CN112816567A - 测定褪黑素和维生素b6的高效液相色谱分析方法 - Google Patents
测定褪黑素和维生素b6的高效液相色谱分析方法 Download PDFInfo
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Abstract
本发明提供一种高效测定单方及复方组分中褪黑素和维生素B6的高效液相色谱分析方法,是以甲醇‑磷酸盐缓冲溶液为流动相进行梯度洗脱,可避免其他组分的干扰,在13分钟内准确灵敏地检测出褪黑素和维生素B6的含量。该方法专属性强、精密度高、稳定性较好、准确度高,操作简单,试剂与耗材价廉易得。适用于含褪黑素、维生素B6的单方以及同时含褪黑素和维生素B6组合物的片剂、粉末、胶囊等剂型中褪黑素和维生素B6的质量控制,可有效地节约检测成本和时间。
Description
技术领域
本发明涉及药物分析技术领域,尤其涉及一种高效快速测定组方中褪黑素和维生素B6含量的液相色谱分析方法。
背景技术
维生素B6(分子式为C8H11NO3.HCl)是一种水溶性维生素,又叫吡哆素,其包括吡哆醇、吡哆醛及吡哆胺,白色或尅白色结晶或结晶性粉末,无臭,遇光渐变质,在空气中稳定;易溶于水,在乙醇中微溶,在酸性水溶中下稳定,碱性溶液中易破坏。维生素B6在人类生长发育过程中,发挥着非常重要的生理作用,为人体内某些辅酶的组成成分,参与多种代谢反应,尤其与氨基酸代谢密切相关。
褪黑素(分子式为C13N2H16O2)又叫褪黑激素,是一种能使皮肤色素颜色变浅的激素物质,是由必需氨基酸色氨酸合成而来,白色类白色结晶粉末,易溶于甲醇,对光线敏感,对空气敏感,易氧化,具有抗氧化、抗炎和抗凋亡等特性。
目前褪黑素和维生素B6的含量测定方法包括放射免疫(RIA)法、酶联免疫(HJSA)法、紫外分光光度(UV)法、气相色谱(GC)法和气相色谱-质谱联用(GC-MS)法、高效液相色谱(HPLC)法、高效液相色谱-质谱联用(HPLC-MS)法、毛细管电泳法、化学发光法、微生物法等。放射免疫(RIA)法、酶联免疫(HJSA)法、微生物法样品前处理相对繁琐,检测周期长;由于褪黑素的挥发性不好,采用气相色谱(GC)法和气相色谱-质谱联用(GC-MS)法检测样品前,需要对样品进行衍生化前处理,操作过程冗长,重复性差;紫外分光光度(UV)法、毛细管电泳法、化学发光法专属性较差;高效液相色谱(HPLC)法或高效液相色谱-质谱联用(HPLC-MS)法在检测褪黑素和维生素B6较常用,但一般是一种方法检测一个组分,复方制剂就需要进行多次检测,才能得到准确结果,对人力物力造成很大浪费;而且检测过程中经常会使用离子对、三乙胺等试剂,严重影响精密仪器和色谱柱的使用寿命;因此,很有必要研究一种高效准确快速低成本且能同时测定复方组分中褪黑素和维生素B6的液相色谱方法。
发明内容
为克服上述问题,本发明提供一种前处理简单,成本较低,准确度高,专属性强、重复性好、快捷的褪黑素和维生素B6的高效液相色谱分析方法,所述方法包括以下步骤:
一种用于测定片剂、粉剂、胶囊剂型产品中褪黑素和维生素B6含量的高效液相色谱分析方法,供试品的提取溶剂为含水溶液,提取方法为超声提取;流动相A为甲醇,流动相B为磷酸盐缓冲溶液,梯度洗脱,流动相A和流动相B梯度变化如下:起始比例为甲醇的体积含量10%,磷酸盐缓冲溶液体积含量90%;2-2.01min,甲醇的体积含量由10%变为50%,磷酸盐缓冲溶液体积含量由90%变为50%;2.01-8min,甲醇的体积含量保持为50%,磷酸盐缓冲溶液体积含量保持为50%;8-8.01min,甲醇的体积含量由50%变为10%,磷酸盐缓冲溶液体积含量由50%变为90%;8.01-13min,甲醇的体积含量保持为10%,磷酸盐缓冲溶液体积含量保持为90%。在本发明中,流速0.8~1.5ml/min,优选0.8-1.2ml/min。在本发明中,紫外检测波长设定为210~279nm,优选260~279nm。
在本发明中,用于供试品的提取溶剂选自甲醇溶液、水或盐酸溶液中的一种,优选盐酸溶液。其中,提取溶剂的浓度选自0.001mol/l-0.1mol/l,优选0.005mol/l-0.05mol/l,更优选0.01mol/l。
在本发明中,提取方法是水浴超声,水浴超声的温度选自25~60℃,优选30~50℃,更优选35~45℃。
