WO2020258710A1 - 一种酸枣仁中黄曲霉毒素的提取检测方法 - Google Patents

一种酸枣仁中黄曲霉毒素的提取检测方法 Download PDF

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WO2020258710A1
WO2020258710A1 PCT/CN2019/121265 CN2019121265W WO2020258710A1 WO 2020258710 A1 WO2020258710 A1 WO 2020258710A1 CN 2019121265 W CN2019121265 W CN 2019121265W WO 2020258710 A1 WO2020258710 A1 WO 2020258710A1
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aflatoxin
aflatoxins
wild jujube
detection
solution
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PCT/CN2019/121265
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French (fr)
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梁鑫淼
金红利
戚华文
刘艳芳
朱小建
徐鑫
袁璐
高德嵩
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泰州医药城国科化物生物医药科技有限公司
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Publication of WO2020258710A1 publication Critical patent/WO2020258710A1/zh

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    • 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
    • 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
    • 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
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • 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
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

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  • the invention relates to the technical field of extraction and detection of traditional Chinese medicines, in particular to a method for extraction and detection of aflatoxins in jujube seeds.
  • Aflatoxin is a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus strains. It is structurally a derivative of dihydrofuranocoumarin. It is much more toxic than cyanide, iodide and organic pesticides. It has been discovered so far The most toxic type of mycotoxins. Aflatoxins are commonly found in plant-derived agricultural and sideline products such as peanuts, grains, and Chinese medicinal materials. They are harmful to human and animal liver tissues. In severe cases, they can cause liver cancer or even death. .
  • Jujube seed has the effects of invigorating the liver, calming the heart, restraining sweat, and promoting fluid production. It is used for sleeplessness, palpitations, dreams, physical weakness, sweating, and thirst, etc.
  • the "Compendium of Materia Medica” lists it as the top grade, but the seed It is prone to mildew and produces a variety of mycotoxins harmful to the human body. Among them, aflatoxin is the most harmful. At present, the determination of aflatoxin in my country mainly focuses on food, peanuts and their products, milk and their products, etc. , The detection method of aflatoxin in Chinese medicinal materials is relatively backward.
  • Common detection methods for aflatoxins include thin layer chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay and so on.
  • Thin-layer chromatography is the earliest and most widely used separation and analysis technique for aflatoxins, but the sample pretreatment is cumbersome and the extract contains many impurities, which cannot meet the needs of modern analysis.
  • High performance liquid chromatography requires samples to be derivatized. The operation is complicated and the analysis time is long; the complex matrix of the enzyme-linked immunosorbent assay is easy to cause interference and false positives; the current "Chinese Pharmacopoeia" 2015 version of the detection methods for aflatoxin all use immunoaffinity columns to purify samples, but this The method is complicated and costly.
  • the present invention expects to provide a method for extraction and detection of aflatoxins in wild jujube seeds, which is simple and fast to operate, reduces the interference of the sample matrix to the target, reduces the matrix effect, has high sensitivity, less pollution, and relatively low detection cost. Low and easy to promote.
  • the present invention provides a method for extracting and detecting aflatoxin in wild jujube kernels, which comprises the following steps:
  • the column is Unitary C18
  • the mobile phase A is formic acid Ammonium or ammonium acetate or formic acid
  • mobile phase B is methanol
  • flow rate is 0.2mL/min ⁇ 0.5mL/min
  • column temperature is 25°C ⁇ 40°C
  • injection volume is 1 ⁇ L-10 ⁇ L
  • ionization method is electrospray positive ionization
  • the detection method is multi-ion reaction monitoring.
  • the pore size of the organic phase filter membrane is 0.2 ⁇ m to 0.5 ⁇ m.
  • the chromatographic column packing is alkylsilane-bonded silica gel
  • the length of the chromatographic column is 100mm-250mm
  • the inner diameter is 2.1 ⁇ m-4.6 ⁇ m
  • the particle size is 1.7 ⁇ m-5 ⁇ m.
