属于本发明的保护范围。
实施例1
一种药物中亚硝胺类杂质的检测方法，包括以下步骤：
(1)分别配制80mg/mL的盐酸二甲双胍供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；
1)80mg/mL盐酸二甲双胍原料药供试品溶液的配制方法：取盐酸二甲双胍适量，精密称定，置离心管中，精密量取甲醇适量，置离心管中，涡旋，摇匀，离心，然后取上清液稀释，过滤，制成浓度为80mg/mL的供试品溶液；
80mg/mL盐酸二甲双胍缓释片供试品溶液的配制方法：取盐酸二甲双胍缓释片适量，研磨成粉末，精密称定粉末适量，置离心管中，精密量取甲醇适量，置离心管中，涡旋，摇匀，离心，然后取上清液稀释，过滤，制成浓度为80mg/mL的供试品溶液；
2)0.5ng/mL-6.5ng/mL亚硝胺类杂质对照品溶液的配制方法：取NDMA和NMBA对照品各1.9mg，精密称定，置100mL量瓶中，加入甲醇稀释至刻度，作为对照品溶液母液-1；取NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA对照品各1.0mg，精密称定，置100mL量瓶中，加入甲醇稀释至刻度，作为对照品溶液母液-2；精密量取对照品溶液母液-1 4.0mL和对照品溶液母液-2 2.0mL，置200mL量瓶中，加入甲醇稀释至刻度，作为对照品溶液母液-3；精密量取对照品溶液母液-3 2.0mL，置200mL量瓶中，加入稀释剂稀释至刻度，作为供试品溶液，其中NDMA和NMBA的浓度为3.8ng/mL，NDEA、NEIPA、NDIPA、NMPA、NDPA和NDBA的浓度为1.0ng/mL。
(2)将步骤(1)得到的盐酸二甲双胍供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并 采用外标法计算盐酸二甲双胍供试品中亚硝胺类杂质的含量；
高效液相色谱的测试条件包括：流动相A为0.1vol％的甲酸水溶液，流动相B为0.1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为350℃，干燥气流量为11L/min，雾化气压力为50psi，毛细管电压为6000V，监测模式为多反应监测，色谱图类型为MRM。
结论：实施例1各杂质色谱峰的信噪比参见表1，MRM图谱参见图1。检测结果显示，各杂质色谱峰的信噪比范围是20.7-198.0，所述检测方法灵敏度高，专属性强，可以达到预期的检测效果。
表1 实施例1各杂质色谱峰的信噪比
Figure PCTCN2021136130-appb-000001
实施例2
一种药物中亚硝胺类杂质的检测方法，具体步骤同对比例1，不同点在于：流动相A为1vol％的甲酸水溶液，流动相B为1vol％的甲酸甲醇溶液。
结论：实施例2各杂质色谱峰的信噪比参见表2，MRM图谱参见图2。实施例2中，除流动相A和流动相B与实施例1不同，其他测试条件均与实施例1相同。由图2和表2可见，各杂质色谱峰的信噪比范围是13.7-213.7，可以达到预期的检测效果。
表2 实施例2各杂质色谱峰的信噪比
Figure PCTCN2021136130-appb-000002
实施例3
一种药物中亚硝胺类杂质的检测方法，具体步骤同对比例1，不同点在于：三重四级杆串联质谱：干燥气温度300℃。
结论：实施例3各杂质色谱峰的信噪比参见表3，MRM图谱参见图3。实施例3中，除干燥气温度与实施例1不同，其他测试条件均与实施例1相同。由图3和表3可见，各杂质色谱峰的信噪比范围是14.9-252.4，没有实施例1好，但是可以达到预期的检测效果。
表3 实施例3各杂质色谱峰的信噪比
Figure PCTCN2021136130-appb-000003
对比例1
一种药物中亚硝胺类杂质的检测方法，具体步骤同实施例1，不同点在于：三重四级杆串联质谱的检测器为ESI离子源。
结论：对比例1中，由图4和表4可见，分析同样浓度的对照品溶液，仅能检测到NMBA，其余七个杂质均无响应。因此，ESI离子源检测器无法检测出全部亚硝胺类杂质，没有达到预期的检测效果。
表4 对比例1各杂质色谱峰的信噪比
Figure PCTCN2021136130-appb-000004
对比例2
一种药物中亚硝胺类杂质的检测方法，具体步骤同对比例1，不同点在于：梯度洗脱程序为：
0-15min，2vol％-65vol％流动相B；15-30min，65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
结论：对比例2中，由图5、图6和表5可见，在此高效液相色谱的检测条件下，APCI离子源检测器可以检测出全部亚硝胺类杂质。但是各杂质色谱峰的信噪比范围是2.9-162.8，色谱峰的响应不佳，没有达到预期的检测效果。
表5 对比例2各杂质色谱峰的信噪比
Figure PCTCN2021136130-appb-000005
综上所述，实施例1-3和对比例1-2的分析如下：
对比例1仅能检测到NMBA，其余七个杂质均无响应；对比例2中，NDMA、NDEA和NMPA色谱峰的信噪比低，响应较差，达不到定量限的要求；实施例1-3中，NDMA、NDEA和NMPA色谱峰的信噪比高，响应较好，灵敏度高，专属性强，可以达到检测的要求；实施例1色谱峰的信噪比最佳，响应最好。检测方法对比见表6和表7。
表6：实施例2、实施例3、对比例1、对比例2与实施例1测试方法的对比
