CN112213411A - 一种双醋瑞因中基因毒性杂质的检测方法 - Google Patents
一种双醋瑞因中基因毒性杂质的检测方法 Download PDFInfo
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Abstract
本发明公开了一种双醋瑞因中基因毒性杂质的检测方法。本方法具体采用高效液相色谱法,使用C18柱;紫外检测器;采用三氟乙酸溶液‑甲醇为流动相梯度洗脱。该检测方法,可迅速准确检测出双醋瑞因中两类杂质的含量,解决了其合成过程两类杂质的检测问题。可有效控制双醋瑞因产品质量。该方法操作简捷,灵敏度高,分离度好,为控制产品质量提供了一种可行有效的分析方法。
Description
技术领域
本发明涉及一种双醋瑞因中基因毒性杂质的检测方法,属于药物分析检测领域。
背景技术
双醋瑞因的化学结构式如下:
化学名为:4,5-二乙酰基-9,10-二氧-9,10-二氢蒽-2-羧酸,属于解热镇痛药,又称二乙酰二氢蒽羧酸、1,8-二乙酰基-3-羧基蒽醌、达赛瑞英、安必丁,是一种新的白介素(IL)-1抑制剂,化学名称为二乙酰大黄酸,属蒽醌类的化合物,本品可诱导软骨生成、具有止痛、抗炎及退热作用,也可显著改善骨关节炎患者的关节功能,延缓病程,减轻疼痛,提高患者的生活质量,具有较好的安全性,临床主要用于治疗骨关节炎(OA)而发挥抗菌作用。
在双醋瑞因在合成过程中,有可能引入杂质A(包括杂质A-1、杂质A-2)和杂质B(包括杂质B-1、杂质B-2、杂质B-3)两类有关物质,这类有关物质显示出基因毒性警示结构,因此,要严格控制其中的含量;具体相关物质如下:
目前还未见有双醋瑞因中检测上述杂质分析方法的文献报道,因此,为了更好地控制双醋瑞因产品的质量,需要建立一套稳定有效的方法来进行本品的有关物质进行检测,解决合成工艺过程中上述杂质对产品纯度测定的干扰。
发明内容
本发明目的在于提供一种双醋瑞因中基因毒性杂质的检测方法,具有高杂质检测灵敏度,同时可以消除双醋瑞因对检测的干扰,同时适用于有类似结构的多个杂质的准确定量,大大提高了杂质的检测可信度和灵敏度。
尤其是,本发明首次公开了检测双醋瑞因中基因毒性杂质的高效液相色谱方法,经过验证,具有高的准确性以及灵敏度。
本发明采用如下技术方案:
双醋瑞因中基因毒性杂质的检测方法,包括以下步骤:
(1)将供试品与溶液A、溶液B混合,然后调整pH值为9.5;再用二氯甲烷萃取,萃取液依次经过溶液C、溶液D洗涤后干燥,再将得到的干燥物溶解在四氢呋喃中,得到供试品溶液;
(2)供试品溶液经过高效液相色谱检测,得到杂质峰,再与对照品溶液的杂质峰比较,完成双醋瑞因中基因毒性杂质的检测。
双醋瑞因中基因毒性杂质的定量方法,包括以下步骤:
(1)将供试品与溶液A、溶液B混合,然后调整pH值为9.5;再用二氯甲烷萃取,萃取液依次经过溶液C、溶液D洗涤后干燥,再将得到的干燥物溶解在四氢呋喃中,得到供试品溶液;
(2)供试品溶液经过高效液相色谱检测,得到杂质峰面积,再与对照品溶液的杂质峰面积比较,得到杂质浓度,完成双醋瑞因中基因毒性杂质的定量。
本发明中,系统溶液的杂质峰面积测试过程中,将杂质A、杂质B溶于四氢呋喃中得到对照品溶液,再经过与供试品溶液同样的高效液相色谱检测,得到杂质峰以及杂质峰面积;其中杂质为杂质A、杂质B:
优选的,杂质A、杂质B、四氢呋喃的用量比为15mg∶15mg∶100mL。
本发明中,高效液相色谱检测时,采用硅胶为填充剂,以三氟乙酸溶液为流动相A,以甲醇为流动相B,流速为0.6~1.5ml/min;优选的,采用十八烷基硅烷键合硅胶为填充剂,以0.1wt%的三氟乙酸溶液为流动相A,以甲醇为流动相B,进行梯度洗脱;具体按表1进行梯度洗脱。
