CN114994210A - 一种植物甾醇总量快速检测的前处理方法 - Google Patents
一种植物甾醇总量快速检测的前处理方法 Download PDFInfo
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Abstract
本发明公开了一种快速检测植物甾醇总量的前处理方法,包括以下步骤:S1:称取待测样品,加入内标溶液和水,在室温下超声波水浴处理;S2:向经过步骤S1处理的溶液加入皂化剂,在室温下超声波水浴处理后,进行加热处理,完成皂化;S3:将经过步骤S2处理的溶液冷却至室温后,加入萃取液进行萃取,萃取完成后,进行离心,提取上层有机液层,向上层有机液层加入脱水剂进行脱水,用氮气吹干;S4:向步骤S3所制得的浓缩液中加入硅烷化试剂,加热处理后,冷却至室温,制得待测样品。采用本发明的前处理方法,使得在进行气相色谱分析的前处理中不需要制备氧化铝柱和配制展开剂,有效地简化了前处理的步骤,从而降低了检测的成本和时间。
Description
技术领域
本发明涉及化学分析技术领域,具体涉及一种植物甾醇总量快速检测的前处理方法。
背景技术
植物甾醇是植物中的一种活性成分,与胆固醇具有同样的环状结构,区别在于其侧链不同。研究表明,植物甾醇具有降低胆固醇,预防动脉粥样硬化、抑制肿瘤细胞增殖等功能活性,被广泛应用于食品、药品领域。人体不能转化合成植物甾醇,只能通过食物摄取。植物甾醇广泛存在于植物的根、茎、叶和果实中,在植物油中含量较高,是人体摄取植物甾醇的重要途径。
目前,我国在植物甾醇检测方面尚未形成强制性标准文件,然而植物甾醇组分多,现有研究方法步骤繁琐,检测成本大,导致植物甾醇的质量控制不能大规模开展。目前检测方法主要有薄层色谱法、高效液相色谱法、气相色谱法和气相质谱法等。
薄层色谱法,检测前的处理是利用不同物质在一定吸附剂与展开剂中吸附和解吸能力的差异,使各个被测组分以不同的速度移动,最后达到分离的一种方法,可用于甾醇的分离、纯化和检测。薄层色谱法应用在甾醇检测领域一般有两个目的,一是定性鉴别,通过良好的分离,根据斑点的比移值(retention factor,Rf)和颜色深浅,判断某甾醇的存在与否,二是定量测定,对上述薄层板进行扫描,利用标准曲线可以准确测出样品中甾醇的含量。薄层色谱法的样品前处理比较繁琐且重复性差。
液相色谱法对样品进行前处理时,在经过皂化之后还需冷冻2h,从而导致前处理比较费时。
现有的气相色谱法适合于分析挥发性强的物质,而对于发挥性弱、热稳定性差的物质往往不能直接进样分析,需将这类物质进行适当的化学处理转化成相应的挥发性衍生物,这类物质才能够得到较好的分离。植物甾醇分子中含有极性羟基,挥发性差,在高温下容易失水或者分解,所以需要对样品进行衍生化处理,才能得到很好的分离。目前一般的衍生化方法包括硅烷化、酯化和酰化。前处理分离甾醇时,一般使用HP-5等非极性柱进行过柱,现有的气相质谱法在前处理阶段同样需要过柱,因此现有的气相色谱法或者气相质谱法的前处理方法均操作繁琐。
由上述可知,现有的植物甾醇检测方法的前处理,操作均较为繁琐,费时费力,从而使得导致植物甾醇的检测不能大规模开展。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种植物甾醇总量快速检测的前处理方法,以解决现有植物甾醇检测方法的前处理操作繁琐,从而导致检测成本高的技术问题。
为解决上述问题,本发明所采用的技术方案如下:
一种植物甾醇总量快速检测的前处理方法,包括以下步骤:
S1:称取待测样品,加入内标溶液和水,在室温下超声波水浴处理;
S2:向经过所述步骤S1处理的溶液加入皂化剂,在室温下超声波水浴处理后,进行加热处理,完成皂化;
S3:将经过所述步骤S2处理的溶液冷却至室温后,加入萃取液进行萃取,萃取完成后,进行离心,提取上层有机液层,向上层有机液层加入脱水剂进行脱水,用氮气吹干,得到浓缩液;
S4:向所述步骤S3所制得的浓缩液中加入硅烷化试剂,加热处理后,冷却至室温,制得待测样品。
作为本发明的优选实施方式,所述步骤S1具体为:称取0.2g待测样品,吸取浓度为4mg/ml的内标溶液100uL,加入待测样品中,再加入100uL-300uL的水,在室温下超声波水浴处理3-5min。
作为本发明的优选实施方式,所述内标溶液为β-胆甾烷醇或胆固醇内标溶液。
作为本发明的优选实施方式,所述步骤S2具体为:加入浓度为2mol/L的皂化剂5mL,在室温下超声波水浴处理5min,置于60℃环境中加热处理1h。