  • step a) after vortexing at 4000 r/min for 3 min, ultrasonicating at 500 W ultrasonic power for 20 min, vortex mixing at 4000 r/min for 3 min, and centrifuging at 4000 r/min for 5 min.
  • N-propyl ethylenediamine was added for purification, vortexed at 4000r/min for 3 minutes, centrifuged at 4000r/min for 3 minutes, and passed through a 0.22 ⁇ m organic phase filter membrane filter.
  • step of determining aflatoxin standard use solution in step c) includes: taking a solution of aflatoxin mixed reference substance of known concentration, diluting with 50% to 90% volume fraction of methanol or ethanol or acetonitrile solution, After the container is stored, it is stored at 2°C to 8°C for 1 to 3 months, and when used, it is prepared with a standard solution of aflatoxin of different concentrations with 50% to 90% volume fraction of methanol.
  • volume fraction of methanol is 70% to 90%.
  • the method of the present invention is simple and fast to operate, reduces the interference of the sample matrix to the target, reduces the matrix effect, has high sensitivity, less pollution, low detection cost, and is easy to popularize.
  • Figure 1 is a schematic flow chart of a method for extraction and detection of aflatoxin in jujube seed according to the present invention
  • Figure 2 is a chromatogram of aflatoxin B1 in the standard used liquid in Example 1 of the present invention.
  • Fig. 3 is a chromatogram of aflatoxin B2 in the standard used liquid in Example 1 of the present invention.
  • Figure 4 is a chromatogram of aflatoxin G1 in the standard used liquid in Example 1 of the present invention.
  • Fig. 5 is a chromatogram of aflatoxin G2 in the standard liquid used in Example 1 of the present invention.
  • Figure 7 is a standard curve diagram of aflatoxin B2 in Example 1 of the present invention.
  • Fig. 8 is a standard curve diagram of aflatoxin G1 in Example 1 of the present invention.
  • Figure 9 is a standard curve diagram of aflatoxin G2 in Example 1 of the present invention.
  • Fig. 1 is a schematic flow chart of a method for extraction and detection of aflatoxin in wild jujube seed according to the present invention. As shown in the figure, it specifically includes the following steps:
  • Step 101 Crush the wild jujube kernels, pass through a No. 2 sieve to obtain wild jujube kernel powder and place it in a centrifuge tube with a stopper, add methanol or acetonitrile, and the material-to-liquid ratio is 1:1 ⁇ 1:100; at 1000r/min ⁇ 3000r/ After vortexing for 1min ⁇ 10min at the speed of 1min, ultrasonic power of 100W ⁇ 500W for 5min ⁇ 30min; add 0.1g ⁇ 20g anhydrous sodium sulfate or anhydrous magnesium sulfate or sodium chloride at 1000r/min ⁇ 3000r/min After vortex mixing for 1min ⁇ 10min, centrifuge at 1500r/min ⁇ 4500r/min for 8min ⁇ 16min, and take the supernatant;
  • the wild jujube seed powder in a 50mL centrifuge tube with a stopper, add acetonitrile with a material-to-liquid ratio of 1:10, vortex at 4000r/min for 3min, sonicate at 500W ultrasonic power for 20min, and add 3 ⁇ 10g of anhydrous sodium sulfate, vortex and mix at 4000r/min for 3min, centrifuge at 4000r/min for 5min, and take the supernatant;
  • Step 102 Take the supernatant into a centrifuge tube, add octadecylsilane-bonded silica gel or N-propyl ethylenediamine for purification, vortex and mix at a speed of 1000r/min ⁇ 3000r/min for 1min ⁇ 10min, then Centrifuge at a speed of 1500r/min ⁇ 4500r/min for 3min ⁇ 10min, filter through an organic phase filter membrane with a pore size of 0.1 ⁇ m ⁇ 0.6 ⁇ m to obtain the test solution;
  • the pore size of the organic phase filter membrane is 0.2 ⁇ m to 0.5 ⁇ m;
  • Step 103 After filtering the standard aflatoxin solution through an organic phase filter with a pore size of 0.1 ⁇ m to 0.6 ⁇ m, the liquid to be tested is detected by a liquid chromatography-tandem mass spectrometer.
