CN113433242B - Molnupiravir含量及有关物质的检测方法 - Google Patents
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Abstract
本发明属分析化学领域,本发明涉及一种Molnupiravir药物含量及有关物质的检测方法,该检测方法包括制备供试品溶液,采用高效液相色谱法,将十八烷基硅烷键合硅胶为色谱柱填料并对供试品溶液进行上样,通过流动相对供试品进行洗脱检测。本发明能快速、有效、准确的检测Molnupiravir的含量。本发明方法操作简单,分析时间短,准确度高,重复性好,为Molnupiravir提供了可靠的检测方法,为药物产品生产工艺及质量研究提供了参考。
Description
技术领域
本发明属于分析化学领域,具体涉及一种用高效液相色谱法分离测定molnupiravir(MK-4482,EIDD-2801)原料药中Molnupiravir药物及有关物质含量的方法。
背景技术
新冠病毒属于冠状病毒科β冠状病毒中的一种单链RNA病毒,与引起非典型肺炎的病毒基因组序列十分相似,同源性达79.5%。单链RNA病毒的结构特征体现在两类蛋白质:结构蛋白质和非结构蛋白质,例如蛋白酶和RNA依赖性RNA聚合酶(RdRp)。RdRp是病毒生命周期中重要的酶,负责从病毒模板RNA合成多个副本的互补RNA。靶向抑制这种酶可以阻止病毒繁殖并导致病毒死亡。
核苷酸每个碱基对都可被细胞良好吸收并被哺乳动物激酶转化为5’-三磷酸形式,并与RdRp酶和RNA模式结合。因此核苷类抗病毒药物是抑制该酶的良好选择。Molnupiravir(MK-4482,EIDD-2801)由美国默克公司研发,是核糖核苷类似物N 4-羟基胞嘧啶核苷(EIDD-1931)的异丁酯前药,现已显示出广泛的流感病毒和多种冠状病毒活性,具有良好的安全性和耐药性。Molnupiravir(MK-4482,EIDD-2801)作为口服制剂,具有很好的便捷性。口服剂量分为单次口服50-1600mg或每日两次口服50-800mg,服用5.5天。Molnupiravir(MK-4482,EIDD-2801)的化学名称为[(2R,3R,4S,5R)-3,4-dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl]methylisobutyrate分子式为C13H19N3O7。[(2R,3R,4S,5R)-3,4-二羟基-5-(4-(羟基氨基)-2-氧嘧啶-1(2H)-基)四氢呋喃-2-基]异丁酸甲酯,其化学结构式如下:
在Molnupiravir(MK-4482,EIDD-2801)的合成工艺过程及保存过程中,存在因去除不完全而影响药物纯度和质量的杂质,即药物质量控制中的有关物质,其化学名称为N4-羟基胞嘧啶核苷酸(NHC,EIDD-1931),分子式为C9H13N3O6,其结构式如下:
对于Molnupiravir(MK-4482,EIDD-2801)合成过程中引入的有关物质,作为原料药时是需要进行质量控制的。因此,实现Molnupiravir及其有关物质的分离,在Molnupiravir的质量控制方面具有重要的现实意义。目前,USP、EP、JP和中国药典及相关文献、专利均未见Molnupiravir含量测定及有关物质的报道。
发明内容
针对现有技术的缺陷和实际需求,本发明的目的在于提供一种能够同时精确检测Molnupiravir药物及有关物质含量的方法,以实现对Molnupiravir的质量控制。该方法需具有专属性强、分离度高、检测结果准备可信、容易操作及检测成本低的优点。
为了实现上述技术目的,本发明提供的方案如下:
一种Molnupiravir药物含量及有关物质检测方法,所述检测方法包括如下步骤:制备供试品溶液,采用高效液相色谱法,将十八烷基硅烷键合硅胶为色谱柱填料并对供试品溶液进行上样,再以有机相水溶液作为流动相进行洗脱检测,色谱条件为检测波长为200~300nm,柱温为25℃~40℃,流速为0.2~2.0mL/min,记录色谱图;所述有关物质为N4-羟基胞嘧啶核苷酸(NHC,EIDD-1931);所述有机相选用甲醇、乙腈、异丙醇、四氢呋喃中的一种或多种的组合。