本发明中,溶液A为氢氧化钠水溶液,溶液B为含有氯化钠、甘氨酸的水溶液,溶液C为溶液A与溶液B混合后调整pH值为9.5后的溶液,溶液D为硫酸水溶液。溶液A中,氢氧化钠、水的用量比为(0.9~1.1)g∶50mL;溶液B中,氯化钠、甘氨酸、水的用量比为(14~15)g∶(18~19)g∶500mL;溶液C中,溶液A的体积百分数为溶液A与溶液B体积和的20~30%;溶液D中,稀硫酸、水的体积比为(0.8~1.2)∶100。优选的,溶液A中,氢氧化钠、水的用量比为1g∶50mL,溶液B中,氯化钠、甘氨酸、水的用量比为(14~15)g∶(18~19)g∶500mL,溶液C中,溶液A的体积百分数为溶液A与溶液B体积和的25~26%,溶液D中,硫酸、水的体积比为1∶100。比如取氢氧化钠10g加水500ml溶解,作为溶液A;取氯化钠14.7g与甘氨酸18.8g加水500ml溶解,作为溶液B;取稀硫酸5ml加水稀释至500ml,作为溶液D。
本发明中,步骤(1)中,供试品为待检测双醋瑞因样品,与溶液A、溶液B的用量比为100mg∶30mL∶70mL;调整pH值为9.5的试剂为氢氧化钠水溶液或者稀硫酸;用二氯甲烷萃取的次数为3次;将得到的干燥物溶解在四氢呋喃中,再加入稀释剂,得到供试品溶液;以体积比为1∶1的流动相A、流动相B的混合物作为稀释剂。
本发明与已有技术相比的积极效果在于:
1、本发明中液相色谱条件的优越性更强,适用于双醋瑞因合成过程中两类杂质的分析,分离度高,可准确定量。
2、本发明的样品配制方法使用该类性杂质的处理,采用水解以后富集的方法,提高了杂质检测的灵敏度,同时可以消除双醋瑞因对检测的干扰,同时适用于有类似结构的多个杂质的准确定量,大大提高了杂质的检测可信度和灵敏度。
3、本发明杂质计算方式为标准曲线法,可以有效避免提取过程中各杂质的损失,保证计算结果的准确可靠。有利于工业优化生产中,双醋瑞因杂质的检测,能更好的控制产品质量,保证药品安全性。
附图说明
图1为对照品溶液色谱图;
图2为杂质A线性关系图;
图3为杂质B线性关系图。
具体实施方式
本发明公开的方法可以定性也可以定量检测双醋瑞因中的杂质(具体结构式见背景技术部分),具有如下步骤:
(1)将待检测双醋瑞因样品与溶液A、溶液B混合,然后调整pH值为9.5;再用二氯甲烷萃取,萃取液依次经过溶液C、溶液D洗涤后干燥,再将得到的干燥物溶解在四氢呋喃中,得到供试品溶液;
(2)供试品溶液经过高效液相色谱检测,得到杂质峰,再与系统溶液的杂质峰比较,可以实现双醋瑞因中基因毒性杂质的定性;
供试品溶液经过高效液相色谱检测,得到杂质峰面积,再与系统溶液的杂质峰面积比较,得到杂质浓度,完成双醋瑞因中基因毒性杂质的定量。
本发明涉及的原料都是本领域常规方法制备或者市售产品,其中稀硫酸的浓度为10wt%。
实施例
色谱条件的选择:
仪器:Thermo Ultimate 3000,柱温30℃,流速1.0ml/min。液相色谱柱用十八烷基硅烷键合硅胶(250mm×4.6mm,5μm),0.1wt%的三氟乙酸水溶液作为流动相A、甲醇作为流动相B的体系,进行梯度洗脱,表1;进样量10μl。在该色谱条件下,杂质A和杂质B保留时间适中,分离度良好。
溶剂配制
①取氢氧化钠10g加水500ml溶解,作为溶剂A;
②取氯化钠14.7g与甘氨酸18.8g加水500ml溶解,作为溶剂B;
③取溶剂A 25.3ml与溶剂B 74.6ml混匀,pH值为9.5(必要时用溶剂A或稀硫酸溶液调节pH至9.5),作为溶剂C;
④取稀硫酸5ml加水稀释至500ml,作为溶剂D;
⑤流动相A-流动相B(v/v,50:50)作为稀释剂。
溶液配制
