WO2020181780A1 - 一种快速准确分析菜籽油中多酚含量的方法 - Google Patents

一种快速准确分析菜籽油中多酚含量的方法 Download PDF

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WO2020181780A1
WO2020181780A1 PCT/CN2019/113576 CN2019113576W WO2020181780A1 WO 2020181780 A1 WO2020181780 A1 WO 2020181780A1 CN 2019113576 W CN2019113576 W CN 2019113576W WO 2020181780 A1 WO2020181780 A1 WO 2020181780A1
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extract
rapeseed oil
polyphenols
polyphenol
extractant
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刘元法
宋军鸽
李进伟
曹晨
徐勇将
翟颖红
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Jiangnan University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0492—Applications, solvents used
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0476—Moving receptacles, e.g. rotating receptacles
    • B01D11/048—Mixing by counter-current streams provoked by centrifugal force, in rotating coils or in other rotating spaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/03—Edible oils or edible fats

Definitions

  • the invention belongs to the field of natural compound analysis, and specifically relates to a method for quickly and accurately analyzing the content of polyphenols in rapeseed oil.
  • Polyphenols are widely found in plants and are an important secondary metabolite in plants. Its molecular structure contains several phenolic hydroxyl groups. Studies have confirmed that polyphenols have physiological functions and biological activities such as anti-tumor, lowering blood sugar, antibacterial, anti-oxidation, and scavenging free radicals, and have broad development prospects.
  • the content of polyphenols in rapeseed is much higher than other oil crops, but during the oil production process, most of the polyphenols are left in the rapeseed meal, and only a small part will be transferred to the rapeseed oil. Since polyphenols can improve the oxidative stability and nutritional quality of rapeseed oil, accurate analysis of the composition and content of polyphenols in rapeseed oil will be of great significance to the processing of rapeseed oil.
  • liquid-liquid extraction and solid-phase extraction are commonly used for the extraction of polyphenols in vegetable oils, but these two methods have disadvantages such as cumbersome operation, large consumption of organic solvents, and high content of matrix co-extracts. Therefore, it is necessary to find a simple, environmentally friendly method that can significantly reduce the content of matrix co-extracts.
  • the present invention provides a new analysis method for polyphenols in rapeseed oil with simple operation, low cost, less matrix interference, and accurate results, which is used to solve the cumbersome steps of the existing detection method and the consumption of organic solvents.
  • the problem of high matrix co-extract content is used to solve the cumbersome steps of the existing detection method and the consumption of organic solvents.
  • QuEChERS extraction and separation technology is a rapid sample preparation technology recently developed in the world in recent years. It has the characteristics of high recovery rate, high precision and accuracy, low solvent usage, simple operation, safety and reliability.
  • the extraction conditions and purification conditions used have a greater impact on the final separation results.
  • QuEChERS extraction and separation technology is mostly used for the residue analysis of pesticides, veterinary drugs, mycotoxins, polycyclic aromatic hydrocarbons and other compounds.
  • Most of the applied substrates are vegetables, fruits, soil, etc.
  • QuEChERS extraction and separation are often used in the detection of pesticide residues in fruits.
  • QuEChERS extraction and separation technology for the extraction of polyphenols from vegetable oils. Therefore, how to apply QuEChERS extraction and separation technology for the extraction of polyphenols from plants is the problem to be overcome by the present invention and is also an important objective of the present invention. one.
  • the first object of the present invention is to provide a method for extracting polyphenols from rapeseed oil, the method comprising:
  • step (2) Add 25 ⁇ 35 mg/mL of adsorbent C 18 to the extract obtained in step (1), and separate and remove the solid components, that is, polyphenol solution.
  • the volume ratio of acetonitrile to water in the extractant is (4-8):1.
  • the mass-volume ratio of rapeseed oil to extractant in the step (1) is 0.25-0.35 g/mL.
  • the step (1) further includes adding anhydrous MgSO 4 and NaCl to cooperate with the extractant.
  • the mass ratio of the anhydrous MgSO 4 and NaCl is 2:1 to 6:1.
  • the step (1) further includes freezing the separated liquid to obtain the extract.
  • the freezing treatment conditions are: temperature -15°C ⁇ -20°C, time 1.5 ⁇ 2.5 h.
  • the step (2) further includes adding anhydrous MgSO 4 in combination with an adsorbent to further remove impurities such as moisture and fat in the extract to obtain a polyphenol solution.
