WO2020125532A1 - 一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法 - Google Patents

一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法 Download PDF

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WO2020125532A1
WO2020125532A1 PCT/CN2019/124737 CN2019124737W WO2020125532A1 WO 2020125532 A1 WO2020125532 A1 WO 2020125532A1 CN 2019124737 W CN2019124737 W CN 2019124737W WO 2020125532 A1 WO2020125532 A1 WO 2020125532A1
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riboflavin
layer chromatography
screening
raman spectroscopy
enhanced raman
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French (fr)
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陈益胜
徐学明
张煌
王萍
金征宇
谢正军
黄彩虹
舒蓝萍
杨哪
金亚美
吴凤凤
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江南大学
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    • 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
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/95—Detectors specially adapted therefor; Signal analysis

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  • the invention relates to a method for screening riboflavin in food and medicine by high-efficiency thin layer chromatography and surface enhanced Raman spectroscopy, which belongs to the technical field of food detection.
  • Riboflavin also known as vitamin B2
  • FMN flavin adenine nucleotide
  • FAD flavin adenine dinucleotide
  • redox cofactors participate in various redox reactions in the body, can promote sugar.
  • the metabolism of fat and protein has a certain effect on maintaining the function of skin, mucous membrane and vision.
  • Riboflavin is synthesized in plants and microorganisms and cannot be synthesized in humans and animals. Therefore
  • riboflavin for American infants and adults is 0.4-1.3 mg/day.
  • riboflavin-fortified foods and riboflavin supplements are increasingly popular.
  • the amount of riboflavin added to animal feed is huge.
  • the detection methods of riboflavin include spectrophotometry, electrochemical method, capillary electrophoresis, high performance liquid chromatography (HPTLC)/gas chromatography (GC) and mass spectrometry (MS).
  • HPTLC high performance liquid chromatography
  • GC gas chromatography
  • MS mass spectrometry
  • the LC-MS (liquid mass spectrometry) analysis method is more commonly used, and its accuracy is higher, and it is generally used as an effective method of analysis technology.
  • the sample preparation process is complicated, the processing requirements are extremely high, the test time is long, and the throughput is low. Multiple samples cannot be tested simultaneously.
  • the ionization interface of the mass spectrometer detector is extremely susceptible to interference from the food matrix material co-extracted with the target, ranging from signal suppression to instrument damage, and the traditional LC/MS method is not suitable for a large number of samples. screening.
  • HPTLC High Performance Thin Layer Chromatography
  • SERS Surface enhanced Raman spectroscopy is an attractive tool for the detection of trace molecules in the field of chemical analysis. It has all the advantages of normal Raman spectroscopy and high sensitivity. Due to its inherent molecular fingerprint specificity and single molecule detection potential, SERS can even replace standard substances for screening, so it is very suitable for determining molecular information at the target site and has potential Combined with HPTLC to replace MS for molecular confirmation.
  • the present invention proposes a new riboflavin screening method: high-efficiency thin layer chromatography-surface enhanced Raman spectroscopy combined for rapid screening of riboflavin, to achieve fast and reliable qualitative and quantitative.
  • the purpose of the present invention is to overcome the above-mentioned shortcomings and provide a method for screening riboflavin in foods and drugs by high-efficiency thin layer chromatography and surface enhanced Raman spectroscopy.
  • the technical solution of the present invention is a method for screening riboflavin in foods and drugs by high-efficiency thin-layer chromatography and surface-enhanced Raman spectroscopy, which quantifies riboflavin by fluorescence scanning after expansion of the high-efficiency thin-layer chromatography plate. Then surface-enhanced Raman spectroscopy was applied to the developed high-efficiency thin-layer chromatography plate to clarify the characteristic Raman fingerprint of riboflavin and to characterize the target site.
  • the specific detection methods are as follows:
  • Riboflavin standard solution is prepared by diluting riboflavin with 1% ammonia water as solvent and diluting with methanol to prepare a sample with a concentration of 0.01 mg/mL.