对照品溶液:取杂质A、杂质B各15mg,置100ml量瓶中,加四氢呋喃溶解并稀释至刻度,摇匀;取1.0ml置100ml量瓶中,加四氢呋喃稀释至刻度,作为对照品溶液。
供试品溶液:取双醋瑞因100mg,加30ml溶剂A,混合10分钟,加70ml溶剂B并用氢氧化钠试液或稀硫酸调节pH值至9.5;用二氯甲烷萃取3次,每次25ml;合并二氯甲烷提取液,用溶剂C洗涤2次,每次8ml;再用10ml溶剂D洗涤1次;取洗涤后的溶液在33℃水浴蒸至近干,用压缩空气完全吹干,残渣加四氢呋喃2ml置40℃水浴超声溶解,加稀释剂稀释至4ml,作为供试品溶液;供试品的处理应进行避光操作,同时整个处理过程应控制在30分钟之内。
供试品溶液Ⅰ:取双醋瑞因100mg,加“对照品溶液”0.5ml后,按“供试品溶液配制方法”进行配制,杂质A、杂质B的添加终含量分别为7.5ppm。
供试品溶液Ⅱ:取双醋瑞因100mg,加“对照品溶液”0.75ml后,按“供试品溶液配制方法”进行配制,杂质A、杂质B的添加终含量分别为11.5ppm。
供试品溶液Ⅲ:取双醋瑞因100mg,加“对照品溶液”1.0ml后,按“供试品溶液配制方法”进行配制,杂质A、杂质B的添加终含量分别为15ppm。
供试品溶液ⅳ:取双醋瑞因100mg,加“对照品溶液”1.25ml后,按“供试品溶液配制方法”进行配制,杂质A、杂质B的添加终含量分别为18.75ppm。
供试品溶液ⅴ:取双醋瑞因100mg,加“对照品溶液”1.5ml后,按“供试品溶液配制方法”进行配制,杂质A、杂质B的添加终含量分别为22.5ppm。
灵敏度测定
取杂质A、杂质B各15mg,置50ml量瓶中,加四氢呋喃溶解并稀释至刻度,摇匀。取1.0ml置100ml量瓶中,加四氢呋喃稀释至刻度。再用四氢呋喃稀释成一系列不同浓度的溶液,分别进样10μl进行液相色谱检测,使之产生主峰为基线噪音三倍的信号。经测试,杂质A的最小定量限度为4ppm(S/N≥10),杂质B的定量限度为6ppm。结果证明,该方法灵敏度高,可以充分满足杂质测定要求,节选数据如下:
溶液稳定性测定
取同一份对照品溶液,分别于0、2、4、8、10小时分别进样测定,其中杂质A、杂质B峰面积在10小时内稳定,五次测试结果没有变化。
以上实验结果表明,本发明方法准确灵敏,重现性好,可较好的检测双醋瑞因中的两类杂质。
准确率测试
取对照品溶液、供试品溶液、供试品溶液Ⅰ、Ⅱ、Ⅲ、ⅳ、ⅴ各10μl,分别注入液相色谱仪,进行同样的测试,对照品溶液测试谱图见图1,通过谱图可以很清晰的看到杂质A、杂质B能够得到很好的分离效果,并且无干扰情况存在,而供试品溶液没有看到杂质A、杂质B的峰,说明采用的双醋瑞因为纯品,杂质低于检测限;供试品溶液Ⅰ、Ⅱ、Ⅲ、ⅳ、ⅴ都得到杂质A、杂质B的峰,位置与对照品溶液峰一致。
以供试品溶液Ⅰ、Ⅱ、Ⅲ、ⅳ、ⅴ中杂质A标准溶液浓度为横坐标(x轴),色谱图中杂质A或杂质B峰面积为纵坐标(y轴),绘制标准曲线(相关系数r≥0.99),见图2;以供试品溶液Ⅰ、Ⅱ、Ⅲ、ⅳ、ⅴ中杂质A或杂质B标准溶液浓度为横坐标(x轴),色谱图中杂质B峰面积为纵坐标(y轴),绘制标准曲线(相关系数r≥0.99),见图3。
以供试品溶液Ⅰ色谱图中杂质A的峰面积/对照品溶液色谱图中杂质A的峰面积,然后乘以对照品溶液中杂质A的浓度,得到供试品溶液Ⅰ中杂质A(包括杂质A-1、A-2)的浓度,为7.8ppm;同样的方法得到供试品溶液Ⅰ中杂质B(包括杂质B-1、B-2、B-3)的浓度,为7.7ppm。
另取双醋瑞因100mg,按照供试品溶液配置,经过同样的检测,其杂质A含量8ppm,杂质B含量13ppm;取该双醋瑞因100mg,按照供试品溶液Ⅰ配置,经过同样的检测,其杂质A含量16ppm,杂质B含量20.1ppm。
以上可以说明本发明方法准确。
