CN116413353A - 一种采用亲水色谱法检测双羟基奥沙利铂的方法 - Google Patents
一种采用亲水色谱法检测双羟基奥沙利铂的方法 Download PDFInfo
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Abstract
本发明提供了一种采用亲水色谱法检测双羟基奥沙利铂的方法,可用于奥沙利铂原料药和制剂中双羟基奥沙利铂杂质的测定。该方法采用以酰胺键合硅胶为填料的色谱柱,流动相为60~80%乙腈水溶液,使用等度洗脱。该方法专属性强、准确度好、灵敏度高、精密度好。
Description
技术领域
本发明涉及药物分析领域,具体涉及奥沙利铂中双羟基奥沙利铂杂质含量的测定方法。
背景技术
双羟基奥沙利铂是奥沙利铂(如式1)的氧化产物,又称(1R -反式)-(1,2-环己二胺-N,N')[ 草酸(2-)O,O'] 二羟基合铂,具体结构如式2:
双羟基奥沙利铂是奥沙利铂的氧化降解产物,各国药典在奥沙利铂原料药/制剂项下均对其含量进行了控制。然而药典方法均是采用普通反相色谱体系,在磷酸水-乙腈的酸性条件下,利用十八烷基硅胶键合硅胶柱对双羟基奥沙利铂及其他杂质进行分离检测。以中国药典2020年版为例,流动相条件为磷酸溶液(取10%磷酸溶液0.6ml,用水稀释成1000ml,用氢氧化钠溶液或磷酸调节pH至3.0):乙腈(99:1),利用奥沙利铂与双羟基奥沙利铂及其他杂质极性的不同,分离检测双羟基奥沙利铂及其他杂质。但采用这种方法进行测定,存在双羟基奥沙利铂与附近其他杂质分离不佳问题;同时因奥沙利铂柱上降解导致主峰前基线抬高,而影响双羟基奥沙利铂及其他杂质检测的问题。
目前国内外关于奥沙利铂的研究主要侧重在临床及不良反应,杂质研究文献较少。且现有杂质研究均以高比例水相为流动相,采用C18柱为分析柱,与中国药典方法类似,未能很好的改善双羟基奥沙利铂与其他杂质分离效果不佳、主峰前基线抬高影响双羟基奥沙利铂杂质检测的问题。采用药典方法的典型图谱见附图1。而双羟基奥沙利铂是奥沙利铂的重要降解杂质,是影响奥沙利铂原料药/制剂质量的重要杂质检查项,因此,开发一种适用于奥沙利铂中双羟基奥沙利铂的检测方法具有重要意义。
发明内容
针对现有药典方法中对奥沙利铂中双羟基奥沙利铂杂质控制方法存在的缺陷和不足,本发明提供了一种检测奥沙利铂中的双羟基奥沙利铂杂质的方法,该方法采用亲水色谱法(HILIC)检测,专属性好、准确度高、重复性好、操作简单、快速。
为了实现上述发明目的,本发明提供了如下方案。
本发明的一种采用亲水色谱法检测双羟基奥沙利铂的方法,其特征在于,该方法包含:色谱柱为酰胺键合硅胶为填料的色谱柱,流动相为60~80%乙腈水溶液和等度洗脱。
上述本发明的方法,流速1.0ml/min,柱温30℃,检测器为紫外检测器,检测波长210nm,进样量为5~10μl。
上述本发明的方法,优选的,色谱柱为XBridge (BEH) Amide柱,流动相为65~75%乙腈水溶液,更优选为70%乙腈水溶液。
上述本发明的方法,进一步包括样品溶液的制备,包含:
(1)奥沙利铂对照品溶液的制备:取奥沙利铂对照品适量,加水配制成对照品溶液;
(2)双羟基奥沙利铂定位溶液的制备:取双羟基奥沙利铂对照品适量,加水配制成双羟基奥沙利铂定位溶液;
(3)供试品溶液的制备:取奥沙利铂注射液原液适量,或,用水适当稀释,作为供试品溶液;或,
取奥沙利铂原料药适量,加入适量水超声溶解配制成供试品溶液;或,
取除奥沙利铂注射液之外的其他奥沙利铂注射剂适量,配制成供试品溶液。
本发明的方法,优选的,可与质谱联用。
本发明的一种采用亲水色谱法检测双羟基奥沙利铂的方法,可用于奥沙利铂原料药和制剂中双羟基奥沙利铂含量的测定。
在一具体实施方案中,本发明的一种采用亲水色谱法检测双羟基奥沙利铂的方法,包括以下步骤:
(1)溶液的制备:
a. 奥沙利铂对照品溶液的制备:取奥沙利铂对照品适量,加水配制成对照品溶液;