  • the separation is centrifugal separation of the extract at a rotation speed of 3000-5000 rpm.
  • the second object of the present invention is to provide a method for detecting polyphenols in vegetable oils.
  • the method is to pre-treat the vegetable oil to obtain a polyphenol solution and then perform the detection; the pre-treatment method is the above method.
  • the method further includes concentrating the polyphenol solution, and then dissolving it with a solvent to obtain a test sample solution, which can be used for detection.
  • the solvent includes methanol.
  • the third object of the present invention is to provide a method for detecting polyphenols in vegetable oil by liquid chromatography.
  • the method is to pre-process the vegetable oil to obtain a liquid sample of polyphenols, and then perform liquid chromatography detection;
  • the method is the above method.
  • the chromatographic column of the liquid chromatography is Thermo Hypersil GOLD (100mm ⁇ 2.1mm, 1.9 ⁇ m), the temperature of the column oven is 30-40°C.
  • the mobile phase A of the liquid chromatography is a 0.1% acetic acid aqueous solution; the mobile phase B is an acetonitrile solution, and the flow rate is 0.2-0.5 mL/min.
  • the invention provides a method for quickly and accurately analyzing the content of polyphenols in rapeseed oil.
  • the polyphenols in rapeseed oil are extracted with acetonitrile-water, and purified by a C18 adsorbent for separation and purification.
  • the average recovery of polyphenols in rapeseed oil is 81.31% ⁇ 102.95%, and the RSDs are 0.86% ⁇ 8.03%, with higher accuracy and precision.
  • the method of the invention not only has a small amount of organic reagents, and has little environmental pollution, but also reduces matrix interference and improves purification efficiency through optimization of adsorbents.
  • the method of the invention not only has simple operation and low cost, but also has less matrix interference and accurate results, and is suitable for the qualitative and quantitative determination of polyphenols in rapeseed oil.
  • Figure 1 is a diagram of the influence of the amount of water on the extraction effect of polyphenols by using Example 1 as a reference;
  • Fig. 2 is a graph of the influence of freezing time on the content of matrix co-extracts by using Example 1 as a reference;
  • Fig. 3 is a graph of the influence of the amount of C 18 on the extraction effect of polyphenols using Example 1 as a reference.
  • Separation treatment accurately weigh 6.00 g rapeseed oil into a 50 mL centrifuge tube, add 3 mL water and 18 mL acetonitrile, and shake for 5 min; add 7.2 g anhydrous MgSO 4 and 1.8 g NaCl, shake for 2 min, and then at 4000 rpm Centrifuge for 5 min under conditions; transfer the supernatant to a centrifuge tube and freeze for 2 h at -20 °C; transfer 9 mL of supernatant to a 15 mL centrifuge tube, add 270 mg C 18 and 1.8 g anhydrous MgSO 4 , Shake for 2 min, then centrifuge at 4000 rpm for 5 min; the supernatant is obtained, which is the polyphenol solution.
  • the liquid chromatography conditions used are: Column: Thermo Hypersil GOLD (100mm ⁇ 2.1mm, 1.9 ⁇ m); column oven temperature: 35°C; injection volume: 2 ⁇ L; flow rate: 0.30mL/min; mobile phase A: 0.1% acetic acid aqueous solution; mobile phase B: acetonitrile solution.
  • Standard addition recovery rate add standard 10 to actual samples ⁇ g/g was measured, repeated 3 times, and the average recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 2.
  • Example 2 the extractant was replaced with 2:1, 4:1, 8:1 acetonitrile/water, and other conditions were unchanged.
  • the obtained sample was tested by liquid chromatography to calculate the average peak area and RSD, and the result was obtained See Table 3.
  • Acetonitrile to water volume ratio Caffeic acid/peak area (% RSD) p-coumaric acid/peak area (% RSD) Sinapinic acid/peak area (% RSD) Ferulic acid/peak area (% RSD) 2:1 226120(10.88) 733015(2.21) 333725(6.38) 533467(5.35) 4:1 379940(11.52) 1021980(6.71) 404765(10.75) 767575(9.67) 6:1 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18) 8:1 531352(12.44) 1128797(4.33) 426528(10.09) 866902(8.02)
  • Example 2 the amount of extractant was replaced with 4.5mL water and 27mL acetonitrile, 2.25mL water and 13.5mL acetonitrile, and other conditions were unchanged.