Figure PCTCN2021136130-appb-000006
Figure PCTCN2021136130-appb-000007
表7：实施例1、实施例2、实施例3、对比例1、对比例2中各杂质色谱峰的信噪比对比
Figure PCTCN2021136130-appb-000008
表8：实施例1、实施例2、实施例3、对比例1、对比例2中NDIPA与NDPA的分离度
Figure PCTCN2021136130-appb-000009
实施例4
对实施例1检测方法进行方法学验证，0.5ng/mL-6.5ng/mL亚硝胺类杂质对照品溶液的配制方法、高效液相色谱和三重四级杆串联质谱的测试条件均与实施例1相同。
1、检测限与定量限
各亚硝胺类杂质的检测限和定量限见表9。
表9 检测限和定量限结果
Figure PCTCN2021136130-appb-000010
2、线性与范围
各亚硝胺类杂质在相应的浓度范围内，待测的浓度与峰面积呈良好的线性关系，r均大于等于0.99。各杂质的线性及范围结果参见表10。
表10 线性与范围结果
杂质 浓度范围 线性方程 r
NDMA 0.00082449-0.0065960μg/mL y＝2.1021E+06x+28 1.00
NMBA 0.00036348-0.0058158μg/mL y＝5.6840E+05x+23 1.00
NDEA 0.00040699-0.0016280μg/mL y＝5.4840E+05x+43 0.99
NEIPA 0.00010220-0.0016353μg/mL y＝2.1173E+06x+26 1.00
NDIPA 0.00010062-0.0016099μg/mL y＝1.0001E+06x+2 1.00
NMPA 0.00042041-0.0016817μg/mL y＝1.2535E+05x+6 1.00
NDPA 0.00010809-0.0017294μg/mL y＝7.3275E+05x-7 1.00
NDBA 0.00021409-0.0017127μg/mL y＝9.9689E+05x+11 1.00
3、精密度与重复性良好
重复性：同一批次样品，重复测定6次的测得结果无明显差异，表明本法重复性良好。
4、回收率与准确度良好
本方法测得本品中各杂质的三个浓度(限度浓度的50％、100％、150％的溶液)，12份样品的回收率均在83.5％～107.7％范围，且其回收率RSD均小于13.2％。表明检测结果符合验证要求，本方法的准确度良好。
5、溶液稳定性良好
有关物质供试品溶液在室温条件下放置一天后进样检测，各杂质检出量无明显变化，且未检出新的杂质，说明本品在室温下放置一天，溶液稳定性良好。
6、耐用性较好
本方法经改变柱温、流速、有机相比例、改变不同厂家色谱柱后， 杂质的检出量及检出的杂质个数均无明显变化。
系统适用性溶液中，杂质与杂质、杂质与主成分之间的分离度良好，耐用性较好。
结论：实施例4中，采用杂质对照品进行完整的方法学验证，亚硝胺类杂质能够准确有效的检出。本发明检测灵敏度高，与亚硝胺类杂质限度相匹配；线性关系良好；精密度和重复性良好；回收率与准确度良好；溶液稳定性良好；耐用性较好。
由上述各实施例可以看出，本发明提供的检测方法，灵敏度高，专属性强，分析快速，抗干扰性强，可准确测定药物中的潜在基因毒性杂质，是药物原料药及制剂工艺改进与创新及质量标准的进一步完善提升的前瞻性方法。

Claims (10)

  1. 一种药物中亚硝胺类杂质的检测方法，其特征在于，所述检测方法包括以下步骤：
    (1)分别配制药物供试品溶液和亚硝胺类杂质对照品溶液；
    (2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
    所述亚硝胺类杂质的信噪比大于10；
    所述亚硝胺类杂质包括N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)、N-亚硝基-N-乙基异丙基胺(NEIPA)、N-亚硝基二异丙基胺(NDIPA)、N-亚硝基二正丙基胺(NDPA)、N-亚硝基-N-甲基苯胺(NMPA)、N-亚硝基二丁基胺(NDBA)和N-亚硝基-N-甲基-4-氨基丁酸(NMBA)中的一种或数种。
  2. 根据权利要求1所述的检测方法，其特征在于，该检测方法同时满足N-亚硝基二甲胺(NDMA)、N-亚硝基二乙胺(NDEA)和N-亚硝基-N-甲基苯胺(NMPA)的信噪比大于10。
  3. 根据权利要求1或2所述的检测方法，其特征在于，所述检测方法包括以下步骤：
    (1)分别配制40-120mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液，在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
    (2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
    其中，高效液相色谱的测试条件包括：流动相A为0.05-1.0vol％的甲酸水溶液，流动相B为0.05-1.0vol％的甲酸甲醇溶液，色谱柱为Agilent Poros hell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.4-0.6mL/min，进样体积为15-25μL，柱温为35-45℃，样品室温度为5-15℃，运行时间为32-40min；