  • the extraction of riboflavin in the sample of multivitamin tablets is made by dissolving 1% ammonia water after milling, taking it out by ultrasound for 20min, centrifuging at 3500r/min for 10min, and obtaining it through a 0.45 ⁇ m water film.
  • the extraction of riboflavin from the breakfast cereal/feed sample is 2.5 g after milling, add 10 mL of 0.1 mol/L hydrochloric acid, and heat in an autoclave at 121°C for 15 min. Take out and cool to room temperature, adjust the pH to 6.8-7.2 with 1 mol/L sodium hydroxide, add 1 mL of peak amylase and place at 37°C for 4 h. After the reaction, the volume was adjusted to 50 mL with methanol. After centrifugation, the supernatant was taken through the membrane to obtain a grain/feed sample.
  • nano-silver glue is prepared according to the classic method of synthesizing nano-silver colloid with sodium borohydride as reducing agent. After centrifuging 4-6mL of original silver colloid (3000-5000r/min, 8-12min), discard the supernatant, put the remaining liquid on the mixer and shake it thoroughly for 1-2min to resuspend it. Double concentrated nano silver colloid.
  • HPTLC-SERS detection After separation by high-efficiency thin-layer chromatography, the rubber plate is marked with visible fluorescent bands by pencil under the irradiation of ultraviolet light. In the marked area, use a capillary pipette to add 2-4 ⁇ L concentrated nano-silver colloid. Immediately scan with a Raman spectrometer to obtain the characteristic fingerprint of the target substance.
  • Detection take the sample to be tested, perform the steps (2)-(5), and compare with the standard curve drawn in step (6) to obtain the content of riboflavin in the sample to be tested.
  • the present invention establishes a method for rapid screening of riboflavin by high-efficiency thin-layer chromatography-surface enhanced Raman spectroscopy, which can realize the simultaneous detection of multiple samples in a single experiment and high-throughput screening .
  • the single screening process takes only 1-2 hours from sample preparation to obtaining the final result, the detection limit can reach 20 ⁇ 50 ⁇ g/kg, the detection method can achieve reproducible RSD ⁇ 10%, which has the advantages of rapid and economic; at the same time based on HPTLC-
  • the establishment of SERS analysis method has opened up new horizons for planar high-efficiency thin-layer chromatography analysis.
  • FIG. 1 is an expanded view (A), a thin layer scanning quantitative chart (B), and a riboflavin standard curve (C) of a riboflavin standard product, a multivitamin sample, a breakfast cereal sample, and a feed sample in Example 1 and Example 2.
  • FIG. 1 is an expanded view (A), a thin layer scanning quantitative chart (B), and a riboflavin standard curve (C) of a riboflavin standard product, a multivitamin sample, a breakfast cereal sample, and a feed sample in Example 1 and Example 2.
  • FIG. 1 is an expanded view (A), a thin layer scanning quantitative chart (B), and a riboflavin standard curve (C) of a riboflavin standard product, a multivitamin sample, a breakfast cereal sample, and a feed sample in Example 1 and Example 2.
  • FIG. 1 is an expanded view (A), a thin layer scanning quantitative chart (B), and a riboflavin standard curve (C) of a riboflavin standard product,
  • Figure 2 is a comparison of SERS fingerprints of the riboflavin standard, multivitamin samples, breakfast cereal samples, and feed samples of Example 3.
  • riboflavin standard solution use ammonia water with a mass concentration of 1% as a solvent and dilute with methanol to prepare a riboflavin standard solution with a concentration of 0.01 mg/mL;
  • silver glue is prepared according to the classic method of synthesizing nano-silver colloid with sodium borohydride as reducing agent. After centrifuging 5mL of the original silver colloid (4000r/min, 10min), the 4.5mL supernatant was discarded, and 0.5mL of the remaining liquid was placed on the mixer and shaken for 1min to re-suspend it to obtain 10-fold concentrated nanoparticles. Silver colloid.
