WO2023124923A1 - 一种马来酸甲麦角新碱原料中成盐率的测定方法 - Google Patents

一种马来酸甲麦角新碱原料中成盐率的测定方法 Download PDF

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WO2023124923A1
WO2023124923A1 PCT/CN2022/138247 CN2022138247W WO2023124923A1 WO 2023124923 A1 WO2023124923 A1 WO 2023124923A1 CN 2022138247 W CN2022138247 W CN 2022138247W WO 2023124923 A1 WO2023124923 A1 WO 2023124923A1
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solution
raw material
maleate
reference substance
maleic acid
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PCT/CN2022/138247
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English (en)
French (fr)
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高燕霞
韩彬
王茉莉
许峰
徐艳梅
周玉岩
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河北省药品医疗器械检验研究院(河北省化妆品检验研究中心)
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Publication of WO2023124923A1 publication Critical patent/WO2023124923A1/zh

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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/64—Electrical detectors
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • G01N2030/047—Standards external

Definitions

  • the invention belongs to the field of drug analysis and detection, in particular to a method for determining the salt-forming rate in methysergide maleate raw materials.
  • Ergot alkaloids are a class of natural alkaloids and their series of derivatives.
  • the basic structure is lysergic acid, which can act on receptors such as serotonin, norepinephrine, and dopamine. It has the functions of stimulating the uterus, constricting blood vessels, and blocking alpha receptors. It can be used clinically to treat various diseases such as uterine bleeding, postpartum uterine recovery, migraine, inhibition of the central nervous system, and cardiovascular and cerebrovascular diseases.
  • the "gold standard" recognized by the world (WHO) - ergot alkaloids, whose mechanism of action is not affected by physiological factors, is the drug of choice for the prevention and treatment of postpartum hemorrhage in pregnant women.
  • Enterprises in our province undertake the national major new drug creation project, take the lead in developing the raw materials and preparations of ergot alkaloid drugs, and for the first time imitate the raw materials of ergonovine maleate, which fills the lack of new treatment methods in the field of postpartum hemorrhage in my country for many years It has very important clinical significance to our country.
  • Methergonovine maleate is a bio-fermentation alkaloid, which is the maleate of methysergide.
  • the fermentation liquid contains many impurities.
  • the production process adopts crystallization to separate and purify. store.
  • the molar ratio of maleic acid to methysergide should be 1:1; Ultrapure water is converted into maleic anhydride.
  • the degradation of impurities can cause renal tubular damage; when methysergide is not completely salted, the free alkaloids specifically bind to plasma proteins, prolonging the onset time of the drug ; At the same time, the salt formation rate has an impact on the acidity, alkalinity, color and related substances of the preparation.
  • the object of the present invention is to above problem, provides a kind of assay method of salinity rate in the methysergide maleate raw material, comprises the following steps:
  • the system suitability solution, the blank solution, the reference substance solution and the test solution were injected into the ion chromatograph respectively, and the chromatogram was recorded, and the maleate content in the methysergide maleate crude drug was calculated by the peak area according to the external standard method.
  • the content of acid is substituted into formula (1) to calculate the salt-forming rate of methysergide maleate raw material;
  • A is the salification rate of methylergide maleate raw material
  • B is the content of maleic acid
  • C is the content of methylergide maleate in the raw material
  • the ion chromatography conditions are:
  • Injection volume 20 ⁇ L
  • Detector conductivity detector
  • Suppressor anion suppressor (ASRS-4mm), suppression current: 50mA;
  • the nitrogen pressure is 27.6kPa.
  • the blank solution is ultrapure ultrapure water.
  • the preparation of system suitability solution get need testing solution and reference substance solution 1:1 mix.
  • eluent concentration 20mmol/L Preferably, during gradient elution: 0-15 minutes, eluent concentration 20mmol/L; 15-22 minutes, eluent concentration 250mmol/L; 22-30 minutes, eluent concentration 20mmol/L.
  • the present invention adopts electric conductance detection-ion chromatographic method, calculates by peak area according to external standard method, measures the content of maleic acid in the methysergide maleate crude drug, and calculates the salt-forming rate.
