CN114236008A - 一种卡培他滨中间体中d-核糖及5-脱氧-d-呋喃核糖含量的检测方法 - Google Patents
一种卡培他滨中间体中d-核糖及5-脱氧-d-呋喃核糖含量的检测方法 Download PDFInfo
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Abstract
本发明公开了一种卡培他滨中间体中D‑核糖及5‑脱氧‑D‑呋喃核糖含量的检测方法。该方法采用LC‑MS检测方法,以Acclaim RSL C120C18为色谱柱,以乙腈‑水(35:65)为流动相,对1,2,3‑三‑O‑乙酰基‑5‑脱氧‑β‑D‑呋喃核糖中D‑核糖及5‑脱氧‑D‑呋喃核糖进行检测,这两种杂质与1,2,3‑三‑O‑乙酰基‑5‑脱氧‑β‑D‑呋喃核糖能够得到很好的分离(D‑核糖RT为1.39min;5‑脱氧‑D‑呋喃核糖RT为1.45min;1,2,3‑三‑O‑乙酰基‑5‑脱氧‑β‑D‑呋喃核糖峰RT为4.33min),方法具有专属性强、快速、灵敏、准确等优势。
Description
技术领域
本发明涉及一种卡培他滨中间体(1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖)中杂质D-核糖及5-脱氧-D-呋喃核糖的检测方法。
背景技术
卡培他滨(Capecitabine)是已上市的第一个口服氟代嘧啶氨基甲酸酯类抗肿瘤药物,是用于治疗乳腺癌、结肠直肠癌的新型靶向药物。该药由罗氏公司开发,商品名为“Xeloda”,1998年4月在美国获准上市,随后陆续在瑞士等国上市。于1999年11月开始在中国进行注册临床试验,由北京、上海、广州等地的5个国家抗肿瘤药物临床试验研究中心进行临床试验,并以商品名“希罗达”上市。卡培他滨口服后经肠黏膜迅速吸收,然后在肝脏被羧基酯酶转化为无活性的中间体5'-脱氧-5'氟胞苷,以后经肝脏和肿瘤组织的胞苷脱氨酶的作用转化为5'-脱氧-5'氟尿苷,最后在肿瘤组织内经胸苷磷酸化酶催化为氟尿嘧啶(5-FU)而起作用。1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖是卡培他滨合成中的关键中间体,分子式:C11H16O7分子量:260.24,其结构式如下:
D-核糖及5-脱氧-D-呋喃核糖是1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖生产过程中的起始物料及中间体(参见文献:宋凯,郑国钧.1,2,3-O-三乙酰基-5-脱氧-D-呋喃核糖的合成研究[J],化学试剂,2020,32(2):171-172),为控制1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖质量,应制定D-核糖及5-脱氧-D-呋喃核糖在1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中的检测方法。但经检索,目前尚无关于D-核糖及5-脱氧-D-呋喃核糖在1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中的含量检测方法的报道。
D核糖,英文名称:D-ribose,分子式:C5H10O5;分子量:150.13,结构式如下所示:
5-脱氧-D-呋喃核糖,英文名称:5-deoxy-d-ribofuran,分子式:C5H10O4;分子量:134.13,结构式如下所示:
发明内容
针对上述问题,本发明首次提供了一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法。该方法采用LC-MS检测方法,以Acclaim RSL C120 C18为色谱柱,以乙腈-水为流动相,对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖进行检测,这两种杂质与1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖能够得到很好的分离,方法具有专属性强、快速、灵敏、准确等优势。
本发明的技术方案是:一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是:
1)将1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖样品用流动相溶解;
2)采用LC-MS检测方法,以Acclaim RSL C120 C18为色谱柱,以乙腈-水(35:65)为流动相,对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖进行检测;
3)采用外标法计算出1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖含量。
其中,色谱柱:Acclaim RSLC120 C18(2.0×100mm,2.2μm),流速:0.2ml/min,柱温:20~50℃,进样量:2μl,流动相:乙腈-水(35:65)。
质谱条件为:离子源:电喷雾电离源(ESI),负离子模式:选择定量(SRM)模式,喷雾电压(IS):2800V;蒸发温度200℃;鞘气(Gas1):35Arb;辅助气(Gas2):7Arb;离子传输管温度(Ion Transfer Tube Temp):325℃;用于定量分析的离子对为m/z184.962→m/z35.167(D-核糖)及m/z168.912→m/z35.250(5-脱氧-D-呋喃核糖)。