  • the column is Unitary C18, and the mobile phase A is Ammonium formate or ammonium acetate or formic acid, mobile phase B is methanol, flow rate is 0.2mL/min ⁇ 0.5mL/min, column temperature is 25°C ⁇ 40°C, injection volume is 1 ⁇ L-10 ⁇ L, ionization method is electrospray positive ion Ionization, the detection method is multi-ion reaction monitoring;
  • the determination step of the aflatoxin standard use solution includes: taking the aflatoxin mixed reference solution with a known concentration, diluting it with a methanol or ethanol or acetonitrile solution with a volume fraction of 50% to 90%, and holding it at 2°C after constant volume. Store at ⁇ 8°C for 1 ⁇ 3 months, use 50% ⁇ 90% volume fraction of methanol to prepare standard solution of aflatoxin with different concentrations;
  • the pore size of the organic phase filter membrane is 0.2 ⁇ m to 0.5 ⁇ m;
  • volume fraction of methanol is preferably 70% to 90%
  • the chromatographic column packing is alkylsilane-bonded silica gel
  • the chromatographic column has a length of 100 mm to 250 mm, an inner diameter of 2.1 ⁇ m to 4.6 ⁇ m, and a particle size of 1.7 ⁇ m to 5 ⁇ m.
  • the calculation method of the aflatoxin content is as follows: draw a standard curve with the mass concentration of aflatoxin as the abscissa and the corresponding peak area as the ordinate. According to the standard curve of each aflatoxin and the sample The peak area is used to calculate the content of each aflatoxin in the sample:
  • X the content of each aflatoxin in the sample, in ⁇ g/kg
  • V The volume of the extract in the sample extraction process, in mL
  • Step 1 Crush the wild jujube seed sample, pass through a No. 2 sieve, weigh 5g wild jujube seed powder and place it in a 50mL centrifuge tube with a stopper, add 20mL acetonitrile, vortex at 4000r/min for 3min, then use 500W ultrasound Ultrasonic power for 20min; add 5g of anhydrous sodium sulfate for dehydration, vortex and mix at 4000r/min for 3min, centrifuge at 4000r/min for 5min, and take the supernatant;
  • Step 2 Take 2mL of supernatant to a 5mL centrifuge tube, add 200mg of N-propyl ethylenediamine adsorbent for purification, vortex and mix at 4000r/min for 3min, centrifuge at 4000r/min for 3min, pass 0.22 ⁇ m Filter the organic phase filter membrane with pore size to obtain the test solution;
  • Step 3 Take the aflatoxin mixed reference solution (the labeled concentrations of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 are 500 ⁇ g/L, 150 ⁇ g/L, 500 ⁇ g/L, 150 ⁇ g/ L) 1mL, dilute with 70% methanol, and dilute to 10mL, to obtain the intermediate standard solution (aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 are respectively 50 ⁇ g/L, 15 ⁇ g/ L, 50 ⁇ g/L, 15 ⁇ g/L), stored at 2°C ⁇ 8°C for three months, when used, it is formulated with methanol to contain aflatoxin B2 and G2 at a concentration of 0.04ng/mL ⁇ 3ng/mL, containing Aspergillus flavus
  • Step 4 Take the standard aflatoxin solution and filter it through a 0.22 ⁇ m pore size organic phase filter membrane, and then use liquid chromatography-tandem mass spectrometer to detect the test solution to obtain Figure 2 to Figure 5 aflatoxin B1, B2 G1, G2 chromatogram, the specific conditions are as follows:
  • Chromatographic column Unitary C18 (100mm ⁇ 3mm, 3 ⁇ m);
  • Mobile phase use 10mmol/L ammonium acetate solution as mobile phase A and methanol as mobile phase B; perform gradient elution as specified in Table 1.