  • the invention establishes an ion chromatography-conductivity detector method for measuring the content of maleic acid in methysergide maleate, and the method has strong specificity, high sensitivity, simple operation and accurate result.
  • Figure 1 is a typical map of system suitability
  • Fig. 2 is reference substance solution chromatogram
  • Fig. 3 is need testing solution chromatogram
  • Fig. 4 is a linear relation diagram of maleic acid.
  • Milli-Q ultrapure ultrapure water system (18.2M' ⁇ /cm3, 25°C, Millipore, USA);
  • Mettler Toledo XS 205 electronic analytical balance (Mettler, Switzerland).
  • Reagent sodium hydroxide solution, purchased from Fluka Sigma-ALDRICH company,
  • Purity level dedicated to ion chromatography, concentration: 50% to 52%;
  • Ultrapure ultrapure water is self-made in the laboratory.
  • Reference substance maleic acid reference substance, purchased from China National Institutes for Food and Drug Control,
  • test product raw material of methysergide maleate, collected from a pharmaceutical factory in Hebei,
  • Injection volume 20 ⁇ L
  • Detector conductivity detector
  • Suppressor anion suppressor (ASRS-4mm), suppression current: 50mA;
  • the nitrogen pressure is 27.6kPa.
  • Preparation of reference substance stock solution Accurately weigh 50.12 mg of maleic acid reference substance, put it in a 50mL measuring bottle, dilute to the mark with ultrapure water, shake well, and make a solution containing 1000 ⁇ g of maleic acid per 1 mL, as a reference substance for storage liquid;
  • Preparation of reference substance solution Accurately measure 1 mL of reference substance stock solution, put it in a 100 mL measuring bottle, dilute to the mark with ultrapure water, shake well, and make a solution containing 10 ⁇ g of maleic acid per 1 mL, as the reference substance solution;
  • test solution take by weighing 10 mg of methysergide maleate raw material, accurately weighed, put in a 250mL measuring bottle, add ultrapure water to dissolve and dilute to the scale, shake up, as the test solution;
  • Preparation of system suitability solution Mix 1:1 of the test solution and the reference solution.
  • Preparation of limit of quantification solution gradually dilute the reference substance solution, inject it into the liquid chromatograph, and record the chromatogram until the signal-to-noise ratio s/n is about 10.
  • the quantification limit solution was continuously measured 6 times, and the chromatogram was recorded.
  • the peak area RSD of the six determinations of the limit of quantification of maleic acid is 1.5%
  • the limit of quantification is 18.107 ⁇ g
  • the limit of detection is 6.036 ⁇ g.
  • Preparation of linear solution stock solution Accurately measure 5.0mL of the reference substance stock solution, place it in a 50mL measuring bottle, add ultrapure water to dilute to the mark, shake well, and make a linear solution stock solution.
  • Preparation of a series of linear solutions Precisely measure 0.1mL, 1.0mL, 2.5mL, 5.0mL, 10.0mL, and 15.0mL of each linear solution stock solution, place them in 50mL measuring bottles, add ultrapure water to dilute to the mark, and shake well , to make a series of reference substance solutions. According to the above-mentioned chromatographic conditions, the sample was injected and determined, and the peak area was recorded.
  • test solution Accurately weigh about 10 mg of this product (batch number: 200424), 9 parts in total, divide into 3 groups, put in a 250mL measuring bottle, add the above-mentioned mixed control solution to dissolve and dilute to the mark, shake well. Take the above solution and inject it into an ion chromatograph, and record the chromatogram; the ratio of the measured amount to the added amount is the recovery rate.
  • the flow rate of eluent was 0.8ml/min, 1.0ml/min, 1.2ml/min; the concentration of eluent was 18mmol/L, 20mmol/L, 22mmol/L and the column temperature was 25°C, 30°C, 35°C.
  • the chromatographic conditions of this method have good durability.
  • the chromatographic retention behaviors of the main peak and its adjacent impurity peaks were investigated under the conditions of eluent flow rates of 0.8ml/min, 1.0ml/min, and 1.2ml/min respectively.
  • the results showed that the main peak of maleic acid ion was stable when the chromatographic conditions were slightly changed, and the adjacent impurity peaks could achieve baseline separation.