作为本发明的优选实施方式,所述皂化剂为氢氧化钾-乙醇溶液。
作为本发明的优选实施方式,所述步骤S3具体为:冷却后用5mL萃取液萃取2次,萃取完成后,进行离心,提取上层有机液层,加入2g脱水剂脱水后,用氮气进行吹干,得到浓缩液。
作为本发明的优选实施方式,所述萃取液为正己烷。
作为本发明的优选实施方式,所述脱水剂为无水硫酸钠。
作为本发明的优选实施方式,所述步骤S4具体为:加入硅烷化试剂100uL,置于105℃环境中反应15min后,取出样液,冷却至室温,完成待测样品的制备。
作为本发明的优选实施方式,所述硅烷化试剂为BSTFA(双(三甲基硅烷基)三氟乙酰胺)和TMCS(三甲基氯硅烷)的混合物,重量比为99:1。
相比现有技术,本发明的有益效果在于:
采用本发明的前处理方法,使得在进行气相色谱分析的前处理中不需要制备氧化铝柱和配制展开剂,有效地简化了前处理的步骤,从而降低了检测的成本和时间,另外不需要进行薄层色谱板分离,从而不会因为薄层色谱板分离效果不明显,导致测试结果不准确,因此本发明可以作为植物油的定级与认证、检测原始脂质的来源以及掺杂打假的指导方法。
下面结合附图和具体实施方式对本发明作进一步详细说明。
附图说明
图1-是本发明实施例的一种采用本发明前处理方法并以β-胆甾烷醇为内标溶液的植物甾醇定性检测结果图。
具体实施方式
本发明所提供的植物甾醇总量快速检测的前处理方法,具体实施例如下:
实施例1:
S1:称取0.2g待测植物油样品放入50ml离心管内,吸取浓度为4mg/ml的β-胆甾烷醇内标溶液100uL,加入到样品后,再加入300uL的水,帮助基质分散,将试管在室温下超声波水浴处理3-5min。
S2:向经过步骤S1处理的混合液加入浓度为2mol/L的氢氧化钾-乙醇溶液5mL,在室温下超声波水浴处理5min后,放入60℃烘箱处理一个小时。
S3:将经过步骤S2处理的混合液冷却至室温后,加入5mL正己烷萃取2次后,进行离心,提取上层有机液层,向提取的上层有机液层加入2g无水硫酸钠进行脱水,再将经过脱水处理后的有机液用氮气吹干,得到浓缩液。
S4:向经过步骤S3处理的浓缩液加入100uL BSTFA(双(三甲基硅烷基)三氟乙酰胺)和TMCS(三甲基氯硅烷)的混合物(重量比为99:1)后,放入105℃烘箱反应15min,取出样液,冷却至室温,完成待测样品的制备。
实施例2:
S1:称取0.2g待测植物油样品放入50ml离心管内,吸取浓度为4mg/ml的胆固醇内标溶液100uL,加入到样品后,再加入300uL的水,帮助基质分散,将试管在室温下超声波水浴处理3-5min。
S2:向经过步骤S1处理的混合液加入浓度为2mol/L的氢氧化钾-乙醇溶液5mL,在室温下超声波水浴处理5min后,放入60℃烘箱处理一个小时。
S3:将经过步骤S2处理的混合液冷却至室温后,加入5mL正己烷萃取2次后,进行离心,提取上层有机液层,漏斗塞棉花后,向提取的上层有机液层加入2g无水硫酸钠进行脱水,再将经过脱水处理后的有机液用氮气吹干,得到浓缩液。
S4:向经过步骤S3处理的浓缩液加入100uL BSTFA(双(三甲基硅烷基)三氟乙酰胺)和TMCS(三甲基氯硅烷)的混合物(重量比为99:1)后,放入105℃烘箱反应15min,取出样液,冷却至室温,完成待测样品的制备。
将待测样品进行气相色谱分析,具体如下:
气相色谱分析条件:
——色谱柱:HP-5毛细管色谱柱(30m×0.320μm×250μm);
——进样器温度:300℃;
——检测器温度:320℃;
——载气:氮气;
——分流比:40:1;
——进样体积:1.0μL;
——分离度(R)应大于1.5。
——程序升温(见表1):
表1程序升温
结果计算:在上述色谱条件下以β-胆甾烷醇、谷甾醇、豆甾醇和菜油甾醇检查保留时间作为定性,结果如图1所示,由结果可知,采用本发明的前处理方法能准确地对不同的植物甾醇进行定性分析。
以β-胆甾烷醇保留时间至1.7*β-胆甾烷醇保留时间区域为植物甾醇总量的峰面积,进行计算,结果如表1所示。
含量最多的谷甾醇、豆甾醇和菜油甾醇的计算:Xj=(Aj×mi)/(Ai×m)
甾醇总量的计算:Xt=[∑(A)×mi]/(Ai×m)
式中:
Xj-试样中甾醇组分j,以100g油中含有的毫克数表示(mg/100g);
Xt-试样中甾醇总量,以100g油中含有的毫克数表示(mg/100g);
∑(A)-单体甾醇峰面积的和;
Ai-胆甾烷醇内标的峰面积;