  • the obtained sample was tested by liquid chromatography to calculate the average peak area and RSD. The results obtained are shown in Table 4.
  • Extracting agent Caffeic acid/peak area (% RSD) p-coumaric acid/peak area (% RSD) Sinapinic acid/peak area (% RSD) Ferulic acid/peak area (% RSD) 2.25mL water and 13.5mL acetonitrile 288232(12.03) 966108(4.70) 344566(9.57) 716155(3.28) 3mL water and 18mL acetonitrile 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18) 4.5mL water and 27mL acetonitrile 462570(10.47) 1110167(2.55) 422409(4.10) 821657(5.87)
  • Example 2 the amount of adsorbent was replaced with 90 mg, 180 mg, 360 mg, 450 mg, and other conditions were unchanged.
  • the obtained sample was tested by liquid chromatography to calculate the average peak area and RSD. The results are shown in Table 5.
  • liquid-liquid extraction technology In sample processing and analysis, liquid-liquid extraction technology is used: accurately weigh 2.50 g rapeseed oil, dissolved in 3 In mL n-hexane, shake 1 min; use 3 respectively mL volume fraction of 80% methanol solution was extracted 3 times, each shaking 5 times min at 4000 Centrifuge at rpm for 5 min; combine the methanol extracts, dry the extracts slowly in a nitrogen blower, and add methanol solution 2.5 mL, use 0.22 The ⁇ m filter membrane was filtered into a sample bottle for HPLC-MS determination; others were the same as in Example 2; the analysis results are shown in Table 6.
  • solid phase extraction technology is used: the glycol-based solid phase extraction column is placed in the solid phase extraction device, and 5 mL methanol and 5 mL n-hexane activated SPE column; accurately weigh 1.50 g rapeseed oil, dissolved in 5mL n-hexane, shake 1 min; Pack the sample on the column, and wash the column with 5 mL n-hexane and 5 mL n-hexane: ethyl acetate (90:10, v/v), and finally use 5 Elute with mL methanol solution, collect the eluate, dry slowly in a nitrogen blowing apparatus, and add 1.5 methanol solution mL, use 0.22
  • the ⁇ m filter membrane was filtered into a sample bottle for HPLC-MS determination; others were the same as in Example 2; the analysis results are shown in Table 6.