b. 双羟基奥沙利铂定位溶液的制备:取双羟基奥沙利铂对照品适量,加水配制成双羟基奥沙利铂定位溶液;
c. 供试品溶液的制备:取奥沙利铂注射液原液适量,或用水适当稀释,作为供试品溶液;或,取奥沙利铂原料药适量,加入适量水超声溶解配制成供试品溶液;或,取除奥沙利铂注射液之外的其他奥沙利铂注射剂适量,配制成供试品溶液。
(2)色谱条件:采用以酰胺键合硅胶为填料的色谱柱,柱温为30℃,流动相为60%~80%乙腈水溶液,进行等度洗脱,进样量为5-10μl,流速为1.0ml/min,检测波长210nm。
(3)样品测定:将步骤(1)的奥沙利铂对照品溶液、双羟基奥沙利铂定位溶液、供试品溶液注入液相色谱仪。按照加校正因子的主成分外标法,计算双羟基奥沙利铂杂质的含量。
在上述具体实施方案中,本发明的方法,优选的,以酰胺键合硅胶为填料的色谱柱为waters XBridge BEH Amide柱,更优选waters XBridge BEH Amide, 250×4.6mm, 5μm或waters XBridge Amide, 250×4.6mm, 3.5μm。
在上述具体实施方案中,本发明的方法,优选的,流动相乙腈水溶液中乙腈比例为65%~75%,更优选为70%。
奥沙利铂为第三代铂类抗肿瘤药,临床应用剂型为注射剂,包括注射用奥沙利铂与奥沙利铂注射液,注射用奥沙利铂辅料为甘露醇、乳糖,不具有紫外吸收,在本发明色谱条件下不干扰双羟基奥沙利铂测定,因此,本发明的方法也同样适用于注射用奥沙利铂中双羟基奥沙利铂含量的测定。
本发明利用奥沙利铂与双羟基奥沙利铂含氨基、羰基与羟基(双羟基奥沙利铂)易与酰胺形成氢键作用的特点,设计以酰胺柱为分析柱的HILIC模式分析方法,同时利用亲水作用色谱特有亲水层,应用亲水分配作用与氢键作用,进行双羟基奥沙利铂含量研究,适用于奥沙利铂原料药/制剂中双羟基奥沙利铂的分析,检测限达到1.52μg/ml,定量限达到3.03μg/ml。与现有方法相比,具有明显优点:无其他杂质干扰,专属性强;弱化了奥沙利铂柱上降解基线抬高对双羟基奥沙利铂检测的影响,准确度高、重复性好;且流动相配制简单、检测成本低。
附图说明
图1 采用中国药典2020中的“双羟基奥沙利铂(杂质III)及其他杂质”分析方法得到的典型图谱(双羟基奥沙利铂出峰位置恰好在基线上抬处,影响双羟基奥沙利铂检测);
图2 实施例1中流动相为50%乙腈溶液时的图谱;
图3 实施例1中流动相为60%乙腈溶液时的图谱;
图4 实施例1中流动相为65%乙腈溶液时的图谱;
图5 实施例1中流动相为70%乙腈溶液时的图谱;
图6 实施例1中流动相为75%乙腈溶液时的图谱;
图7 实施例1中流动相为80%乙腈溶液时的图谱;
图8 实施例1中流动相为90%乙腈溶液时的图谱;
图9 实施例2中未破坏供试品图谱;
图10 实施例2中酸破坏供试品图谱;
图11 实施例2中热破坏供试品图谱;
图12 实施例2中氧化破坏供试品图谱;
图13 实施例2中碱破坏供试品图谱;
图14 实施例2中光破坏供试品图谱。
具体实施方式
以下实施例用于进一步说明和理解本发明的精神实质,但不以任何方式限制本发明的保护范围。
实施例 1 流动相比例考察
检测器与色谱条件
HPLC仪:岛津,LC-20AD,PDA检测器;
色谱柱:Waters XBridge BEH Amide(250×4.6mm, 5μm);柱温30℃;进样盘不控温;采集时间:20min;流速为1.0ml/min;检测波长:210nm;进样量5μl。
实验步骤:
(1)样品配制:取奥沙利铂注射液样品适量,作为供试品溶液(含奥沙利铂约5mg/ml)。
(2)检测:取上述样品溶液5μl注入液相色谱仪,记录色谱图。典型色谱图见图2-图8。
表1 流动相比例考察结果