在上述实施例的基础上,将流动相替换为水&甲醇或者0.1wt%的三氟乙酸水溶液&乙腈,经过同样的方法检测对照品溶液,发现两种实验下,杂质A和B未能有效分开,且杂质A和B拖尾,更无法定量。
本发明采用普通色谱柱(C18柱),检测波长254nm,柱温30℃,流速为1.0ml/min,色谱条的流动相以0.1%的三氟乙酸溶液为流动相A,以甲醇为流动相B,按表1进行梯度洗脱。有效地对双促瑞因中杂质进行检测,可准确有效的检测出双醋瑞因杂质,灵敏度高,可较好地控制产品质量。实现了双醋瑞因的杂质测定,保证了双醋瑞因的质量控制,在合成过程的质量控制方面具有重要的现实意义。
Claims (10)
1.双醋瑞因中基因毒性杂质的检测方法,其特征在于,包括以下步骤:
(1)将供试品与溶液A、溶液B混合,然后调整pH值为9.5;再用二氯甲烷萃取,萃取液依次经过溶液C、溶液D洗涤后干燥,再将得到的干燥物溶解在四氢呋喃中,得到供试品溶液;
(2)供试品溶液经过高效液相色谱检测,得到杂质峰,再与对照品溶液的杂质峰比较,完成双醋瑞因中基因毒性杂质的检测。
2.如权利要求1所述双醋瑞因中基因毒性杂质的检测方法,其特征在于,将基因毒性杂质溶解于四氢呋喃,得到对照品溶液;对照品溶液经过所述高效液相色谱检测,得到杂质峰;所述基因毒性杂质为杂质A、杂质B。
3.如权利要求1所述双醋瑞因中基因毒性杂质的检测方法,其特征在于,高效液相色谱检测时,采用硅胶为填充剂,以三氟乙酸溶液为流动相A,以甲醇为流动相B;流速为0.6~1.5ml/min。
4.如权利要求1所述双醋瑞因中基因毒性杂质的检测方法,其特征在于,溶液A为氢氧化钠水溶液,溶液B为含有氯化钠、甘氨酸的水溶液,溶液C为溶液A与溶液B混合后调整pH值为9.5后的溶液,溶液D为硫酸水溶液。
5.如权利要求5所述双醋瑞因中基因毒性杂质的检测方法,其特征在于,溶液A中,氢氧化钠、水的用量比为(0.9~1.1)g∶50mL;溶液B中,氯化钠、甘氨酸、水的用量比为(14~15)g∶(18~19)g∶500mL;溶液C中,溶液A的体积百分数为溶液A与溶液B体积和的20~30%;溶液D中,稀硫酸、水的体积比为(0.8~1.2)∶100。
6.双醋瑞因中基因毒性杂质的定量方法,其特征在于,包括以下步骤:
(1)将供试品与溶液A、溶液B混合,然后调整pH值为9.5;再用二氯甲烷萃取,萃取液依次经过溶液C、溶液D洗涤后干燥,再将得到的干燥物溶解在四氢呋喃中,得到供试品溶液;
(2)供试品溶液经过高效液相色谱检测,得到杂质峰面积,再与对照品溶液的杂质峰面积比较,得到杂质浓度,完成双醋瑞因中基因毒性杂质的定量。
7.如权利要求6所述双醋瑞因中基因毒性杂质的定量方法,其特征在于,将得到的干燥物溶解在四氢呋喃中,再加入稀释剂,得到供试品溶液;高效液相色谱检测时,采用硅胶为填充剂,以三氟乙酸溶液为流动相A,以甲醇为流动相B;以流动相A、流动相B的混合物作为稀释剂;将基因毒性杂质溶解于四氢呋喃,得到对照品溶液;对照品溶液经过所述高效液相色谱检测,得到杂质峰面积;所述基因毒性杂质为杂质A、杂质B。
8.如权利要求6所述双醋瑞因中基因毒性杂质的定量方法,其特征在于,对照品溶液中,杂质A、杂质B、四氢呋喃的用量比为15mg∶15mg∶100mL。
9.如权利要求6所述双醋瑞因中基因毒性杂质的定量方法,其特征在于,溶液A为氢氧化钠水溶液,溶液B为含有氯化钠、甘氨酸的水溶液,溶液C为溶液A与溶液B混合后调整pH值为9.5后的溶液,溶液D为硫酸水溶液。
10.如权利要求6所述双醋瑞因中基因毒性杂质的定量方法,其特征在于,供试品与溶液A、溶液B的用量比为100mg∶30mL∶70mL。
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