  • Mass spectrometry conditions ionization method, electrospray positive ionization;
  • Detection mode multiple reaction monitoring (MRM); spray pressure: 7.0Bar; heating gas flow: 1000L/Hr; drying gas flow: 1000L/Hr; drying gas temperature: 500°C; capillary voltage: 3.1kV; cone voltage: 68V ; Cone flow rate: 150L/Hr.
  • MRM multiple reaction monitoring
  • spray pressure 7.0Bar
  • heating gas flow 1000L/Hr
  • drying gas flow 1000L/Hr
  • drying gas temperature 500°C
  • capillary voltage 3.1kV
  • cone voltage 68V
  • Cone flow rate 150L/Hr.
  • CE collision voltage
  • the peak area corresponding to aflatoxin B1 is 468652
  • the peak area corresponding to aflatoxin B2 is 240081
  • the peak area corresponding to aflatoxin G1 is 351048, and the peak area corresponding to aflatoxin G2 is 45290.
  • the preliminary extraction process takes about 30 minutes, which takes a short time, is simple to operate, easy to use, and involves less organic solvents, so there is less environmental pollution.
  • the existing immunoaffinity column to purify the sample (China Pharmacopoeia), lower cost and more economical and applicable.

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Abstract

一种酸枣仁中黄曲霉毒素的提取检测方法，包括将酸枣仁粉碎，过二号筛，得酸枣仁粉末并置于具塞离心管中，加入甲醇或乙腈，涡旋振荡后，以100W～500W的超声功率超声5min～30min；加入无水硫酸钠或无水硫酸镁或氯化钠，涡旋混合后，转速离心，得到上清液；取上清液至离心管中，加入十八烷基硅烷键合硅胶或N-丙基乙二胺净化，涡旋混合后，转速离心，经有机相滤膜过滤，得待测液；取黄曲霉毒素标准使用液经有机相滤膜过滤后，采用液相色谱-串联质谱仪检测待测液。该方法操作简单、快速，降低了样品基质对目标物的干扰，减少了基质效应，灵敏度高，污染少，检测成本较低，易于推广。

Description

一种酸枣仁中黄曲霉毒素的提取检测方法 技术领域
本发明涉及中药提取检测技术领域，具体涉及一种酸枣仁中黄曲霉毒素的提取检测方法。
背景技术
黄曲霉毒素是黄曲霉和寄生曲霉产毒菌株的次级代谢产物，结构上是一类二氢呋喃香豆素的衍生物，毒性远高于氰化物、碘化物和有机农药，是迄今为止发现毒性最强的一类真菌毒素，黄曲霉毒素常见于花生、谷物和中药材等植物源性农副产品中，其危害性在于对人及动物肝脏组织有破坏作用，严重时，可导致肝癌甚至死亡。
酸枣仁具有补肝、宁心、敛汗、生津的作用，用于虚烦不眠，惊悸多梦，体虚多汗，津伤口渴等，《本草纲目》将其列为上品，但酸枣仁易发生霉变，从而产生多种对人体有害的霉菌毒素，其中以黄曲霉毒素危害最大，当前，我国关于黄曲霉毒素的测定主要集中在对粮食、花生及其制品、奶类及其制品等，对于中药材中黄曲霉毒素的检测方法相对比较落后。