  • the final eluent flow rate was 1.0ml/min.
  • the chromatographic retention behaviors of the main peak and its adjacent impurity peaks were investigated under the conditions of eluent concentrations of 18mmol/L, 20mmol/L, and 22mmol/L. The results showed that the main peak of maleic acid ion was stable when the chromatographic conditions were changed slightly, and the adjacent impurity peaks could achieve baseline separation. The final eluent concentration was 20mmol/L.
  • the chromatographic retention behavior of the main peak and its adjacent impurity peaks were investigated under the conditions of chromatographic column temperature 25°C, 30°C and 35°C respectively. The results showed that the main peak of maleic acid ion was stable when the chromatographic conditions were slightly changed, and the adjacent impurity peaks could achieve baseline separation.
  • the final column temperature was 30°C.
  • the molecular formula of methysergide is C 20 H 25 N 3 O 2
  • the relative molecular weight is 339.4278
  • the molecular formula of maleic acid is C 4 H 4 O 4
  • the relative molecular weight is 116.0722, which can be calculated to obtain methylergonovine maleate
  • the theoretical maleic acid content is 25.48% when containing 1 molecule of maleic acid.
  • the maleic acid content of metergide maleate is controlled at 80.0% to 120.0% of the theoretical value, then the maleic acid content limit should be 20.4% ⁇ 30.6%.
  • the content of the active ingredient of methysergide maleate is determined. Since the reference substance is calculated according to C20H25N3O2.C4H4O4, the content result measured is the methysergide maleate content.
  • the established method was used to test the collected 3 batches of samples, the content of maleic acid was between 25.18% and 25.33%, the average content was 25.24%, and the salt formation rate was 0.97, which met the limit requirements, suggesting that the production process of the domestic production enterprise Stable, there is no too much free maleic acid in the product, and the quality is safe and controllable.