  • HPTLC-SERS detection placed on the DD70 imaging system (Biostep), the image of the silica gel plate was acquired under the illumination of 366nm, and the visible fluorescent band was marked with a pencil according to the Rf value developed by the riboflavin standard. In the marked area, use a capillary pipette to drop 3 ⁇ L concentrated silver colloids in sequence, and immediately scan with a Raman spectrometer. Each Raman scattering signal is integrated three times on average, and each integration acquisition time is 10s, to obtain the riboflavin standard SERS fingerprint Atlas.
  • Pretreatment of multi-vitamin tablets pulverize the tablets, weigh 0.5g in 10mL of 1% ammonia water, take out after 20min of ultrasound, centrifuge at 3500r/min for 10min, obtain a sample of the tablets through a 0.45 ⁇ m water film, 4°C Refrigerate medium.
  • Pre-treatment of breakfast cereals grind the cereals separately, weigh 2.5g, add 10mL of 0.1mol/L hydrochloric acid, put them in an autoclave and heat at 121°C for 15min. Take out and cool to room temperature, adjust the pH to 6.8-7.2 with 1mol/L sodium hydroxide, add 1mL of peak amylase and place at 37°C for 4h. After the end, make up to 50 mL with methanol. After centrifugation, the supernatant was taken through the membrane to obtain a grain sample, which was refrigerated at 4°C.
  • Feed pretreatment is the same as breakfast cereals.

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Abstract

一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，属于食品检测技术领域。该方法将展开后的高效薄层色谱板通过荧光扫描进行核黄素的定量，采用表面增强拉曼光谱应用于展开后的高效薄层色谱板，明确核黄素的特征拉曼指纹图谱，进行目标位点的定性。该方法可实现单次实验同时检测多种样品，实现高通量筛检。单次筛检过程从样品准备到获得最终结果仅需1～2h，检测限可达到20～50μg/kg，检测方法可实现重复性RSD<10％，具有快速、经济的优点；同时基于HPTLC-SERS的分析方法的建立为平面高效薄层色谱分析提供了新的方法。

Description

一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法 技术领域