  • Example 7 the extractant was replaced with pure acetonitrile, and other conditions were unchanged.
  • the obtained sample was tested by liquid chromatography to calculate the average peak area and RSD. The results are shown in Table 7.
  • Extracting agent Caffeic acid/peak area (% RSD) p-coumaric acid/peak area (% RSD) Sinapinic acid/peak area (% RSD) Ferulic acid/peak area (% RSD) Acetonitrile 154868(9.34) 674863(4.80) 227807(9.01) 519482(6.05) Acetonitrile and water 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)

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Abstract

一种快速准确分析菜籽油中多酚含量的方法，分离方法利用乙腈-水作为萃取剂来提取菜籽油中多酚，并配合C 18吸附剂净化，然后进行分离提纯。与传统液液萃取和固相萃取相比，该方法对菜籽油中多酚的平均回收率为81.31％～102.95％，RSDs为0.86％～8.03％，准确度和精密度更高。本方法不仅有机试剂用量少，对环境污染小，而且通过对吸附剂的优化，降低了基质干扰，提高了净化效率，本方法操作简便，成本低，基质干扰少，结果准确，适合菜籽油中多酚的定性和定量测定。

Description

一种快速准确分析菜籽油中多酚含量的方法 技术领域
本发明属于天然化合物分析领域，具体涉及一种快速准确分析菜籽油中多酚含量的方法。
 背景技术
多酚广泛存在于植物体中，是植物体内一种重要的次生代谢产物，其分子结构中含有若干个酚羟基。研究证实，多酚具有抗肿瘤、降血糖、抑菌、抗氧化、清除自由基等生理功能和生物活性，具有广阔的开发前景。菜籽中的多酚含量远高于其他油料作物，但是在制油过程中，大部分多酚都残留在菜籽饼粕中，仅有少部分会转移至菜籽油中。由于多酚可改善菜籽油的氧化稳定性和营养品质，若能精确的分析菜籽油中多酚的组成及含量，对菜籽油的加工具有重要的意义。
目前，植物油中多酚的提取普遍采用液液萃取和固相萃取，但这两种方法存在操作繁琐，有机溶剂消耗大，基质共提物含量高等弊端。因此，寻求一种简便、环境友好且能够显著降低基质共提物含量的方法是非常必要的。
发明内容
本发明基于QuEChERS分离技术提供了一种操作简便、成本低，而且基质干扰少、结果准确的菜籽油中多酚新的分析方法，用以解决现有检测方式步骤繁琐，有机溶剂消耗大，基质共提物含量高的问题。
QuEChERS萃取分离技术是近年来国际上最新发展起来的一种快速样品前处理技术，具有回收率高、精确度和准确度高、溶剂使用量少、操作简便、安全可靠等特点。然而在QuEChERS萃取分离技术中，由于不同基质及目标物性质的千差万别，所用的萃取条件和净化条件对最终分离结果影响较大。目前，QuEChERS萃取分离技术多用于农药、兽药、真菌毒素、多环芳烃等化合物的残留分析，应用的基质大多为蔬菜、水果、土壤等，比如QuEChERS萃取分离常用于水果中农残检测，所用萃取剂基本为乙腈，其他溶剂与其存在明显的效果差异，且所用吸附剂也基本选用PSA。目前还未有将QuEChERS萃取分离技术用于植物油中多酚提取的报道，因此，如何将QuEChERS萃取分离技术应用于植物中多酚的提取是本发明所要克服的问题，也是本发明的重要发明目的之一。
本发明的第一个目的是提供一种菜籽油中多酚的提取方法，所述方法包括：
（1）乙腈与水的混合溶液作为萃取剂对菜籽油进行萃取，分离萃取液，得到提取液；
（2）向步骤（1）所得提取液中加入吸附剂C 1825~35 mg/mL，分离除去固体成分，即得多酚溶液。
本发明的一种实施方式中，所述萃取剂中乙腈与水的体积比为（4-8）：1。
本发明的一种实施方式中，所述步骤（1）中菜籽油与萃取剂的质量体积比为0.25~0.35g/mL。
本发明的一种实施方式中，所述步骤（1）还包括加入无水MgSO 4和NaCl，与萃取剂配合作用。
本发明的一种实施方式中，所述无水MgSO 4和NaCl的质量比为2:1~6:1。
本发明的一种实施方式中，所述步骤（1）还包括将分离后的液体冷冻处理，进而得到提取液。
本发明的一种实施方式中，所述冷冻处理的条件为：温度-15℃~-20℃，时间1.5~2.5 h。
本发明的一种实施方式中，所述步骤（2）还包括加入无水MgSO 4与吸附剂配合使用，进一步除去水分和提取液中的脂肪等杂质，得到多酚溶液。