在本发明中,流动相B的磷酸盐缓冲溶液为0.05~0.1M磷酸二氢钾和0.005~0.01M磷酸氢二钾的混合溶液,优选0.1M磷酸二氢钾和0.01M磷酸氢二钾的混合溶液。混合溶液的pH选自3.00~5.00,优选3.50~4.50,更优选4.00。
在本发明实施方案中,进行上述色谱分析的色谱仪器及条件为:
检测仪器:高效液相色谱仪(Thermo U3000),配备紫外检测器;
色谱条件:
用十八烷基硅烷键合硅胶为填充剂(推荐色谱柱Welch Ximate C18,4.6mm×150mm,5μm)或效能相当的色谱柱,流动相:磷酸盐缓冲液-甲醇;检测波长:210~279nm;流速:0.8~1.5ml/min;柱温:25~30℃,最佳柱温:30℃;运行时间:13min。
本发明中,采用高效液相色谱对褪黑素维生素B6片溶液中的褪黑素和维生素B6进行定量检测,需要对该方法进行专属性、准确度等多方面的确认,具体为:
专属性:配制褪黑素维生素B6片溶液和不含褪黑素维生素B6的空白辅料溶液,分别进样,结果显示表明空白溶剂、空白辅料对供试品溶液的主峰出峰无干扰,且能与相邻峰有效的分离开,说明本发明提供的方法能够准确测定褪黑素维生素B6片功效成分褪黑素维生素B6的含量。
准确度:采用本发明的高效液相色谱方法对褪黑素维生素B6片溶液中的褪黑素维生素B6进行检测,结果显示表明其回收率在合格标准范围,准确度良好。
线性:采用本发明的高效液相色谱方法对褪黑素维生素B6片中的褪黑素维生素B6对照品溶液进行检测,在线性范围40%~160%内,结果显示其主成分线性相关系数不小于0.99,表明检测结果与褪黑素维生素B6的浓度具有良好的线性关系。
本发明中,采用高效液相色谱对不同剂型样品配制成的样品溶液中的褪黑素维生素B6进行定量检测。
附图说明
图1示对照品溶液的HPLC检测的色谱图;
图2示实施例1的色谱条件下HPLC检测的色谱图;
图3示实施例2的色谱条件下HPLC检测的色谱图;
图4空白辅料示实施例1的色谱条件下HPLC检测的色谱图;
具体实施方式
下面通过实施例对本发明作进一步详细的说明,分别以片剂、胶囊的选择为例。试剂:甲醇(SINENCE,批号1803M06362),磷酸二氢钾、磷酸氢二钾(天津市大茂化学试剂厂,AR),磷酸(天津市富宇精细化工有限公司,AR),褪黑素对照品(北京北纳创联生物技术研究院),维生素B6对照品(中国食品药品检定研究院),样品(深圳芙莱特营养与健康有限公司)
实施例1:
第一步:提取溶剂,量取盐酸0.9ml,用水稀释至1000ml,摇匀,即得。
第二步:磷酸盐缓冲溶液:称取磷酸二氢钾13.6g,磷酸氢二钾1.72g,加水至1000ml,搅拌使溶解,磷酸调节pH至3.50,抽滤,即得。
第三步:对照品溶液的配制,精密称取褪黑素对照品11.0mg,维生素B6对照品24.0mg[约相当于维生素B6(吡哆醇)20mg],置100ml量瓶中,加0.01mol/L盐酸溶液50ml,超声(45℃)使溶解,冷却至室温,加0.01mol/L盐酸溶液稀释至刻度,摇匀,作为褪黑素、维生素B6混合对照品贮备液;精密量取褪黑素、维生素B6混合对照品贮备液5.0ml,置于50ml量瓶中,加0.01mol/L盐酸溶液稀释至刻度,摇匀。
第四步:样品溶液配制,取本品20片,研细,精密称取适量(约相当于褪黑素1.12mg、维生素B6(以吡哆醇计)2mg),置100ml量瓶中,加0.01mol/L盐酸溶液50ml,超声(45℃)10min,冷却至室温后,再加0.01mol/L盐酸溶液稀释至刻度,摇匀,滤过,取续滤液即得。
第五步:液相色谱条件的具体内容如下:
色谱柱:Welch-Ximate C18150mmX4.6mm,5μm
流动相:甲醇-磷酸盐缓冲溶液梯度洗脱,洗脱梯度如下:
检测波长:210nm
流速:1.0ml/min
柱温:30℃
进样体积:10μL
运行时间:13min
检测色谱图如图2,检测结果如表1
表1检测结果
名称 | 保留时间/min | 理论塔板数 | 拖尾因子 | 含量% |
褪黑素 | 2.732 | 40271 | 0.99 | 93.21 |
维生素B<sub>6</sub> | 6.560 | 11826 | 1.02 | 97.40 |