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Abstract

本发明涉及药物分析检测领域，提供了一种马来酸甲麦角新碱原料中成盐率的测定方法。本发明包括以下步骤：分别将系统适用性溶液、空白溶液、对照品溶液和供试品溶液加注入到离子色谱仪中，记录色谱图，按外标法以峰面积计算马来酸甲麦角新碱原料药中马来酸的含量，代入公式(1)计算得出马来酸甲麦角新碱原料成盐率。本发明方法专属性强，灵敏度高，操作简单，结果准确。

Description

一种马来酸甲麦角新碱原料中成盐率的测定方法 技术领域
本发明属于药物分析检测领域，具体是一种马来酸甲麦角新碱原料中成盐率的测定方法。
背景技术
麦角生物碱是一类天然生物碱及其系列衍生物，基本结构是麦角酸，可以作用于5-羟色胺、去甲肾上腺素以及多巴胺等受体，具有兴奋子宫、收缩血管、阻断α受体等药理作用，临床上可用于治疗子宫出血、产后子宫复原、偏头痛、抑制中枢、心脑血管等多种疾病。
目前，国际(WHO)公认的“金标准”——麦角生物碱，作用机理不受生理因素的影响，是孕产妇预防和治疗产后出血的首选药。我省企业承担国家重大新药创制课题，率先对麦角生物碱类药品的原料及其制剂进行开发，首次仿制出马来酸麦角新碱原料，填补了我国在治疗产后出血领域多年来缺乏新的治疗手段的空白，对我国具有非常重要的临床意义。
马来酸甲麦角新碱是生物发酵类生物碱，是甲麦角新碱的马来酸盐，发酵液中含有较多杂质，生产工艺中采用结晶方式分离、纯化，成盐后性质稳定，易于储存。
查阅文献，英国药典(BP2020)、欧洲药典(EP10.0)及日本药局方(JP17)收载该本品种原料药质量标准，美国药典(USP44)收载了原料药及片剂和注射剂质量标准，对比发现，现行质量标准项目设置无法全面体现生产过程中的产品质量。美国药典和日本药局方收载了马来酸甲麦角新碱的含量测定方法分别是高效液相色谱法(HPLC)和紫外吸光光谱法(UV)，其原理均为检测有紫外吸收官能团的甲麦角新碱响应值，却无法区分是成盐型还是游离型，
当马来酸甲麦角新碱完全成盐时，马来酸与甲麦角新碱的摩尔比应为1:1；当甲麦角新碱成盐过剩的时候，游离的马来酸受热后会脱超纯水成成顺丁烯二酸酐，有实验表明，该降解杂质会导致肾小管损伤；当甲麦角新碱成盐不完全的时候，游离的生物碱特异结合血浆蛋白，延长药物起效时间；同时，成盐率对制剂的酸碱性、颜色及有关物质都有影响。
Figure PCTCN2022138247-appb-000001
马来酸脱水反应
本研究，建立了离子色谱-电导检测器法测定马来酸甲麦角新碱原料中马来酸的含量，同时按照美国药典(USP43)收载的马来酸甲麦角新碱的含量测定方法，测定马来酸甲麦角新碱的含量，通过计算马来酸与马来酸甲麦角新碱的摩尔比，得出马来酸麦角新碱原料中马来酸成盐率，为后续研究提供准确有效的数据，也为生产企业在工艺中控制工业投料稳定性，保障后续产品质量稳定提供技术支持。
发明内容
本发明的目的是针对以上问题，提供了一种马来酸甲麦角新碱原料中成盐率的测定方法，包括以下步骤：
分别将系统适用性溶液、空白溶液、对照品溶液和供试品溶液加注入到离子色谱仪中，记录色谱图，按外标法以峰面积计算马来酸甲麦角新碱原料药中马来酸的含量，代入公式(1)计算得出马来酸甲麦角新碱原料成盐率；
A＝(B/116.0722)/(C/445.5)            (1)
其中，A为马来酸甲麦角新碱原料成盐率，B为马来酸的含量，C为原料 中马来酸甲麦角新碱的含量；
离子色谱条件为：
色谱柱：Dionex IonPac AS19(250mm×4mm，5μm)；
保护柱：Dionex IonPac AG19(50mm×4mm，5μm)；
淋洗液：100mmol/L氢氧化钠溶液；
洗脱方式：梯度洗脱；
淋洗液流速：1.0mL/min；
色谱柱温度：30℃；
检测池温度：35℃；
进样量：20μL；
检测器：电导检测器；
抑制器：阴离子抑制器(ASRS-4mm)，抑制电流：50mA；
氮气压力27.6kPa。
优选的，所述空白溶液为超纯超纯水。
优选的，所述对照品溶液的制备：
(1)精密称取马来酸对照品50.12mg，置50mL量瓶中，用超纯水稀释至刻度，摇匀，制成每1mL含马来酸1000μg溶液，作为对照品贮备液；
(2)精密量取对照品贮备液1mL，置100mL量瓶中，用超纯水稀释至刻度，摇匀，制成每1mL含马来酸10μg溶液，作为对照品溶液。