本发明的一种实施方式中，所述分离是将萃取液离心分离，转速3000~5000 rpm。
本发明的第二个目的是提供一种植物油中多酚的检测方法，所述方法是预先将植物油作前处理得到多酚溶液，然后进行检测；所述前处理方法为上述方法。
本发明的一种实施方式中，所述方法还包括将多酚溶液进行浓缩，然后用溶剂溶解得到检测样液，即可用于检测。
本发明的一种实施方式中，所述溶剂包括甲醇。
本发明的第三个目的是提供一种植物油中多酚的液相色谱检测方法，所述方法是预先将植物油作前处理得到多酚的液体样品，然后进行液相色谱检测；所述前处理方法为上述方法。
本发明的一种实施方式中，所述液相色谱的色谱柱为Thermo Hypersil GOLD (100mm×2.1mm, 1.9μm)，柱温箱温度为30-40℃。
本发明的一种实施方式中，所述液相色谱的流动相A为0.1%乙酸水溶液；流动相B为乙腈溶液，流速为0.2-0.5mL/min。
本发明有益效果：
本发明提供了一种快速准确分析菜籽油中多酚含量的方法，用乙腈-水提取菜籽油中多酚，利用C 18吸附剂净化，进行分离纯化。与传统液液萃取和固相萃取相比，该方法对菜籽油中多酚的平均回收率为81.31%~102.95%，RSDs为0.86%~8.03%，准确度和精密度更高。本发明方法不仅有机试剂用量少，对环境污染小，而且通过对吸附剂的优化，降低了基质干扰，提高了净化效率。本发明方法不仅操作简便、成本低，而且基质干扰少、结果准确，适合菜籽油中多酚的定性和定量测定。
附图说明
图1为以实施例1作为参照，考察水的用量对多酚提取效果的影响图；
图2为以实施例1作为参照，考察冷冻时间对基质共提物含量的影响图；
图3为以实施例1作为参照，考察C 18用量对多酚提取效果的影响图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：
分离处理：准确称取6.00 g菜籽油于50 mL离心管中，加入3 mL水和18 mL乙腈，振荡5 min；加入7.2 g无水MgSO 4和1.8 gNaCl，振荡2 min，然后于4000 rpm条件下，离心5min；移取上清液至离心管中，-20 ℃冷冻2 h；移取9 mL上清液至15 mL离心管中，加入270 mg C 18和1.8 g无水MgSO 4，振荡2 min，然后于4000 rpm条件下，离心5 min；得到上清液，即为多酚溶液。
实施例2：
采用的液相色谱条件为：色谱柱：Thermo Hypersil GOLD (100mm×2.1mm, 1.9μm)；柱温箱温度：35℃；进样量：2μL；流速为0.30mL/min；流动相A：0.1%乙酸水溶液；流动相B：乙腈溶液。
（1）标准溶液的制备：使用色谱级甲醇制备浓度为200μg /mL的多酚混合标准溶液，然后在使用前在-20℃下储存。通过用甲醇稀释储备溶液来制备工作溶液。
（2）用甲醇稀释标准溶液成不同浓度的工作溶液后分别进行测定：每个浓度平行测定3次，得出多酚的线性方程和相关系数。以3倍信噪比计算得出方法检出限，10倍信噪比计算得出方法定量限，结果见表1。
表1线性范围、检出限和定量限的测定
化合物 回归方程 相关系数R2 线性范围 (μg/mL) LOD (μg/mL) LOQ (μg/mL)
原儿茶酸 Y=3.0872x+0.0976 0.9961 0.04-10 0.010 0.033
对羟基苯甲酸 Y=5.0126x+1.2662 0.9955 0.04-10 0.010 0.033
咖啡酸 Y=0.1727x-0.0290 0.9977 0.30-10 0.080 0.267
香草酸 Y=0.6213x+0.3024 0.9964 0.03-10 0.008 0.027
丁香酸 Y=1.0651x+0.2823 0.9935 0.03-10 0.008 0.027
对香豆酸 Y=3.0005x+0.9064 0.9925 0.02-10 0.006 0.020
芥子酸 Y=0.2064x+0.0778 0.9902 0.03-10 0.008 0.027
阿魏酸 Y=2.7085x+1.2602 0.9945 0.02-10 0.004 0.013
肉桂酸 Y=4.7007x+2.4276 0.9924 0.01-10 0.002 0.007
芥子碱 Y=9.9452x+6.1696 0.9923 0.02-10 0.004 0.013
（3）准确移取4.5 mL上清液（实施例1所得多酚溶液）至氮吹管中，在氮吹仪中缓缓吹干，加入甲醇溶液1.5 mL，用0.22 μm滤膜过滤至样品瓶中，进行HPLC-MS测定。
（4）加标回收率：对实际样品加标10 μg/g进行测定，重复3次，计算平均回收率与相对标准偏差(RSD)，结果见表2。
表2加标回收率测定
化合物 加标回收率
原儿茶酸 86.17a±8.03b
对羟基苯甲酸 99.23±4.83
咖啡酸 91.06±4.13
香草酸 99.53±3.33
丁香酸 100.34±0.86
对香豆酸 99.71±1.48