Aj-甾醇组分j的峰面积;
mi-胆甾烷醇的质量,单位为毫克(mg);
m-试样的质量,单位为克(g)。
对比例(薄层色谱法):
采用现有GB/T25223-2010动植物油脂甾醇组成和甾醇总量的测定气相色谱法,前处理方法操作步骤具体如下:
皂化:称取0.2g(精确至0.0001g)的待测植物油样品于平底烧瓶中,加入1mL 1mg/L的胆固醇标准溶液,混匀,加入5mL 1.2moL/L氢氧化钾一乙醇溶液,混匀,置于70℃水浴锅中搅拌皂化至溶液澄清透明(约15min)。
氧化铝层析提取不皂化物:向层析柱倒入10mL乙醇,并称取10g 1级氧化铝分次倒入层析柱中,再用10mL乙醇将层析柱中的氧化铝洗入层析柱中。用5mL乙醇稀释皂化液,并移取5mL皂化液,到装好的层析柱中,用250mL平底烧瓶收集流出液,流速为2mL/min,待皂化液流完后,加入5mL乙醇洗脱,待流尽,加入30mL乙醚继续洗脱。将收集的洗脱液旋转浓缩至干,用少量乙醚溶解平底烧瓶的目标物,此样品称为层析样品。此步骤提取不皂化物大约需要2-3小时。
薄层色谱分离甾醇:将氧化铝层析后提取的不皂化物乙醚溶液,用平头微量注射器吸取溶液(共200μL)点在薄层板下边缘2cm处,样液点成圆点,但直径不超过3mm,圆点两端距离边缘至少留出3cm间隙。吸取50μL胆固醇标准溶液在距离边缘1.5cm处,左右各点一点。在展开槽中加入大约100mL展开剂。将板放入展开槽中展开,直到溶剂到达上边缘。取出薄层板,在通风橱中挥干溶剂,而后将薄层板放入含有碘单质的烧杯中,用盖子盖好,用吹风机加热加快碘单质挥发,待薄层板中的物质完全显色后,用铅笔快速标记出所需要的甾醇范围,用刀片刮下标记的硅胶层,并收集到已准备过滤的滤纸上,在收集的硅胶中加入0.5mL乙醇,用5mL乙醚对烧杯中的硅胶洗提三次,并滤入烧瓶中,用旋转蒸发器将乙醚提取物浓缩至约1ml,将浓缩液转移至反应瓶中,用氮气流吹干反应瓶中的溶剂。薄层色谱分离甾醇大约耗时约2-3小时。
甾醇三甲基硅醚的制备:
取100uL硅烷化试剂加于浓缩液的反应瓶中。密封反应瓶,置于105℃烘箱中加热15min。取出并冷却至室温后,将溶液直接注入到气相色谱仪进行分析。甾醇三甲基硅醚的制备约半小时。
现有的前处理需要制备氧化铝柱和配制展开剂等,使得整个前处理时间大约需要6h。采用实施例1、实施例2以及对比例的方法进行植物油样品植物甾醇含量的检测,每个样品检测两次,分为第一组和第二组,取其平均值,进行分析。
从表2至表4可以看出,对比例的植物甾醇含量平均值明显小于实施例1和2,RSD(相对标准偏差)值明显大于实施例1和2,由此可以得出,对比例由于薄层色谱板分离效果不明显,导致每次的检测结果波动较大,难以获得准确的检测结果。实施例1和实施例2两者进行对比,无论是植物甾醇含量平均值还是RSD值差距均不大,而且两个实施例的组内对比,两次植物甾醇含量的检测值较为接近,由此可以得出,采用本发明前处理方法对待测样品进行处理后,进行植物甾醇含量的检测,得到的检测结果较为准确。
表2实施例1的检测结果
表3实施例2的检测结果
表4对比例的检测结果
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (10)
1.一种快速检测植物甾醇总量的前处理方法,其特征在于,包括以下步骤:
S1:称取待测样品,加入内标溶液和水,在室温下超声波水浴处理;
S2:向经过所述步骤S1处理的溶液加入皂化剂,在室温下超声波水浴处理后,进行加热处理,完成皂化;
S3:将经过所述步骤S2处理的溶液冷却至室温后,加入萃取液进行萃取,萃取完成后,进行离心,提取上层有机液层,向上层有机液层加入脱水剂进行脱水,用氮气吹干,得到浓缩液;
S4:向所述步骤S3所制得的浓缩液中加入硅烷化试剂,加热处理后,冷却至室温,制得待测样品。
2.根据权利要求1所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述步骤S1具体为:称取0.2g待测样品,吸取浓度为4mg/ml的内标溶液100uL,加入待测样品中,再加入100uL-300uL的水,在室温下超声波水浴处理3-5min。
3.根据权利要求2所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述内标溶液为β-胆甾烷醇或胆固醇内标溶液。