实施例2:
深圳芙莱特营养与健康有限公司生产的褪黑素维生素B6软胶囊样品中褪黑素维生素B6含量的测定。具体步骤:
第一步:提取溶剂,量取盐酸0.9ml,用水稀释至1000ml,摇匀,即得。
第二步:磷酸盐缓冲溶液:称取磷酸二氢钾6.8g,磷酸氢二钾0.86g,加水至1000ml,搅拌使溶解,磷酸调节pH至4.00,抽滤,即得。
第三步:对照品溶液的配制,精密称取褪黑素对照品11.0mg,维生素B6对照品24.0mg[约相当于维生素B6(吡哆醇)20mg],置100ml量瓶中,加0.01mol/L盐酸溶液50ml,超声(35℃)使溶解,冷却至室温,加0.01mol/L盐酸溶液稀释至刻度,摇匀,作为褪黑素、维生素B6混合对照品贮备液;精密量取褪黑素、维生素B6混合对照品贮备液5.0ml,置于50ml量瓶中,加0.01mol/L盐酸溶液稀释至刻度,摇匀。第四步:样品溶液配制,取本品20粒,剪破胶囊皮,取均匀内容物适量(约相当于褪黑素1.12mg、维生素B6(以吡哆醇计)2mg),精密称定,置100ml量瓶中,加0.01mol/L盐酸溶液50ml,超声(35℃)10min,冷却至室温后,再加0.01mol/L盐酸溶液稀释至刻度,摇匀,滤过,取续滤液即得。
第五步:液相色谱条件的具体内容如下:
色谱柱:C18150mmX4.6mm,5μm
流动相:甲醇-磷酸盐缓冲溶液梯度洗脱,洗脱梯度如下:
检测波长:278nm
流速:1.1ml/min
柱温:30℃
进样体积:10μL
运行时间:13min
检测色谱图如图3,检测结果如表2
表2检测结果
名称 | 保留时间/min | 理论塔板数 | 拖尾因子 | 含量% |
褪黑素 | 6.468 | 40874 | 1.00 | 105.81 |
维生素B<sub>6</sub> | 2.545 | 12326 | 1.02 | 91.95 |
实施例1~2显示,本发明对褪黑素维生素B6口服片剂和软胶囊样品进行检测,褪黑素维生素B6均能在10min以内出峰,峰型好,与其他成分达到基线分离,说明该方法适用于不同剂型的复方组分中褪黑素维生素B6的检测。
以上所述的具体实施例,对本发明的解决的技术问题、技术方案以及有益效果作了一系列的详细说明。但该说明仅仅是针对本发明的具体实施例而已,凡没有脱离本发明技术基础、原则和精神而作的等效实施例或变更,均应包括在本发明的保护范围之内。
Claims (7)
1.一种测定褪黑素和维生素B6含量高效液相色谱分析方法,其特征在于,供试品的提取溶剂为含水溶液,提取方法为超声提取;流动相A为甲醇,流动相B为磷酸盐缓冲溶液,梯度洗脱,紫外检测波长设定为210~279nm。
2.根据权利要求1所述的分析方法,其特征在于,流动相梯度洗脱程序为:0-2min,甲醇的体积含量保持为10%,磷酸盐缓冲液的体积含量保持为90%;2-2.01min,甲醇的体积含量由10%变为50%,磷酸盐缓冲液的体积含量由90%变为50%;2.01-8min,甲醇的体积含量保持为50%,磷酸盐缓冲液的体积含量保持为50%;8-8.01min,甲醇的体积含量由50%变为10%,磷酸盐缓冲液的体积含量由50%变为90%;8.01-13min,甲醇的体积含量为10%,磷酸盐缓冲液的体积含量为90%。
3.根据权利要求1所述的分析方法,其特征在于,所述用于供试品的提取溶剂选自甲醇溶液、水或盐酸溶液中的一种,优选盐酸溶液。
4.根据权利要求3所述的分析方法,其特征在于,所述提取溶剂的浓度选自0.001mol/L-0.1mol/L,优选0.005mol/L-0.05mol/L,更加优选0.01mol/L。
5.根据权利要求1所述的分析方法,其特征在于,所述提取方法是水浴超声,温度选自25~60℃,优选30~50℃,更加优选35~45℃。
6.根据权利要求1所述的分析方法,其特征在于,作为流动B的磷酸盐缓释液选自0.05~0.1M磷酸二氢钾和0.005~0.01M磷酸氢二钾的混合溶液,优选0.1M磷酸二氢钾和0.01M磷酸氢二钾的混合溶液。
7.根据权利要求6所述的分析方法,其特征在于,所述混合溶液的pH值选自3.00~5.00,优选3.50~4.50,更优选4.00。
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