    所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
    其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为200-300℃，蒸发室温度为300-400℃，干燥气流量为3-5L/min，雾化气压力为30-40psi，毛细管电压为1400-1600V，电晕针电流为5-7μA，监测模式为多反应监测，色谱图类型为MRM。
  4. 根据权利要求1-2任意一项所述的检测方法，其特征在于，所述检测方法包括以下步骤：
    (1)分别配制80mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
    (2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
    其中，高效液相色谱的测试条件包括：流动相A为0.1vol％的甲酸水溶液，流动相B为0.1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
    所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
    0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
    其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为250℃，蒸发室温度为350℃，干燥气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
  5. 根据权利要求1-2任意一项所述的检测方法，其特征在于，所述检测方法包括以下步骤：
    (1)分别配制80mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
    (2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
    其中，高效液相色谱的测试条件包括：流动相A为1vol％的甲酸水溶液，流动相B为1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
    所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
    0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
    其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为250℃，蒸发室温度为350℃，干燥气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
  6. 根据权利要求1-2任意一项所述的检测方法，其特征在于，
    (1)分别配制80mg/mL的药物供试品溶液和0.5ng/mL-6.5ng/mL的亚硝胺类杂质对照品溶液；在配置过程中所述药物供试品溶液和对照品溶液经多次稀释制备，所述稀释剂独立地选自甲醇、乙醇、异丙醇或水中的任意一种或多种的，其中最后一次使用的稀释剂为甲醇和水的体积比为(0.5-2):1的混合溶剂；
    (2)将步骤(1)得到的药物供试品溶液和亚硝胺类杂质对照品溶液分别注入高效液相色谱-三重四级杆串联质谱仪进行检测，并采用外标法计算药物供试品中亚硝胺类杂质的含量；
    其中，高效液相色谱的测试条件包括：流动相A为0.1vol％的甲酸水溶液，流动相B为0.1vol％的甲酸甲醇溶液，色谱柱为Agilent Poroshell 120 SB-AQ，内径为4-5mm，柱长为150mm，粒径为2.7-4μm，流速为0.5mL/min，进样体积为20μL，柱温为40℃，样品室温度为10℃，运行时间为35min；
    所述高效液相色谱的检测中采用梯度程序进行洗脱，所述梯度程序为：
    0-15min，2vol％-50vol％流动相B；15-30min，50vol％-65vol％流动相B；30-31min，65vol％-2vol％流动相B；31-35min，2vol％流动相B；
    其中，三重四级杆串联质谱的测试条件包括：0-5.4分钟流动相排废，离子源为APCI，极性为正，干燥气温度为300℃，蒸发室温度为350℃，干燥 气流量为4L/min，雾化气压力为35psi，毛细管电压为1500V，电晕针电流为6μA，监测模式为多反应监测，色谱图类型为MRM。
  7. 根据权利要求1-6中任意一项所述的检测方法，高效液相色谱-三重四级杆串联质谱仪为Agilent 1260高效液相色谱或Agilent 6410三重串联四极杆质谱液质联用质谱仪。
  8. 根据权利要求1-7中任意一项所述的检测方法，其特征在于，所述药物为易溶于水的药学上可接受的盐，在色谱条件下的保留时间小于等于5.4分钟。
  9. 根据权利要求1-8中任意一项所述的检测方法，其特征在于，所述药物为二甲双胍可药用盐及其中间体或制剂。
  10. 根据权利要求1-8中任意一项所述的检测方法，其特征在于，所述药物为盐酸二甲双胍及其中间体或制剂。
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