本发明的优势是:
1、D-核糖及5-脱氧-D-呋喃核糖是1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖生产过程中的起始物料和中间体。本发明首次建立了1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖的检测方法,便于对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖的质量进行控制,从而提高了卡培他滨的用药安全性。
2、分离效果好、灵敏度高
在高效液相色谱法中D-核糖及5-脱氧-D-呋喃核糖灵敏度不高,不能在1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中有效检出,所以我们采用灵敏度极高的质谱分析法来控制1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖含量。本发明首次建立了LC-MS的检测方法,并对方法及D-核糖及5-脱氧-D-呋喃核糖进行了优化,D-核糖及5-脱氧-D-呋喃核糖优化结果见图1~2,方法优化使这两种成分与1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖能够得到很好的分离。从图3~6可以看出:D-核糖RT为1.39min;5-脱氧-D-呋喃核糖RT为1.45min;1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖峰RT为4.33min,D-核糖及5-脱氧-D-呋喃核糖与1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖分离效果好。
3、方法的专属性强、快速、灵敏、准确
经实验证明,该方法具有专属性强(专门针对D-核糖及5-脱氧-D-呋喃核糖)、快速、灵敏(检测限分别为0.04140μg/ml及0.04240μg/ml,定量限分别为0.08280μg/ml及0.08480μg/ml)、准确(100.16%~103.62%及101.37%~104.15%)等优点,能够可靠地对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中的D-核糖及5-脱氧-D-呋喃核糖含量进行定性定量分析。
附图说明
图1为D-核糖优化子离子和碰撞能图;
图2为5-脱氧-D-呋喃核糖优化子离子和碰撞能图;
图3为D-核糖(RT:1.39min)及5-脱氧-D-呋喃核糖质谱图(RT:1.45min);
图4为样品谱图(RT:4.33min);
图5为D-核糖峰提取图;
图6为5-脱氧-D-呋喃核糖峰提取图。
具体实施方式
实施例1
1仪器与材料
1.1仪器:三重四级杆液质联用仪(Thermo Ultimate 3000-TSQ Quantiva);
1.2试剂:乙腈为色谱级,水为超纯水。
2方法与结果
2.1色谱和质谱条件
色谱柱:Acclaim RSLC120 C18(2.0×100mm,2.2μm),流速:0.2ml/min,柱温:20~50℃,进样量:2μl,流动相:乙腈-水(35:65)。
质谱条件为:离子源:电喷雾电离源(ESI),负离子模式:选择定量(SRM)模式,喷雾电压(IS):2800V;蒸发温度200℃;鞘气(Gas1):35Arb;辅助气(Gas2):7Arb;
离子传输管温度(Ion Transfer Tube Temp):325℃;用于定量分析的离子对为m/z184.962→m/z35.167(D-核糖)及m/z168.912→m/z35.250(5-脱氧-D-呋喃核糖),质谱条件如表1所示。
表1质谱条件表
2.2溶液的制备
2.21对照品溶液的制备
分别取D-核糖及5-脱氧-D-呋喃核糖对照各约20mg,精密称定,置同一100ml量瓶中,加流动相(乙腈-水=35:65)溶解并稀释至刻度,摇匀;精密量取1ml,置100ml量瓶中,用流动相稀释至刻度,摇匀。
2.22线性溶液的制备
分别取D-核糖及5-脱氧-D-呋喃核糖对照各约20mg,精密称定,置同一100ml量瓶中,加流动相溶解并稀释至刻度,摇匀;精密量取1ml,置10ml量瓶中,用流动相稀释至刻度,摇匀,分别取0.2ml、0.5ml、0.8ml、1.0ml、1.2ml、1.5ml、1.8ml稀释至10ml,各溶液分别为0.02%、0.05%、0.08%、0.10%、0.12%、0.15%、0.18%。
2.22样品溶液的制备
供试品溶液:取1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖约20mg共六份,精密称定,置10ml量瓶中,加流动相溶解并稀释至刻度,摇匀。计算方法的重复性。不同日期对同一样品进行测定,计算方法的中间精密度。配制样品加不同浓度D-核糖及5-脱氧-D-呋喃核糖对照品溶液的供试品溶液,计算方法的准确度。
3分析方法的验证
3.1线性关系
取系列稀释对照品溶液进行LC-MS测定,所得结果以D-核糖及5-脱氧-D-呋喃核糖的浓度为横坐标,面积为纵坐标,绘制标准工作曲线。D-核糖线性方程为y=84766.9174x-13984.9979,r=0.9967;5-脱氧-D-呋喃核糖线性方程为y=53161.4534x+7733.4983,r=0.9957。