优选的，置250mL量瓶中，加超纯水溶解并稀释至刻度，摇匀，作为供试品溶液。
优选的，系统适用性溶液的制备：取供试品溶液和对照品溶液1:1混合。
优选的，梯度洗脱时：0～15分钟，淋洗液浓度20mmol/L；15～22分钟，淋洗液浓度250mmol/L；22～30分钟，淋洗液浓度20mmol/L。
与现有技术相比，本发明的有益效果如下：
本发明采用电导检测-离子色谱法，按外标法，以峰面积计算，测定马来酸甲麦角新碱原料药中马来酸的含量，并计算成盐率。本发明建立了离子色谱-电导检测器法测定马来酸甲麦角新碱中马来酸的含量，方法专属性强，灵敏度高，操作简单，结果准确。
附图说明
图1为系统适用性典型图谱；
图2为对照品溶液色谱图；
图3为供试品溶液色谱图；
图4为马来酸线性关系图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
1、仪器与试药
仪器：Dionex ICS 3000离子色谱仪(美国Thermo公司)；
ASRS-4mm抑制器(美国Thermo公司)；
Milli-Q超纯超纯水系统(18.2M′Ω/cm3，25℃，美国Millipore公司)；
Mettler Toledo XS 205型电子分析天平(瑞士Mettler公司)。
试剂：氢氧化钠溶液，购买自Fluka Sigma-ALDRICH公司，
纯度级别：离子色谱专用，浓度：50％～52％；
超纯超纯水为实验室自制。
对照品：马来酸对照品，购买自中国食品药品检定研究院，
批号：190015-202003，含量：99.6％。
供试品：马来酸甲麦角新碱原料，征集自河北某药厂，
批号：190826、200424、180322。
2、方法
2.1色谱条件
色谱柱：Dionex IonPac AS19(250mm×4mm，5μm)；
保护柱：Dionex IonPac AG19(50mm×4mm，5μm)；
淋洗液：100mmol/L氢氧化钠溶液；
洗脱方式：梯度洗脱(见下表)；
淋洗液流速：1.0mL/min；
色谱柱温度：30℃；
检测池温度：35℃；
进样量：20μL；
检测器：电导检测器；
抑制器：阴离子抑制器(ASRS-4mm)，抑制电流：50mA；
氮气压力27.6kPa。
表1淋洗液梯度洗脱表
Figure PCTCN2022138247-appb-000002
2.2溶液的配制
空白溶液的配制：超纯超纯水；
对照品贮备液的配制：精密称取马来酸对照品50.12mg，置50mL量瓶中，用超纯水稀释至刻度，摇匀，制成每1mL含马来酸1000μg溶液，作为对照 品贮备液；
对照品溶液的配制：精密量取对照品贮备液1mL，置100mL量瓶中，用超纯水稀释至刻度，摇匀，制成每1mL含马来酸10μg溶液，作为对照品溶液；
供试品溶液的配制：称取马来酸甲麦角新碱原料10mg，精密称定，置250mL量瓶中，加超纯水溶解并稀释至刻度，摇匀，作为供试品溶液；
系统适用性溶液的配制：取供试品溶液和对照品溶液1:1混合。
3、方法学验证
3.1专属性实验
取系统适用性溶液、空白溶液、对照品溶液和供试品溶液各分别注入离子色谱仪，记录色谱图，结果如附图1-4所示。
结果表明，在该色谱条件下，溶剂不干扰样品测定，马来酸离子峰与前后杂质峰均能达到基线分离。
3.2定量限和检出限
定量限溶液的配制：逐级稀释对照品溶液，注入液相色谱仪，记录色谱图，至信噪比s/n约10。
照拟建立方法，连续测定6次定量限溶液，记录色谱图。
表2定量限试验
Figure PCTCN2022138247-appb-000003
精密量取对照品溶液，逐级稀释，注入液相色谱仪，记录色谱图(附图5)直至信噪比s/n约3，即为检出限溶液。
由上述试验结果可知：马来酸定量限6次测定结果的峰面积RSD为1.5％，定量限分别为18.107μg；检出限为6.036μg。
3.3线性和范围
线性溶液贮备液的制备：精密量取对照品贮备液5.0mL，置于50mL量瓶中，加超纯水稀释至刻度，摇匀，制成线性溶液贮备液。
系列线性溶液的制备：精密量取线性溶液贮备液各0.1mL，1.0mL，2.5mL，5.0mL，10.0mL，15.0mL，分别置于50mL量瓶中，加超纯水稀释至刻度，摇匀，制成系列对照品溶液。按上述色谱条件进样测定，记录峰面积。
表3马来酸线性关系试验结果
Figure PCTCN2022138247-appb-000004
以峰面积为纵坐标，以浓度为横坐标进行线性回归，线性关系图如附图6所示：
马来酸在0.2μg/ml～30μg/ml范围内，线性方程为y＝0.045x+0.0078，相关系数R2＝0.9998，峰面积A与浓度C(μg/ml)呈显著线性关系。