本发明涉及一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，属于食品检测技术领域。
背景技术
核黄素，又称维生素B2，是一种具备重要生理功能的水溶性维生素。核黄素的两种活性形式：黄素腺嘌呤核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)，作为氧化还原辅助因子，参与体内多种氧化还原反应，能促进糖、脂肪和蛋白质的代谢，对维持皮肤、黏膜和视觉的机能有一定的作用。研究表明，核黄素对于偏头痛、心脑血管疾病及中风等有抑制作用。核黄素在植物和微生物中合成，无法在人类和动物体内合成，因此，人类和动物需要从体外获取必需的核黄素。美国婴儿及成人的核黄素参考摄入量为0.4-1.3mg/天。除日常饮食外，核黄素强化食物及核黄素补充剂越来越受到广泛欢迎。同时，核黄素在动物饲料中的添加量占比巨大。
目前核黄素的检测方法有分光光度法、电化学法、毛细管电泳法及高效液相色谱法(HPTLC)/气相色谱(GC)与质谱法(MS)联用等。其中LC–MS(液质联用)分析法比较常用，其准确度较高，被普遍作为分析技术的有效方法，但样品前处理过程复杂，处理要求极高，测试时间长，通量低，无法实现多个样品同时检测。此外，质谱检测器的离子化界面极易受到与目标物共萃取出的食品基质物质的干扰，轻则引起信号抑制，重则造成仪器损坏，因此传统的液质联用法不适合对大量样品进行筛检。
近几年来，高效薄层色谱(HPTLC)快速发展，实现了一整套完备的分析体系，进一步拓宽了薄层色谱的应用领域。与其他分离技术相比，HPTLC系统的初始成本、维护成本以及单个样品测定成本相对较低，并可以灵活的与不同的技术联用，进行被分离化合物的结构鉴定、定性或定量。目前，HPTLC实现了与多种新型检测器的联用，生物传感器、原子吸收光谱、傅里叶变换红外光谱、质谱联用(HPTLC-MS)、表面增强拉曼联用(SERS)等。其与SERS的联用更是广受关注。表面增强拉曼光谱作为化学分析领域中痕量分子检测的一个很有吸引力的工具。其具有正常拉曼光谱的所有优点并且灵敏度高，由于其固有的分子指纹 特异性和单分子检测潜力，SERS甚至可以取代标准物质进行筛选，因此非常适合在目标位点确定分子信息，并有潜力与HPTLC联用取代MS进行分子确认。
因此，本发明提出一种新型核黄素筛检方法：高效薄层色谱-表面增强拉曼光谱联用进行核黄素快速筛检，实现快速可靠的定性与定量。
发明内容
本发明的目的是克服上述不足之处，提供一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法。
本发明的技术方案，一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其将展开后的高效薄层色谱板通过荧光扫描进行核黄素的定量，然后采用表面增强拉曼光谱应用于展开后的高效薄层色谱板，明确核黄素的特征拉曼指纹图谱，进行目标位点的定性。
具体检测方法如下：
1、样品制备：核黄素标准液是将核黄素用1％的氨水为溶剂，用甲醇稀释制备浓度为0.01mg/mL的样品。
复合维生素药片样品中核黄素的提取是磨粉后用1％的氨水溶解，超声20min取出，3500r/min离心10min，过0.45μm水膜得到。
早餐谷物/饲料样品中核黄素的提取是磨粉后称取2.5g，加入10mL 0.1mol/L的盐酸，放入高压灭菌锅中121℃加热15min。取出冷却至室温，用1mol/L的氢氧化钠调pH至6.8-7.2之间，加入1mL高峰淀粉酶置于37℃中反应4h。反应结束后，用甲醇定容至50mL，离心后取上清液过膜得到谷物/饲料样品。
2、纳米银胶的制备：依据以硼氢化钠为还原剂合成纳米银胶体的经典方法制备银胶。取4-6mL的原银胶体离心(3000-5000r/min，8-12min)后，弃上清液，将剩余液再置于混匀器上充分振荡1-2min，使其重新悬浮，得到10倍浓缩的纳米银胶体。
3、高效薄层色谱分离：将2-8μL核黄素的标准液和复合维生素片/早餐谷物/饲料样品用Linomat 5进行精确点样，点样完成后用展开液(甲醇/乙酸乙酯/三乙胺/水＝2/8/1.5/1(v/v))展开，上行展开60mm取出硅胶板置于45-55℃平板加热器上充分干燥2-4min。
4、薄层色谱扫描仪扫描定量：将经过高效薄层色谱板分离后所得的胶板置于薄层扫描仪中进行扫描定量。