4.根据权利要求1所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述步骤S2具体为:加入浓度为2mol/L的皂化剂5mL,在室温下超声波水浴处理5min,置于60℃环境中加热处理1h。
5.根据权利要求4所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述皂化剂为氢氧化钾-乙醇溶液。
6.根据权利要求1所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述步骤S3具体为:冷却后用5mL萃取液萃取2次,萃取完成后,进行离心,提取上层有机液层,加入2g脱水剂脱水后,用氮气进行吹干,得到浓缩液。
7.据权利要求6所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述萃取液为正己烷。
8.根据权利要求6或7所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述脱水剂为无水硫酸钠。
9.根据权利要求1所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述步骤S4具体为:加入硅烷化试剂100uL,置于105℃境中反应15min后,取出样液,冷却至室温,完成待测样品的制备。
10.根据权利要求9所述的植物甾醇总量快速检测的前处理方法,其特征在于,所述硅烷化试剂为BSTFA和TMCS的混合物,重量比为99:1。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060135794A1 (en) * | 2002-06-19 | 2006-06-22 | Tibor Czuppon | Process for recovery of plant sterols from by-product of vegetable oil refining |
CN105842372A (zh) * | 2016-06-08 | 2016-08-10 | 江西恒时生物科技有限公司 | 一种测定植物油脱臭馏出物中不同形式植物甾醇含量的方法 |
CN114280172A (zh) * | 2021-12-06 | 2022-04-05 | 南京诺齐生物科技有限公司 | 一种植物甾醇的检测分析方法 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060135794A1 (en) * | 2002-06-19 | 2006-06-22 | Tibor Czuppon | Process for recovery of plant sterols from by-product of vegetable oil refining |
CN105842372A (zh) * | 2016-06-08 | 2016-08-10 | 江西恒时生物科技有限公司 | 一种测定植物油脱臭馏出物中不同形式植物甾醇含量的方法 |
CN114280172A (zh) * | 2021-12-06 | 2022-04-05 | 南京诺齐生物科技有限公司 | 一种植物甾醇的检测分析方法 |
Non-Patent Citations (4)
Title |
---|
ELIZA M. ROTTA 等: "Ultrasound-Assisted Saponification Coupled with Gas Chromatography-Flame Ionization Detection for the Determination of Phytosterols from Passion Fruit Seed Oil", J. BRAZ. CHEM. SOC., vol. 29, no. 3, pages 579 - 586 * |
倪芳妍等: "植物油中总甾醇测定前处理方法的研究", 中国油脂, vol. 36, no. 02, pages 74 - 76 * |
彭祖茂等: "超高效液相色谱-串联质谱同时测定植物油中14种营养成分", 色谱, vol. 36, no. 11, pages 1140 - 1146 * |
杨虹等: "食用油中植物甾醇测定方法的优化及含量分析", 中国粮油学报, vol. 26, no. 02, pages 120 - 123 * |
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