本发明采用高效液相色谱法时,色谱柱以十八烷基硅烷键合硅胶为填料,选自Phenomenex Gemini C18柱或ZORBAX SB C18柱或Diamonsil C18柱;优选PhenomenexGemini C18柱,长度250mm,内径4.6mm,粒径5μ。
优选地,本发明上述所说的方法中,所述有机相为甲醇;优选地,流动相为甲醇体积百分数为20%~60%的甲醇水溶液,甲醇体积百分数优选为30%。
作为本发明可选择的实施例,本发明上述所说的方法中,所述流动相的流速为0.9~1.1ml/min,优选1.0ml/min。
作为本发明可选择的实施例,本发明上述所说的方法中,所述色谱柱的柱温为25℃~35℃,优选30℃。
作为本发明可选择的实施例,本发明的上述方法中,检测波长为235nm;进样体积为10μL。
本发明提供的一种Molnupiravir药物含量检测方法及有关物质测定方法的优点在于:本发明能快速、有效、准确的检测Molnupiravir药物的含量及有关物质,***适应性分离度达到2.0以上,一法双测,节约成本,为合成研究及其他工艺研究提供了参考依据。
附图说明
图1为波长选择试验中的对照品溶液的光谱扫描图;
图2为***适用性试验中的对照品色谱图,其中峰1为杂质-I(NHC)色谱峰,峰2为杂质-Ⅱ(未知杂质)色谱峰,峰3为主成分色谱峰;
图3为线性关系试验中的Molnupiravir含量线性图;
图4为实施例3中的供试品溶液色谱图,各色谱峰编号与图2一致;
图5为对比例1中的供试品溶液色谱图,各色谱峰编号与图2一致;
图6为对比例2中的供试品溶液色谱图,各色谱峰编号与图2一致;
图7为对比例3中的供试品溶液色谱图,各色谱峰编号与图2一致。
具体实施方式
下面通过具体实施例来进一步说明本发明。应当理解为:本发明的实施例仅用于进一步解释本发明,但不限于本实施的范围。
实施例1:方法学验证
1.波长选择试验
对照品溶液:取Molnupiravir对照品适量,精密称定,加水溶解并稀释制成每1ml中含10μg的溶液。
照紫外-可见分光光度法,在200nm~400nm范围内对对照品溶液进行光谱扫描。如图1所示,图1为波长选择试验中的对照品溶液的光谱扫描图。对照品溶液在200.0nm、235.2nm、271.6nm处有最大吸收,从而在200nm-300nm均能够进行一定检测,选235nm为检测波长。
2.专属性试验
空白溶剂:30%甲醇水溶液。
供试品溶液的制备:精密称定含Molnupiravir 10mg的样品,置50ml量瓶中,加溶剂溶解并稀释至刻度,摇匀,作为供试品溶液。采用优选色谱条件测定,精密量取上述溶液各10μL,分别注入液相色谱仪,记录色谱图,如图2所示,图2为专属性试验中的供试品溶液与杂质溶液的色谱图。
在该色谱条件下空白溶剂不干扰主峰测定;Molnupiravir与相邻峰之间的分离度大于2.0,DAD检测器下最大吸收波长为235nm,主峰峰纯度合格;本方法分离度高、专属性强。
3.线性关系试验:
线性关系贮备液的制备:取Molnupiravir对照品约40mg,精密称定,置100ml容量瓶中,加溶剂溶解并稀释至刻度,摇匀,即得。线性关系系列溶液的制备:精密量取线性储备液适量,逐步稀释为系列梯度浓度标准溶液,摇匀,分别作为限度的5%、12.5%、25%、50%、100%、200%线性溶液。
分别量取线性关系溶液各10μL,注入液相色谱仪,记录色谱图,计算线性方程和相关系数。
其结果如图3所示,图3为线性关系试验中的Molnupiravir含量线性图,由图可知,Molnupiravir在浓度为10.17~406.60ug/ml范围内,峰面积与浓度呈良好的线性关系。
4.耐用性试验
供试品溶液:同专属性试验样品溶液。