黄曲霉毒素常见的检测方法有薄层层析法、高效液相色谱法、酶联免疫吸附法等。薄层层析法是黄曲霉毒素最早、应用最广的分离分析技术，但样品前期处理繁琐、提取液杂质较多，不能满足于现代分析的需求；高效液相色谱法需要对样品进行衍生，操作复杂且分析时间较长；酶联免疫吸附法的复杂基质易造成干扰，出现假阳性；目前《中国药典》2015版针对黄曲霉毒素的检测方法均采用免疫亲和柱来净化样品，但该方法操作复杂且成本较高。
发明内容
有鉴于此，本发明期望提供一种酸枣仁中黄曲霉毒素的提取检测方法，操作简单、快速，降低了样品基质对目标物的干扰，减少了基质效应，灵敏度高，污染少，检测成本较低，易于推广。
为达到上述目的，本发明的技术方案是这样实现的：
本发明提供了一种酸枣仁中黄曲霉毒素提取检测方法，包括以下步骤：
a)将酸枣仁粉碎，过二号筛，得酸枣仁粉末并置于具塞离心管中，加入甲醇或乙腈，料液比为1:1～1:100；以1000r/min～3000r/min的转速涡旋振荡1min～10min后，以100W～500W的超声功率超声5min～30min；加入0.1g～20g无水硫酸钠或无水硫酸镁或氯化钠，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心8min～16min，取上清液；
b)取上清液至离心管中，加入十八烷基硅烷键合硅胶或N-丙基乙二胺净化，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心3min～10min，经0.1μm～0.6μm孔径的有机相滤膜过滤，得待测液；
c)取黄曲霉毒素标准使用液经0.1μm～0.6μm孔径的有机相滤膜过滤后，采用液相色谱-串联质谱仪检测所述待测液，色谱柱为Unitary C18，流动相A为甲酸铵或醋酸铵或甲酸，流动相B为甲醇，流速为0.2mL/min～0.5mL/min，柱温为25℃～40℃，进样体积为1μL-10μL，电离方式为电喷雾正离子电离，检测方式为多离子反应监测。
进一步地，所述有机相滤膜孔径为0.2μm～0.5μm。
进一步地，所述色谱柱填料为烷基硅烷键合硅胶，所述色谱柱长度为 100mm～250mm，内径为2.1μm～4.6μm，粒径为1.7μm～5μm。
进一步地，所述步骤a)中以4000r/min的转速涡旋振荡3min后，以500W的超声功率超声20min，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心5min。
进一步地，所述步骤b)中加入N-丙基乙二胺净化，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心3min后，经0.22μm孔径的有机相滤膜过滤。
进一步地，所述步骤c)中黄曲霉毒素标准使用液测定步骤包括：取已知浓度的黄曲霉毒素混合对照品溶液，用50％～90％体积分数的甲醇或乙醇或乙腈溶液稀释、定容后在2℃～8℃下保存1～3个月，使用时用50％～90％体积分数的甲醇配制成不同浓度的黄曲霉毒素标准使用液。
进一步地，所述甲醇体积分数为70％～90％。
本发明有益效果如下：本发明方法操作简单、快速，降低了样品基质对目标物的干扰，减少了基质效应，灵敏度高，污染少，检测成本较低，易于推广。
附图说明
图1为本发明一种酸枣仁中黄曲霉毒素的提取检测方法流程示意图；
图2为本发明实施例1中标准使用液黄曲霉毒素B1色谱图；
图3为本发明实施例1中标准使用液黄曲霉毒素B2色谱图；
图4为本发明实施例1中标准使用液黄曲霉毒素G1色谱图；
图5为本发明实施例1中标准使用液黄曲霉毒素G2色谱图；
图6为本发明实施例1中黄曲霉毒素B1标准曲线图；
图7为本发明实施例1中黄曲霉毒素B2标准曲线图；
图8为本发明实施例1中黄曲霉毒素G1标准曲线图；
图9为本发明实施例1中黄曲霉毒素G2标准曲线图。
具体实施方式
为了能够更加详尽地了解本发明的特点与技术内容，下面对本发明的实现进行详细阐述。
图1为本发明一种酸枣仁中黄曲霉毒素的提取检测方法流程示意图，如图所示，具体包括以下步骤：
