CN103175904B - 保健食品中原花青素的测定方法 - Google Patents
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Abstract
本发明公开了一种保健食品中原花青素的测定方法,包括如下步骤:(1)称取原料0.25g置于容量瓶,加入甲醇,超声处理,冷却至室温,以甲醇定容至刻度,摇匀后过滤膜,滤液供高效液相色谱-串联质谱测定;(2)精确称取原花青素标准品10mg于容量瓶中,用色谱纯甲醇超声溶解定容至刻度,为标准储备液;将所述标准储备液用甲醇稀释,配成1.0μg/mL,2.0μg/mL,5.0μg/mL和10.0μg/mL的标准溶液;分别注入高效液相色谱-串联质谱测定,得到标准曲线;(3)依据标准曲线计算待测样品中原花青素浓度;本发明的方法准确度好、精度高、线性良好,且便于开展保健食品中原花青素含量的测定。
Description
技术领域
本发明涉及一种保健食品中原花青素的测定方法。
技术背景
原花青素,简称OPC,是一种有着特殊分子结构的生物类黄酮,是目前国际上公认的清除人体内自由基最有效的天然抗氧化剂。由于其特殊的健康功能,目前多种抗氧化类保健食品中均以原花青素作为主要功效成分,其来源有葡萄、蓝莓、樱桃、李子和松树皮等,主要成分是一类双黄酮衍生物的天然多酚化合物。
研究表明原花青素不仅具有很强的抗氧化活性,同时还具有良好的抗肿瘤活性、血管内皮细胞保护作用、调节血压功能、抗炎症和过敏功能,是多种功能性食品的有效成分,其成分鉴别和含量测定是评价保健食品功能的重要指标。
目前对原花青素的检测方法主要有:
一、分光光度法:
此方法的利用其原理是原花青素在无机酸存在和加热的条件下被降解,产生红色花青素,在525nm处有最大吸收。原花青素的含量与吸光度值符合朗伯一比尔定律。原花青素在正丁醇一盐酸溶液中氧化降解,生成红色物质,可用于测定原花青素类化合物。此方法具有代表性的是2007版的《保健食品评价与技术规范》。此法采用分光法测定,准确度较低。
二、直接HPLC分析法
此法利用HPLC良好的分离性能,利用有机溶剂提取后进行色谱分析,该方法要求色谱条件能够良好的分离原花青素中单体、二聚体和其他寡聚体,对低分子量的花青素效果较好。
但考虑到保健食品中花青素的组成比较复杂,单一等度色谱条件难以适用于广泛产品的检测。
发明内容
本发明的目的是克服现有技术的问题,提供一种保健食品中原花青素的测定方法。
本发明的技术方案概述如下:
一种保健食品中原花青素的测定方法,包括如下步骤:
(1)称取原料0.25g置于25mL容量瓶,加入甲醇20mL,超声处理30min,冷却至室温,以甲醇定容至刻度,摇匀后过0.22μm滤膜,滤液供高效液相色谱-串联质谱测定;
(2)精确称取原花青素标准品10mg于10mL容量瓶中,用色谱纯甲醇超声溶解定容至刻度,为标准储备液;将所述标准储备液用甲醇稀释,配成1.0μg/mL,2.0μg/mL,5.0μg/mL和10.0μg/mL的标准溶液;分别注入高效液相色谱-串联质谱测定,得到标准曲线;
(3)依据标准曲线计算待测样品中原花青素浓度;
LC测定条件
a)色谱柱:XTerra MS C18,150mm×2.1mm,3.5μm;
b)流动相A为甲醇,流动相B为水,流动相C为5%的甲酸水溶液;
0~20分钟:流动相A从82%到72%,流动相B从14%到24%,流动相C保持4%;
20~50分钟:流动相A从72%到61%,流动相B从24%到35%,流动相C保持4%;
50~55分钟:流动相A从61%到11%,流动相B从35%到85%,流动相C保持4%;
55~65分钟:流动相A保持11%,流动相B保持85%,流动相C保持4%;
65~70分钟,流动相A从11%到82%,流动相B从85%到14%,流动相C保持4%;
70~80分钟,流动相A保持82%,流动相B保持14%,流动相C保持4%;
c)进样量:5μL;
d)柱温:35℃;
e)流速:0.2mL/min;
MS/MS参考条件:
a)电离源:电喷雾负离子源;
b)毛细管电压:-3.5kV;
c)源电压:130V;
d)扫描范围:200~1200amu;
e)脱溶剂气:氮气,700L/Hr,温度400℃;
f)碰撞气:氩气;
g)扫描模式:多反应检测(MRM)模式。
本发明的有益效果是:HPLC-MS-MS技术灵敏度高,选择性和特异性好,利用梯度流动相替代等度流动相,使样品峰有效分离,可对样品中低浓度的定量分析。本发明的方法准确度好、精度高、线性良好,且便于开展保健食品中原花青素含量的测定。
附图说明
图1为实施例1的原花青素素标准品液相色谱质谱图。
图2为实施例1的样品液相色谱质谱图。
具体实施方式
下面通过实例对本发明做进一步的描述,这些描述并不是对本发明内容进一步的限定。本领域的技术人员应理解,对本发明内容所做的等同替换或相应的改进,仍属于本发明的保护范围之内。