取浓度为2.07μg/ml的D-核糖溶液及浓度为2.12μg/ml的5-脱氧-D-呋喃核糖连续进样六次,D-核糖得到的峰面积RSD=1.05%,保留时间RSD=0%;5-脱氧-D-呋喃核糖得到的峰面积RSD=1.63%,保留时间RSD=0.28%,数据及结果见表2。
表2仪器精密度测试数据及结果
3.2重复性与中间精密度
取同一1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖样品配置的六份溶液进行重复性实验,结果见表3-1及表3-2。
表3-1 D-核糖重复性结果
表3-2 5-脱氧-D-呋喃核糖重复性结果
不同日期取重复试验的同一1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖样品配置的六份溶液进行重复性实验,结果见表4-1及表4-2。
表4-1 D-核糖中间精密度结果
表4-2 5-脱氧-D-呋喃核糖中间精密度结果
3.3准确度与耐用性
配制样品加不同浓度D-核糖及5-脱氧-D-呋喃核糖对照品溶液的供试品溶液,计算方法的准确度。结果见表5-1及表5-2。
表5-1 D-核糖准确度结果
表5-2 5-脱氧-D-呋喃核糖准确度结果
3.4耐用性:取对照品溶液,供试品溶液,在18小时内,重复进样,结果显示两者均稳定。
3.5检测限:方法的检测限(LOD)为取信噪比S/N>3倍时的D-核糖及5-脱氧-D-呋喃核糖的进样浓度,通过计算分别为0.04140μg/ml及0.04240μg/ml。
3.6定量限:方法的定量限(LOQ)为取信噪比S/N>10倍时的D-核糖及5-脱氧-D-呋喃核糖的进样浓度通过计算分别为0.08280μg/ml及0.08480μg/ml。
4讨论
在高效液相色谱仪中使用紫外检测器、示差折光检测器均不能准确控制1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖的含量。本文建立了LC-MS的检测方法,并对方法进行了优化,使这两种成分与供试品能够得到很好的分离(如图3-6)。经实验证明,该方法具有专属性强、快速、灵敏、准确等优点,能够可靠地对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖的含量进行定性定量分析。
D-核糖及5-脱氧-D-呋喃核糖均无警示结构。根据ICH Q3A(R2)的要求,我们将控制限度订为≤0.10%。
Claims (8)
1.一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,
1)将待测1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖样品用流动相溶解;
2)采用LC-MS检测方法,以Acclaim RSL C120 C18为色谱柱,以乙腈-水为流动相,对1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖进行检测;
3)计算1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖含量。
2.如权利要求1所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,所述流动相中乙腈-水的体积比为35:65。
3.如权利要求1所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,色谱柱的流速:0.2ml/min,柱温:20~50℃,进样量:2μl。
4.如权利要求1所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,质谱条件为:离子源:电喷雾电离源ESI,负离子模式:选择定量SRM模式,喷雾电压:2800V;蒸发温度200℃。
5.如权利要求4所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,鞘气Gas1:35Arb;辅助气Gas2:7Arb;离子传输管温度:325℃。
6.如权利要求4所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,用于定量分析的离子对为:D-核糖m/z184.962→m/z35.167,5-脱氧-D-呋喃核糖m/z168.912→m/z35.250。
7.如权利要求6所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,D-核糖RT为1.39min;5-脱氧-D-呋喃核糖RT为1.45min;1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖峰RT为4.33min。
8.如权利要求7所述的一种卡培他滨中间体中D-核糖及5-脱氧-D-呋喃核糖的检测方法,其特征是,所述步骤3)采用外标法计算1,2,3-三-O-乙酰基-5-脱氧-β-D-呋喃核糖中D-核糖及5-脱氧-D-呋喃核糖含量。
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