色谱条件保持其它参数优选条件色谱条件一致,分别将流速设为0.9ml/min、1.1ml/min、柱温设为28℃、32℃、波长设为233nm、237nm,记录各条件下的Molnupiravir的含量。试验结果见表1。
表1耐用性结果
实施例2:分离测定不同批次molnupiravir原料药
样品前处理:
溶剂:30%甲醇水溶液,即甲醇-水(30:70,v/v)。
供试品溶液:取Molnupiravir原料药10mg,精密称定,置于50ml量瓶中,加溶剂溶解、稀释、定容至刻度,摇匀,作为供试品溶液。
含量测定对照品溶液:取Molnupiravir对照品10mg,制备方法同供试品溶液,制成含Molnupiravir约200μg/ml的对照品溶液。
有关物质检查对照溶液:取供试品溶液,精密量取1ml置100ml容量瓶中,加30%甲醇水溶液稀释至刻度,摇匀,即得1%对照溶液。高效液相色谱条件:
色谱柱:Phenomenex Gemini C18(250mm×4.6mm,5.0μm);流动相:甲醇-水(30:70,v/v),等度洗脱;检测波长:235nm;流速:1.0mL/min;柱温:30℃;进样体积:10μL。
分别取供试品溶液、对照品溶液及1%对照溶液各10μL,注入高效液相色谱仪中,按照上述色谱条件进行检测,记录色谱图及峰面积。对于含量测定,按外标法以峰面积计算得到供试品中molnupiravir含量;对于有关物质检查,各杂质含量按主成分自身对照法计算。
按相同方法,对多批次molnupiravir原料药进行检测。检测结果如下表2所示,其 中,杂质-Ⅰ为N4-羟基胞嘧啶核苷酸(NHC,EIDD-1931),杂质-Ⅱ为不明杂质。
表2多批次Molnupiravir原料药测定结果
批号 | 杂质-Ⅰ,% | 杂质-Ⅱ,% | 总杂质,% | Molnupiravir,% |
101616 | 0.11 | 0.06 | 0.17 | 100.8 |
102427 | 0.20 | 0.06 | 0.26 | 99.56 |
102529 | 0.18 | 0.06 | 0.24 | 99.37 |
实施例3:
样品前处理:
溶剂:20%甲醇水溶液,即甲醇-水(20:80,v/v)。
供试品溶液、含量测定对照品溶液及有关物质检查对照溶液:除溶剂外,制备方法同实施例2中样品前处理方法。
高效液相色谱条件:
色谱柱:Phenomenex Gemini C18(250mm×4.6mm,5.0μm);流动相:甲醇-水(20:80,v/v),等度洗脱;检测波长:235nm;流速:1.0mL/min;柱温:30℃;进样体积:10μL;进样浓度:200ug/ml;稀释剂:20%甲醇水溶液。
精密量取上述溶液各10μL,注入液相色谱仪,记录色谱图,如图4所示。在该色谱条件下,供试品色谱峰个数与优选色谱条件一致,各峰面积百分比与优选色谱条件下测定结果无明显差异。
对比例1:
样品前处理:
溶剂:纯水。
供试品溶液、含量测定对照品溶液及有关物质检查对照溶液:除溶剂外,制备方法同实施例2中样品前处理方法。
高效液相色谱条件:
色谱柱:Phenomenex Gemini C18(250mm×4.6mm,5.0μm);流动相:A相为纯水,B相为甲醇,梯度洗脱,洗脱程序见表3;检测波长:235nm;流速:1.0mL/min;柱温:30℃;进样体积:10μL;进样浓度:200ug/ml;稀释剂:纯水。
表3梯度洗脱程序表
时间(min) | 流动相A(%) | 流动相B(%) |
0 | 5 | 95 |
5 | 5 | 95 |
60 | 0 | 100 |
65 | 0 | 100 |
65.01 | 5 | 95 |
75 | 5 | 95 |
精密量取上述溶液各10μL,注入液相色谱仪,记录色谱图,如图5所示。在该色谱条件下,供试品色谱峰个数与优选色谱条件一致,测定结果与优选色谱条件无明显差异。对比例1在流动相梯度洗脱下并没有更多的杂质峰检出,且分析方法运行时间较长,而本发明方法可以有效分离检测有关物质的同时大大缩短分析时间。