芥子酸 101.90±3.24
阿魏酸 102.95±1.74
肉桂酸 81.31±2.95
芥子碱 97.96±7.90
a平均回收率，%； b相对标准偏差（RSD，%）。
实施例3：
参照实施例1，将萃取剂分别替换为2：1、4：1、8：1乙腈/水，其他条件不变，得到的样液经液相色谱检测，计算平均峰面积与RSD，所得结果见表3。
表3 实施例3制得样品的检测结果
乙腈与水体积比 咖啡酸/峰面积(% RSD) p-香豆酸/峰面积(% RSD) 芥子酸/峰面积(% RSD) 阿魏酸/峰面积(% RSD)
2:1 226120(10.88) 733015(2.21) 333725(6.38) 533467(5.35)
4:1 379940(11.52) 1021980(6.71) 404765(10.75) 767575(9.67)
6:1 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)
8:1 531352(12.44) 1128797(4.33) 426528(10.09) 866902(8.02)
实施例4：
参照实施例1，将萃取剂的用量分别替换为4.5mL水和27mL乙腈，2.25mL水和13.5mL乙腈，其他条件不变，得到的样液经液相色谱检测，计算平均峰面积与RSD，所得结果见表4。
表4 实施例4制得样品的检测结果
萃取剂 咖啡酸/峰面积(% RSD) p-香豆酸/峰面积(% RSD) 芥子酸/峰面积(% RSD) 阿魏酸/峰面积(% RSD)
2.25mL水和13.5mL乙腈 288232(12.03) 966108(4.70) 344566(9.57) 716155(3.28)
3mL水和18mL乙腈 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)
4.5mL水和27mL乙腈 462570(10.47) 1110167(2.55) 422409(4.10) 821657(5.87)
实施例5：
参照实施例1，将吸附剂的用量分别替换为90mg、180mg、360mg、450mg，其他条件不变，得到的样液经液相色谱检测，计算平均峰面积与RSD，所得结果见表5。
表5实施例5制得样品的检测结果
C18用量/mg 咖啡酸/峰面积 (% RSD) p-香豆酸/峰面积(% RSD) 芥子酸/峰面积(% RSD) 阿魏酸/峰面积 (% RSD)
90 497706(4.82) 1104624(2.35) 281526(10.04) 845011(2.86)
180 502562(9.83) 1126695(2.69) 284957(11.98) 857426(2.05)
270 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)
360 415247(2.43) 1011740(2.39) 360893(11.75) 769054(2.45)
450 339775(6.87) 993345(4.56) 222556(11.03) 734897(7.35)
对照例1：
样品处理和分析中，采用液液萃取技术：准确称取2.50 g菜籽油，溶于3 mL正己烷中，振荡1 min；分别用3 mL体积分数80%甲醇溶液提取3次，每次振荡5 min，于4000 rpm条件下离心5min；合并甲醇提取液，将提取液在氮吹仪中缓缓吹干，加入甲醇溶液2.5 mL，用0.22 μm滤膜过滤至样品瓶中，进行HPLC-MS测定；其它同实施例2；分析结果见表6。
对照例2
样品处理和分析中，采用固相萃取技术：将二醇基固相萃取柱置于固相萃取装置中，分别用5 mL甲醇和5 mL正己烷活化SPE柱；准确称取1.50 g菜籽油，溶于5mL正己烷中，振荡1 min；将样品装柱，先后用5mL正己烷和5mL正己烷:乙酸乙酯（90:10，v/v）过柱清洗，最后用5 mL甲醇溶液洗脱，收集洗脱液，在氮吹仪中缓缓吹干，加入甲醇溶液1.5 mL，用0.22 μm滤膜过滤至样品瓶中，进行HPLC-MS测定；其它同实施例2；分析结果见表6。
表6 对照例1、2制得样品的检测结果情况
化合物 对照例1 对照例2 实施例1
原儿茶酸 82.53±5.26 65.85±3.05 86.17±8.03
对羟基苯甲酸 76.90±7.82 77.95±10.13 99.23±4.83
咖啡酸 81.28±8.81 56.98±9.23 91.06±4.13
香草酸 62.16±4.05 52.32±8.51 99.53±3.33
丁香酸 101.11±7.62 71.54±4.81 100.34±0.86
对香豆酸 77.88±9.03 61.31±5.74 99.71±1.48
芥子酸 75.39±7.54 83.79±5.58 101.90±3.24
阿魏酸 66.04±9.65 65.29±7.73 102.95±1.74
肉桂酸 81.41±5.44 67.73±3.57 81.31±2.95