实施例1
一种保健食品中原花青素的测定方法,包括如下步骤:
(1)称取原料葡萄籽提取物0.25g置于25mL容量瓶,加入甲醇20mL,超声处理30min,冷却至室温,以甲醇定容至刻度,摇匀后过0.22μm滤膜,滤液供高效液相色谱-串联质谱测定;
(2)精确称取原花青素标准品10mg于10mL容量瓶中,用色谱纯甲醇超声溶解定容至刻度,为标准储备液;将所述标准储备液用甲醇稀释,配成1.0μg/mL,2.0μg/mL,5.0μg/mL和10.0μg/mL的标准溶液;分别注入高效液相色谱-串联质谱测定,用反相C18色谱柱进行色谱分离,质谱仪进行检测;得到标准曲线;
(3)依据浓度与响应值绘制标准曲线计算待测样品中原花青素浓度;
LC测定条件
a)色谱柱:XTerra MS C18,150mm×2.1mm,3.5μm;
b)流动相A为甲醇,流动相B为水,流动相C为5%的甲酸水溶液;
0~20分钟:流动相A从82%到72%,流动相B从14%到24%,流动相C保持4%;
20~50分钟:流动相A从72%到61%,流动相B从24%到35%,流动相C保持4%;
50~55分钟:流动相A从61%到11%,流动相B从35%到85%,流动相C保持4%;
55~65分钟:流动相A保持11%,流动相B保持85%,流动相C保持4%;
65~70分钟,流动相A从11%到82%,流动相B从85%到14%,流动相C保持4%;
70~80分钟,流动相A保持82%,流动相B保持14%,流动相C保持4%;
c)进样量:5μL;
d)柱温:35℃;
e)流速:0.2mL/min;
MS/MS参考条件:
a)电离源:电喷雾负离子源;
b)毛细管电压:-3.5kV;
c)源电压:130V;
d)扫描范围:200~1200amu;
e)脱溶剂气:氮气,700L/Hr,温度400℃;
f)碰撞气:氩气;
g)扫描模式:多反应检测(MRM)模式。
质谱参数
表1各种化合物的离子对和电压
Claims (1)
1.一种保健食品中原花青素的测定方法,其特征是包括如下步骤:
(1)称取原料0.25g置于25mL容量瓶,加入甲醇20mL,超声处理30min,冷却至室温,以甲醇定容至刻度,摇匀后过0.22μm滤膜,滤液供高效液相色谱-串联质谱测定;
(2)精确称取原花青素标准品10mg于10mL容量瓶中,用色谱纯甲醇超声溶解定容至刻度,为标准储备液;将所述标准储备液用甲醇稀释,配成1.0μg/mL,2.0μg/mL,5.0μg/mL和10.0μg/mL的标准溶液;分别注入高效液相色谱-串联质谱测定,得到标准曲线;
(3)依据标准曲线计算待测样品中原花青素浓度;
LC测定条件
a)色谱柱:XTerra MS C18,150mm×2.1mm,3.5μm;
b)流动相A为甲醇,流动相B为水,流动相C为5%的甲酸水溶液;
0~20分钟:流动相A从82%到72%,流动相B从14%到24%,流动相C保持4%;
20~50分钟:流动相A从72%到61%,流动相B从24%到35%,流动相C保持4%;
50~55分钟:流动相A从61%到11%,流动相B从35%到85%,流动相C保持4%;
55~65分钟:流动相A保持11%,流动相B保持85%,流动相C保持4%;
65~70分钟,流动相A从11%到82%,流动相B从85%到14%,流动相C保持4%;
70~80分钟,流动相A保持82%,流动相B保持14%,流动相C保持4%;
c)进样量:5μL;
d)柱温:35℃;
e)流速:0.2mL/min;
MS/MS参考条件:
a)电离源:电喷雾负离子源;
b)毛细管电压:-3.5kV;
c)源电压:130V;
d)扫描范围:200~1200amu;
e)脱溶剂气:氮气,700L/Hr,温度400℃;
f)碰撞气:氩气;
g)扫描模式:多反应检测(MRM)模式。
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US7566401B2 (en) * | 2005-10-03 | 2009-07-28 | Kelm Mark A | Process for separating and isolating xanthines, individual polar protic monomers, and polar protic oligomers |
CN101936966B (zh) * | 2010-09-22 | 2012-07-25 | 中国烟草总公司郑州烟草研究院 | 同时分析烟用香原料山楂酊中三种低聚原花青素的方法 |
CN102818863B (zh) * | 2012-06-01 | 2014-02-05 | 浙江康恩贝制药股份有限公司 | 一种鉴别银杏叶制剂中原花青素的方法 |
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