结果显示:双羟基奥沙利铂对流动相中水相比例敏感,如图2所示,50%乙腈水溶液作为流动相时,双羟基奥沙利铂落在奥沙利铂峰上,影响双羟基奥沙利铂积分;如图8所示,90%乙腈水溶液作为流动相时,双羟基奥沙利铂出峰时间过于长,峰形严重展宽致使其掩埋与基线中,无法积分。以60%~80%乙腈水溶液作为流动相,奥沙利铂与双羟基奥沙利铂分离良好,同时双羟基奥沙利铂峰形良好,适用于奥沙利铂中双羟基奥沙利铂的含量测定,其中优选比例为65%~75%乙腈水溶液,进一步优选比例为70%乙腈水溶液。
实施例2 专属性考察
检测器与色谱条件:
HPLC仪:Waters,e2695,PDA检测器;
色谱柱:Waters XBridge BEH Amide(250×4.6mm, 3.5μm);柱温30℃;进样盘不控温;流速为1.0ml/min;检测波长:210nm;进样量10μl;
流动相比例:70%乙腈。
实验步骤:
(1)样品配制:
定位溶液:取双羟基奥沙利铂对照品约1.5mg,置100ml量瓶中,加水溶解并稀释至刻度,摇匀,即得(含双羟基奥沙利铂约15μg/ml);
未破坏供试品溶液:取奥沙利铂原料药约50mg,置10ml量瓶中,加水稀释至刻度,摇匀即得(含奥沙利铂约5mg/ml);
光破坏供试品溶液:取奥沙利铂注射液一支,置4500±500lux强度光下放置5天,取原液作为光破坏供试品溶液(含奥沙利铂约5mg/ml);
热破坏供试品溶液:取奥沙利铂原料药约50mg,精密称定,置10ml量瓶,加水超声溶解并稀释至刻度,摇匀,于70℃水浴放置4h,即得(含奥沙利铂约5mg/ml);
酸破坏供试品溶液:取奥沙利铂原料药约50mg,精密称定,置10ml量瓶,精密加入8ml水超声溶解后,精密加入0.1mol/L盐酸溶液0.5ml,室温放置2h后,精密加入0.1mol/L氢氧化钠溶液0.5ml,加水稀释至刻度,摇匀,即得(含奥沙利铂约5mg/ml);
碱破坏供试品溶液:取奥沙利铂原料药约50mg,精密称定,置10ml量瓶,精密加入8ml水超声溶解后,精密加入0.1mol/L氢氧化钠溶液0.5ml,室温放置2h后,精密加入0.1mol/L盐酸溶液0.5ml,加水稀释至刻度,摇匀,即得(含奥沙利铂约5mg/ml);
氧化破坏供试品溶液:取奥沙利铂原料药约50mg,精密称定,置10ml量瓶,加适量水超声溶解后,精密加入0.3%过氧化氢溶液0.2ml,加水稀释至刻度,摇匀,室温放置2h,即得(含奥沙利铂约5mg/ml)。
(2)检测:取上述各样品溶液10μl注入液相色谱仪,记录色谱图。典型色谱图见图9-图14。
表2专属性试验结果
结果显示:在各破坏条件下,其他杂质均不干扰奥沙利铂原料药/注射液中双羟基奥沙利铂的检测;对比图9-图14,在氧化条件下(图12)双羟基奥沙利铂峰面积显著增加,奥沙利铂极易氧化成双羟基奥沙利铂。
实施例3 线性考察
检测器与色谱条件:
HPLC仪:Aglient,1260,VWD检测器;
流动相比例:70%乙腈,其余色谱条件同实施例1。
试验步骤:
(1)样品配制:
双羟基奥沙利铂储备液:取双羟基奥沙利铂对照品约20mg,精密称定,置20ml量瓶中,加水超声使溶解并稀释至刻度,摇匀,即得(含双羟基奥沙利铂约1mg/ml);
线性储备液:取奥沙利铂原料药10mg,精密称定,置20ml量瓶中,再精密量取双羟基奥沙利铂对照品储备液3ml至同一量瓶中,加水超声溶解并稀释至刻度,摇匀,即得(含奥沙利铂约500μg/ml、双羟基奥沙利铂约150μg/ml);
取线性储备液照下表用稀释剂稀释制成系列浓度的线性溶液:
表3 线性溶液配制方法
(2)检测:取各线性线性溶液5μl注入液相色谱仪,记录色谱图。
结果显示,在10.19μg/ml ~101.89μg/ml范围内,奥沙利铂线性方程为y =4079.7581 x - 770.9024(y为峰面积,x为浓度),相关性系数r为1.0000;在3.03μg/ml ~30.30μg/ml范围内,双羟基奥沙利铂线性方程为y = 16845.1805 x - 3438.4634(y为峰面积,x为浓度),相关性系数r为1.0000,表明方法线性良好。