5、HPTLC-SERS检测：经过高效薄层色谱分离后，胶板在紫外光的照射下用铅笔标记出可见的荧光条带。在标记区域，用毛细移液管滴加2-4μL浓缩纳米银胶体。立即用拉曼光谱仪进行扫描，获得目标物质的特征指纹图谱。
6、标准曲线的绘制：根据上述结果绘制核黄素标准曲线和添加了核黄素的空白样品标准曲线；
7、检测：取待测样品，进行步骤(2)-(5)检测，与步骤(6)绘制所得标准曲线进行对比，得到待测样品中核黄素的含量。
本发明的有益效果：本发明建立了一种高效薄层色谱-表面增强拉曼光谱联用快速筛检核黄素的方法，可实现单次实验同时检测多种样品，实现高通量筛检。单次筛检过程从样品准备到获得最终结果仅需1-2h，检测限可达到20～50μg/kg，检测方法可实现重复性RSD<10％，具有快速、经济的优点；同时基于HPTLC-SERS的分析方法的建立为平面高效薄层色谱分析开辟了新的视野。
附图说明
图1是实施例1、实施例2中核黄素标准品、复合维生素样品、早餐谷物样品及饲料样品展开图(A)、薄层扫描定量图(B)和核黄素标准曲线(C)。
图2是实施例3核黄素标准品、复合维生素样品、早餐谷物样品、饲料样品的SERS指纹图谱对照。
具体实施方式
实施例1
(1)核黄素标准液的制备：用质量浓度为1％的氨水为溶剂，用甲醇稀释制备浓度为0.01mg/mL的核黄素标准溶液；
(2)纳米银胶的制备：依据以硼氢化钠为还原剂合成纳米银胶体的经典方法制备银胶。取5mL的原银胶体离心(4000r/min，10min)后，弃4.5mL上清液，将0.5mL剩余液再置于混匀器上充分振荡1min，使其重新悬浮，得到10倍浓缩的纳米银胶体。
(3)高效薄层色谱分离：将2-8μL核黄素的标准液用Linomat 5进行精确点样，点样完成后用展开液(甲醇/乙酸乙酯/三乙胺/水＝2/8/1.5/1(v/v))展开，上行展开60mm取出硅胶板置于50℃平板加热器上充分干燥3min；
(4)薄层扫描仪扫描定量：将步骤(2)高效薄层色谱板氧化后所得的胶板置于薄层扫描仪中进行扫描定量，光源为氘灯或钨灯，扫描波长为300-400nm。扫描结束后，以扫描面积为y轴，核黄素质量为x轴，制作标准曲线；
结果如图1B、1C所示，随后通过所得标准曲线计算样品核黄素含量。
(5)HPTLC-SERS检测：置于DD70成像系统(Biostep)上，在366nm的照明下获取硅胶板的图像，并根据核黄素标品展开的Rf值，用铅笔标记出可见的荧光条带。在标记区域，用毛细移液管依次滴加3μL浓缩银胶体，立即用拉曼光谱仪进行扫描，每个拉曼散射信号平 均积分三次，各积分采集时间为10s，得到核黄素标准品SERS指纹图谱。
实施例2
分别对复合维生素药片、早餐谷物和饲料中的核黄素进行检测。
(1)复合维生素药片的预处理：将药片磨粉，称取0.5g溶于10mL 1％的氨水中，超声20min后取出，3500r/min离心10min，过0.45μm水膜得到药片样品，4℃中冷藏。
早餐谷物的预处理：分别将谷物磨粉，称取2.5g，加入10mL 0.1mol/L的盐酸，放入高压灭菌锅中121℃加热15min。取出冷却至室温，用1mol/L的氢氧化钠调pH至6.8-7.2之间，加入1mL高峰淀粉酶置于37℃反应4h。结束后，用甲醇定容至50mL。离心后取上清液过膜得到谷物样品，4℃中冷藏。
饲料的预处理与早餐谷物的处理方法相同。
(2)薄层色谱分离与检测：采用0.5MPa氮气为载体，用100μl注射器(CAMAG)将样品和核黄素标准液用Linomat 5进行精确点样于20×10cm的薄层板上，点样的条带长6mm，条带距离底部8mm，距离左端12mm，条带间距1.7mm。点样完成后用ADC-2(CAMAG)展开仪展开，展开前，通过在另一槽注入10mL的流动相使缸内达到饱和状态。取10mL优化后的展开液(甲醇/乙酸乙酯/三乙胺/水＝2/8/1.5/1(v/v))，上行展开60mm取出，置于50℃平板加热器上充分干燥3min。然后用薄层色谱扫描仪进行扫描定量，扫描条件与实施例1中(4)相同。
结果如图1A所示，含有核黄素的不同样品均能够获得清晰的成像，图1B结果显示，根据检测结果可计算获得核黄素的标准曲线(与图1C相同)，将维生素药片、早餐谷物和饲料的色谱峰值代入标准曲线，经计算，各样品中核黄素含量分别为0.35mg/mL，0.043mg/mL，0.67mg/mL，与HPLC检测结果一致。扫描结束后，进行SERS测定，测定条件与实施例1中(5)相同，得到样品的SERS指纹图谱，与标准品进行比较(图2)。