3.4准确度试验
标准加入溶液的配制：精密量取对照品贮备液8.0mL、10.0mL、12.0mL置1000mL量瓶中，加超纯水稀释至刻度，摇匀，即得标准加入液(1)8μg/mL、(2)10μg/mL、(3)12μg/mL；
供试品溶液的配制：精密称取本品(批号：200424)约10mg，共9份， 分3组，置250mL量瓶中，分别加入上述混合对照溶液溶解并稀释至刻度，摇匀。取上述溶液注入离子色谱仪，记录色谱图；测得量与加入量的比值即为回收率。
表4回收率试验
Figure PCTCN2022138247-appb-000005
由以上试验结果可知：马来酸回收率范围为96.30％～101.15％(RSD％＝1.92)，回符合验证要求，本法准确性良好。
3.5精密度试验
3.5.1进样精密度
取对照品溶液注入离子色谱仪，重复进样6次，记录色谱图，计算RSD，考察本法的仪器精密度。
表5进样精密度试验
Figure PCTCN2022138247-appb-000006
Figure PCTCN2022138247-appb-000007
由试验结果可知：马来酸保留时间RSD为0.1％，马来酸峰面积RSD为1.7％，本方法进样精密度良好。
3.5.2重复性实验
取马来酸甲麦角新碱原料1批(批号：200424)和对照品溶液，照拟定方法，平行制备6份，分别注入离子色谱仪，记录色谱图。考察本法的重复性。
表6重复性试验结果
Figure PCTCN2022138247-appb-000008
结论：同一实验人员重复测定6次，6次测定结果马来酸的检出量基本一致。方法重复性良好。
3.5.3中间精密度试验
不同实验人员按对照品溶液和供试品溶液操作，配制对照品溶液6份和供试品(批号：200424)溶液6份。分别注入离子色谱仪，记录色谱图。
表7中间精密度试验结果
Figure PCTCN2022138247-appb-000009
Figure PCTCN2022138247-appb-000010
结论：不同实验人员重复测定6次，6次测定结果比较，马来酸检出量一致。方法重复性良好。
3.6耐用性试验
分别考察了淋洗液流速0.8ml/min、1.0ml/min、1.2ml/min；淋洗液浓度18mmol/L、20mmol/L、22mmol/L和色谱柱温度25℃、30℃、35℃，条件下主峰与其相邻杂质峰色谱保留行为。结果表明，在各色谱条件小幅度改变情况下马来酸离子主峰出峰稳定，与其相邻杂质峰均可达到基线分离，本方法色谱条件耐用性良好。
3.7稳定性试验
按对照品溶液配制方法制备，于室温放置0、1、2、3、4、5、6小时，取不同放置时间的上述溶液注入离子色谱仪，记录色谱图。
表8溶液稳定性试验结果
Figure PCTCN2022138247-appb-000011
由试验结果可知：室温放置6小时，对照品溶液面积RSD为1.2％(n＝6)，对照品溶液在6小时内稳定。
4、样品测定
取原料3批，批号：190826、200424、180322，每份10mg，按拟定方法进样测定，记录峰面积并计算马来酸含量。结果见下表。
表9样品测定结果
Figure PCTCN2022138247-appb-000012
讨论
5.1淋洗液流速的确认
分别考察了淋洗液流速0.8ml/min、1.0ml/min、1.2ml/min条件下主峰与其相邻杂质峰色谱保留行为。结果表明，在各色谱条件小幅度改变情况下马来酸离子主峰出峰稳定，与其相邻杂质峰均可达到基线分离，最终选择淋洗液流速为1.0ml/min。
表10淋洗液流速考察试验结果
Figure PCTCN2022138247-appb-000013
5.2淋洗液浓度的确认
分别考察了淋洗液浓度为18mmol/L、20mmol/L、22mmol/L条件下主峰与其相邻杂质峰色谱保留行为。结果表明，在各色谱条件小幅度改变情况下马来酸离子主峰出峰稳定，与其相邻杂质峰均可达到基线分离，最终选择淋洗液浓度为20mmol/L。
表11淋洗液浓度考察试验结果
Figure PCTCN2022138247-appb-000014
5.3色谱柱温度的确认
分别考察了色谱柱温度25℃、30℃、35℃，条件下主峰与其相邻杂质峰色谱保留行为。结果表明，在各色谱条件小幅度改变情况下马来酸离子主峰出峰稳定，与其相邻杂质峰均可达到基线分离，最终选择色谱柱温度为30℃。
表12色谱柱温度考察试验结果
Figure PCTCN2022138247-appb-000015
6结论
6.1马来酸限度确定