实施例4 检测限与定量限考察
检测器与色谱条件同实施例3
(1)溶液配制:
定量限溶液:同实施例3中线性溶液1(含双羟基奥沙利铂约3μg/ml);
检测限溶液:精密量取定量限溶液5ml,置10ml量瓶中,用水稀释至刻度,摇匀,即得(双羟基奥沙利铂约1.5μg/ml)。
(2)检测:取定量限溶液连续进样6次,检测限溶液进样1次,进样量5μl,记录色谱图。
结果显示,6针定量限溶液中,双羟基奥沙利铂峰S/N分别为56、71、61、84、69、58,均大于10,峰面积RSD为2.0%(表明仪器进样精密度良好);检测限溶液中,双羟基奥沙利铂峰S/N为28,大于3。双羟基奥沙利铂检测限为1.52μg/ml,定量限为3.03μg/ml。
实施例5 精密度和准确度考察
检测器与色谱条件同实施例3
(1)溶液配制:
定位溶液:照实施例2项下配制;
对照品溶液:取奥沙利铂原料药约20mg,精密称定,置20ml量瓶中,加水超声使溶解并稀释至刻度,摇匀;精密量取上述溶液1ml,置20ml量瓶中,用水稀释至刻度,摇匀,即得(奥沙利铂约0.05mg/ml);
准确度储备液:同实施例3中,线性储备液(含双羟基奥沙利铂约150μg/ml);
空白供试品溶液:取奥沙利铂原料药50mg,精密称定,置10ml量瓶中,加水超声使溶解并稀释至刻度,摇匀,即得(平行配制3份);
低浓度水平溶液:取奥沙利铂原料药50mg,精密称定,置10ml量瓶中,再精密量取准确度储备液0.2ml,加水超声使溶解并稀释至刻度,摇匀,即得(平行配制3份)(含双羟基奥沙利铂约3μg/ml);
中浓度水平溶液:取奥沙利铂原料药50mg,精密称定,置10ml量瓶中,再精密移取准确度储备液1ml,加水超声使溶解并稀释至刻度,摇匀,即得(平行配制3份)(含双羟基奥沙利铂约15μg/ml);
高浓度水平溶液:取奥沙利铂原料药50mg,精密称定,置10ml量瓶中,再精密移取准确度储备液1.5ml,加水超声使溶解并稀释至刻度,摇匀,即得(平行配制3份)(含双羟基奥沙利铂约22.5μg/ml);
(2)检测:分别取各浓度水平溶液5μl注入液相色谱仪,记录色谱图。
(3)计算:计算9份各浓度水平溶液的回收率及回收率RSD。
表4 准确度结果
结果显示,9份供试品溶液中回收率为91.9%~102.4%,平均回收率为97.6%,均在90.0%~110.0%范围内,回收率的RSD为3.1%,小于10.0%,表明本方法准确可靠,精密度好,适用于奥沙利铂原料药/制剂中双羟基奥沙利铂含量。
Claims (7)
1.一种采用亲水色谱法检测双羟基奥沙利铂的方法,其特征在于,该方法包含:色谱柱为酰胺键合硅胶为填料的色谱柱,流动相为60~80%乙腈水溶液,等度洗脱。
2.根据权利要求1所述的方法,流速1.0ml/min,柱温30℃,检测器为紫外检测器,检测波长210nm,进样量为5~10μl。
3.根据权利要求1或2所述的方法,所述色谱柱为XBridge (BEH) Amide柱。
4.根据权利要求1或2所述的方法,所述流动相为65~75%乙腈水溶液。
5.根据权利要求4所述的方法,所述流动相为70%乙腈水溶液。
6.根据权利要求1~5任一所述的方法,进一步包括样品溶液的配制,包含:
(1)奥沙利铂对照品溶液的制备:取奥沙利铂对照品适量,加水配制成对照品溶液;
(2)双羟基奥沙利铂定位溶液的制备:取双羟基奥沙利铂对照品适量,加水配制成双羟基奥沙利铂定位溶液;
(3)供试品溶液的制备:取奥沙利铂注射液原液适量,或,用水适当稀释,作为供试品溶液;
或,取奥沙利铂原料药适量,加入适量水超声溶解配制成供试品溶液;
或,取除奥沙利铂注射液之外的其他奥沙利铂注射剂适量,配制成供试品溶液。
7.根据权利要求1~5任一所述方法,所述方法可与质谱联用。
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