芥子碱 105.80±4.80 67.55±8.06 97.96±7.90
由表6结果可知，所开发的方法对菜籽油中多酚的平均回收率为81.31%~102.95%，RSDs为0.86%~8.03%。与传统液液萃取和固相萃取相比，本专利开发的方法准确度和精密度更高，提取效果更好。而且，相比于液液萃取和固相萃取，该方法不仅操作简便、成本低，而且通过采用C 18吸附剂净化，降低了基质干扰，结果更准确。
对照例3：
参照实施例1，将萃取剂替换为纯乙腈，其他条件不变，得到的样液经液相色谱检测，计算平均峰面积与RSD，所得结果见表7。
表7对照例3所得样品的检测结果
萃取剂 咖啡酸/峰面积(% RSD) p-香豆酸/峰面积(% RSD) 芥子酸/峰面积(% RSD) 阿魏酸/峰面积 (% RSD)
乙腈 154868(9.34) 674863(4.80) 227807(9.01) 519482(6.05)
乙腈和水 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)
对照例4：
参照实施例1，将吸附剂分别替换为PSA或者GCB，其他条件不变，得到的样液经液相色谱检测，所得结果见表8。
表8对照例4所得样品的检测结果
吸附剂 咖啡酸/峰面积(% RSD) p-香豆酸/峰面积(% RSD) 芥子酸/峰面积(% RSD) 阿魏酸/峰面积(% RSD)
PSA 138529(8.78) 478271(3.70) 378770(4.04) 606039(3.64)
GCB 182310(3.86) 563629 (4.53) 412811(6.74) 641664 (4.81)
C18 564683(9.93) 1132224(2.74) 439369(7.74) 868811(2.18)
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (17)

  1. 一种菜籽油中多酚的提取方法，其特征在于，所述方法包括：
    （1）乙腈与水的混合溶液作为萃取剂对菜籽油进行萃取，分离萃取液，得到提取液；所述萃取剂中乙腈与水的体积比为（5-8）：1；菜籽油与萃取剂的质量体积比为0.15~0.35g/mL；
    （2）向步骤（1）所得提取液中加入吸附剂C 1820~35mg/mL，固液分离后即得多酚溶液。
  2. 一种菜籽油中多酚的提取方法，其特征在于，所述方法包括：
    （1）乙腈与水的混合溶液作为萃取剂对菜籽油进行萃取，分离萃取液，得到提取液；
    （2）向步骤（1）所得提取液中加入吸附剂C 1820~35mg/mL，固液分离后即得多酚溶液。
  3. 根据权利要2所述的方法，其特征在于，所述萃取剂中乙腈与水的体积比为（5-8）：1。
  4. 根据权利要2所述的方法，其特征在于，所述步骤（1）中菜籽油与萃取剂的质量体积比为0.15~0.35g/mL。
  5. 根据权利要1~4任一所述的方法，其特征在于，所述步骤（1）还包括加入无水MgSO 4和NaCl，与萃取剂配合作用。
  6. 根据权利要5所述的方法，其特征在于，所述无水MgSO 4和NaCl的质量比为2:1~6:1。
  7. 根据权利要1~4任一所述的方法，其特征在于，所述步骤（1）还包括将分离后的液体冷冻处理，得到提取液。
  8. 根据权利要5所述的方法，其特征在于，所述步骤（1）还包括将分离后的液体冷冻处理，得到提取液。
  9. 根据权利要6所述的方法，其特征在于，所述步骤（1）还包括将分离后的液体冷冻处理，得到提取液。
  10. 根据权利要7-9任一所述的方法，其特征在于，所述冷冻处理的条件为：温度-15℃~ -20℃，时间1.5~2.5 h。
  11. 根据权利要1~4任一所述的方法，其特征在于，所述步骤（2）还包括加入无水MgSO 4与吸附剂配合使用，进一步除去水分和提取液中的脂肪类杂质，得到多酚溶液。
  12. 根据权利要5所述的方法，其特征在于，所述步骤（2）还包括加入无水MgSO 4与吸附剂配合使用，进一步除去水分和提取液中的脂肪类杂质，得到多酚溶液。
  13. 根据权利要6所述的方法，其特征在于，所述步骤（2）还包括加入无水MgSO 4与吸附剂配合使用，进一步除去水分和提取液中的脂肪类杂质，得到多酚溶液。
  14. 一种植物油中多酚的检测方法，所述方法是利用权利要求1~4任一所述的方法对植物油作前处理，得到多酚溶液，然后进行检测。
  15. 一种植物油中多酚的检测方法，所述方法是利用权利要求6所述的方法对植物油作前处理，得到多酚溶液，然后进行检测。
  16. 一种植物油中多酚的液相色谱检测方法，所述方法是利用权利要求1~4任一所述的方法对植物油作前处理，得到多酚的液体样品，然后进行液相色谱检测。
  17. 一种植物油中多酚的液相色谱检测方法，所述方法是利用权利要求6所述的方法对植物油作前处理，得到多酚的液体样品，然后进行液相色谱检测。
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