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 (9)

  1. 一种高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于：其将展开后的高效薄层色谱板通过荧光扫描进行核黄素的定量，采用表面增强拉曼光谱应用于展开后的高效薄层色谱板，明确核黄素的特征拉曼指纹图谱，进行目标位点的定性。
  2. 如权利要求1所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于具体步骤如下：
    (1)纳米银胶的制备：取4-6mL的原银胶体以3000-5000r/min离心8-12min后，弃去上清液，将剩余液体置于混匀器上充分振荡1-2min，使其重新悬浮，得到浓缩的纳米银胶体；
    (2)高效薄层色谱分离：将2-8μL核黄素的标准液和添加了核黄素标准液的待测空白样品用薄层色谱半自动点样仪进行精确点样，点样完成后用展开液展开，上行展开60mm取出硅胶板置于45-55℃平板加热器上充分干燥2-4min；
    (3)薄层色谱扫描仪扫描定量：将经过步骤(2)高效薄层色谱板分离后所得的胶板置于薄层扫描仪中进行扫描定量；
    (4)HPTLC-SERS检测：经过步骤(2)高效薄层色谱分离后，胶板在紫外光的照射下用铅笔标记出可见的荧光条带；在标记区域，用毛细移液管滴加步骤(1)制备所得2-4μL浓缩纳米银胶体；立即用拉曼光谱仪进行扫描，获得目标物质的特征指纹图谱；
    (5)标准曲线的绘制：根据上述结果绘制核黄素标准曲线和添加了核黄素的空白样品标准曲线；
    (6)检测：取待测样品，进行步骤(1)-(4)检测，与步骤(5)绘制所得标准曲线进行对比，得到待测样品中核黄素的含量。
  3. 如权利要求2所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于：将步骤(2)所得的胶板置于薄层色谱扫描仪中进行扫描定量；步骤(3)薄层色谱扫描条件为汞灯、氘灯或钨灯，其测定波长为200-500nm之间，最后通过测定的标准曲线进行含量计算。
  4. 如权利要求2所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于：在展开后的硅胶板上标记带有荧光的核黄素，直接进行SERS测定，成功将SERS直接运用在高效薄层色谱板上。
  5. 如权利要求2所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于：步骤(2)所述点样展开液具体为体积比甲醇：乙酸乙酯：三乙胺：水＝2：8：1.5：1。
  6. 如权利要求2所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于：步骤(2)所述样品为复合维生素、早餐谷物或饲料样品。
  7. 如权利要求6所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于所述复合维生素制作样品的前处理过程如下：将复合维生素药片磨粉，称取0.5g溶于10mL 1％的氨水中，超声20min后取出，3500r/min离心10min，过0.45μm水膜得到药片样品，4℃中冷藏。
  8. 如权利要求6所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于所述早餐谷物或饲料样品的前处理过程如下：分别将早餐谷物或饲料磨粉，称取2.5g，加入10mL 0.1mol/L的盐酸，放入高压灭菌锅中121℃加热15min；取出冷却至室温，用1mol/L的氢氧化钠调pH至6.8-7.2，加入1mL高峰淀粉酶置于37℃反应4h；结束后，用甲醇定容至50mL；离心后取上清液过膜得到谷物或饲料样品，4℃中冷藏。
  9. 如权利要求2所述高效薄层色谱和表面增强拉曼光谱联用筛检食品药品中核黄素的方法，其特征在于所述核黄素的标准液是将核黄素用1％的氨水为溶剂，用甲醇稀释制备得到。
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