对比例2:
样品前处理:
溶剂:30%乙腈水溶液,即乙腈-水(30:70,v/v)。
供试品溶液、含量测定对照品溶液及有关物质检查对照溶液:除溶剂外,制备方法同实施例2中样品前处理方法。
高效液相色谱条件:
色谱柱:Phenomenex Gemini C18(250mm×4.6mm,5.0μm);流动相:乙腈-水(30:70,v/v),等度洗脱;检测波长:235nm;流速:1.0mL/min;柱温:30℃;进样体积:10μL;进样浓度:200ug/ml;稀释剂:30%乙腈水溶液。
精密量取上述溶液各10μL,注入液相色谱仪,记录色谱图,如图6所示。在该色谱条件下,杂质峰1与杂质峰2分离度降低,主峰拖尾严重,不适用于molnupiravir原料药的分析检测。
对比例3:
样品前处理:
溶剂:磷酸盐缓冲液(pH7.0)-水(30:70,v/v)。
供试品溶液、含量测定对照品溶液及有关物质检查对照溶液:除溶剂外,制备方法同实施例2中样品前处理方法。
高效液相色谱条件:
色谱柱:Phenomenex Gemini C18(250mm×4.6mm,5.0μm);流动相:磷酸盐缓冲液(pH7.0)(取0.02mol/L磷酸二氢钠溶液与0.02mol/L磷酸氢二钠溶液等体积混合后,用0.1mol/L氢氧化钠溶液或0.1mol/L磷酸溶液调节pH值至7.0)-甲醇(30:70,v/v),等度洗脱;检测波长:235nm;流速:1.0mL/min;柱温:30℃;进样体积:10μL;进样浓度:200ug/ml;稀释剂:流动相。
精密量取上述溶液各10μL,注入液相色谱仪,记录色谱图,如图6所示。在该色谱条件下,供试品色谱峰个数与优选色谱条件一致,测定结果与优选色谱条件无明显差异;对比例2中杂质分离度没有明显优化,但流动相配制更为复杂,因此本发明方法在实现相同分离度的条件下实验操作更为简便。
综上所述,本发明所述方法操作简单,灵敏度高,专属性好,可准确测定Molnupiravir的含量及有关物质,从而有效控制Molnupiravir的产品质量,为Molnupiravir原料药合成及质量标准的进一步完善提升提供了参考方法。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,所述检测方法包括如下步骤:制备供试品溶液,采用高效液相色谱法,将十八烷基硅烷键合硅胶为色谱柱填料并对供试品溶液进行上样,再以有机相水溶液作为流动相进行洗脱检测,色谱条件为检测波长为200~300 nm,柱温为25℃~40℃,流速为0.2~2.0 mL/min,记录色谱图;所述有关物质为N4-羟基胞嘧啶核苷酸;
所述有机相为甲醇,所述流动相由甲醇和水按体积比30:70混合而得。
2.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,采用高效液相色谱法时,色谱柱选自品牌为Phenomenex Gemini C18柱、ZORBAX SB C18柱或Diamonsil C18柱。
3.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,所述检测波长为235 nm。
4.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,所述流动相的流速为0.9~1.1 ml/min;所述色谱柱的柱温为25℃~35℃。
5.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,所述流动相的流速为1.0 ml/min。
6.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,所述柱温为30℃。
7.根据权利要求1所述一种Molnupiravir药物含量及有关物质的检测方法,其特征在于,进样量为10~50μL。
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