已知甲麦角新碱分子式C 20H 25N 3O 2，相对分子量为：339.4278，马来酸分子式：C 4H 4O 4，相对分子量为：116.0722，可以计算得到马来酸甲麦角新碱中含有1分子马来酸酸时的理论马来酸含量为25.48％。参照《中国药典》2020年版四部9003药品杂质检查指导原则，将马来酸甲麦角新碱的马来酸含量控制在理论值的80.0％～120.0％，则马来酸的含量限度应为20.4％～30.6％。
6.2成盐率考察
照USP43中HPLC法测定马来酸甲麦角新碱活性成分含量，由于对照品是按照C20H25N3O2·C4H4O4计，因此测得的含量结果即为马来酸甲麦角新碱含量。用摩尔浓度计算成盐率，以马来酸含量除以马来酸相对分子量与马来酸甲麦角新碱含量除以马来酸甲麦角新碱相对分子量的比值，公式：马来酸成盐率＝(马来酸％/116.0722)/(马来酸甲麦角新碱％/445.5)[9]。
表13马来酸甲麦角新碱成盐率
Figure PCTCN2022138247-appb-000016
6.2产品质量评价
对比各国药典，对于成盐药物中盐的检查，通常只有化学鉴别，很少有专属性强的成盐离子定量检查[10]，本研究建立了离子色谱-电导检测器法测定马来酸甲麦角新碱中马来酸的含量，方法专属性强，灵敏度高，操作简单，结果准确。
应用建立的方法对征集到的3批样品进行检测，马来酸的含量在25.18％～25.33％之间，平均含量为25.24％，成盐率0.97，符合限度要求，提示国内该生产企业生产工艺稳定，产品中没有过多游离的马来酸，质量安全、可控。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明 确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (6)

  1. 一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，包括以下步骤：
    分别将系统适用性溶液、空白溶液、对照品溶液和供试品溶液加注入到离子色谱仪中，记录色谱图，按外标法以峰面积计算马来酸甲麦角新碱原料药中马来酸的含量，代入公式(1)计算得出马来酸甲麦角新碱原料成盐率；
    A＝(B/116.0722)/(C/445.5)  (1)
    其中，A为马来酸甲麦角新碱原料成盐率，B为马来酸的含量，C为原料中马来酸甲麦角新碱的含量；
    离子色谱条件为：
    色谱柱：Dionex IonPac AS19(250mm×4mm，5μm)；
    保护柱：Dionex IonPac AG19(50mm×4mm，5μm)；
    淋洗液：100mmol/L氢氧化钠溶液；
    洗脱方式：梯度洗脱；
    淋洗液流速：1.0mL/min；
    色谱柱温度：30℃；
    检测池温度：35℃；
    进样量：20μL；
    检测器：电导检测器；
    抑制器：阴离子抑制器(ASRS-4mm)，抑制电流：50mA；
    氮气压力27.6kPa。
  2. 根据权利要求1所述的一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，所述空白溶液为超纯超纯水。
  3. 根据权利要求1所述的一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，所述对照品溶液的制备：
    (1)精密称取马来酸对照品50.12mg，置50mL量瓶中，用超纯水稀释至 刻度，摇匀，制成每1mL含马来酸1000μg溶液，作为对照品贮备液；
    (2)精密量取对照品贮备液1mL，置100mL量瓶中，用超纯水稀释至刻度，摇匀，制成每1mL含马来酸10μg溶液，作为对照品溶液。
  4. 根据权利要求1所述的一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，供试品溶液的制备：称取马来酸甲麦角新碱原料10mg，精密称定，置250mL量瓶中，加超纯水溶解并稀释至刻度，摇匀，作为供试品溶液。
  5. 根据权利要求1所述的一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，系统适用性溶液的制备：取供试品溶液和对照品溶液1:1混合。
  6. 根据权利要求1所述的一种马来酸甲麦角新碱原料中成盐率的测定方法，其特征在于，梯度洗脱时：0～15分钟，淋洗液浓度20mmol/L；15～22分钟，淋洗液浓度250mmol/L；22～30分钟，淋洗液浓度20mmol/L。
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