步骤101：将酸枣仁粉碎，过二号筛，得酸枣仁粉末并置于具塞离心管中，加入甲醇或乙腈，料液比为1:1～1:100；以1000r/min～3000r/min的转速涡旋振荡1min～10min后，以100W～500W的超声功率超声5min～30min；加入0.1g～20g无水硫酸钠或无水硫酸镁或氯化钠，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心8min～16min，取上清液；
这里，优选将酸枣仁粉末置于50mL具塞离心管中，加入乙腈，料液比为1:10，以4000r/min的转速涡旋振荡3min后，以500W的超声功率超声20min，加入3～10g无水硫酸钠，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心5min，取上清液；
步骤102：取上清液至离心管中，加入十八烷基硅烷键合硅胶或N-丙基乙二胺净化，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心3min～10min，经0.1μm～0.6μm孔径的有机相滤膜过滤，得待测液；
这里，优选有机相滤膜孔径为0.2μm～0.5μm；
这里，优选取1mL～5mL上清液至5mL离心管中，加入100mg～500mg N-丙基乙 二胺净化，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心3min后，经0.22μm孔径的有机相滤膜过滤，得待测液；
步骤103：取黄曲霉毒素标准使用液经0.1μm～0.6μm孔径的有机相滤膜过滤后，采用液相色谱-串联质谱仪检测所述待测液，色谱柱为Unitary C18，流动相A为甲酸铵或醋酸铵或甲酸，流动相B为甲醇，流速为0.2mL/min～0.5mL/min，柱温为25℃～40℃，进样体积为1μL-10μL，电离方式为电喷雾正离子电离，检测方式为多离子反应监测；
这里，所述黄曲霉毒素标准使用液测定步骤包括：取已知浓度的黄曲霉毒素混合对照品溶液，用50％～90％体积分数的甲醇或乙醇或乙腈溶液稀释、定容后在2℃～8℃下保存1～3个月，使用时用50％～90％体积分数的甲醇配制成不同浓度的黄曲霉毒素标准使用液；
这里，优选有机相滤膜孔径为0.2μm～0.5μm；
这里，所述甲醇体积分数优选为70％～90％；
这里，所述色谱柱填料为烷基硅烷键合硅胶，所述色谱柱长度为100mm～250mm，内径为2.1μm～4.6μm，粒径为1.7μm～5μm。
本发明中，所述黄曲霉毒素含量的计算方法如下：以黄曲霉毒素的质量浓度为横坐标，对应的峰面积为纵坐标绘制标准曲线，根据每种黄曲霉毒素的标准曲线及样品中的峰面积计算样品中每种黄曲霉毒素含量：
X＝(A×V×1000)/m
其中：X—样品中每种黄曲霉毒素含量，单位为μg/kg；
A—试样按外标法在标准曲线中对应的浓度，单位为μg/mL；
V—样品提取过程中提取液的体积，单位为mL；
m—样品的取样量，单位为g。
下面将通过具体实施例来进一步详细阐述。
实施例
步骤1：将酸枣仁样品粉碎，过二号筛，称取5g酸枣仁粉末并置于50mL具塞离心管中，加入20mL乙腈，以4000r/min的转速涡旋振荡3min后，以500W的超声功率超声20min；加入5g无水硫酸钠脱水，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心5min，取上清液；
步骤2：取2mL上清液至5mL离心管中，加入200mgN-丙基乙二胺吸附剂净化，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心3min，经0.22μm孔径的有机相滤膜过滤，得待测液；
步骤3：取黄曲霉毒素混合对照品溶液(黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1和黄曲霉毒素G2的标示浓度分别为500μg/L、150μg/L、500μg/L、150μg/L)1mL，用70％甲醇稀释、定容至10mL，得中间标准溶液(黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1和黄曲霉毒素G2的标示浓度分别为50μg/L、15μg/L、50μg/L、15μg/L)，在2℃～8℃下保存三个月，使用时用甲醇配制成含黄曲霉毒素B2、G2浓度为0.04ng/mL～3ng/mL，含黄曲霉毒素B1、G1浓度为0.12ng/mL～10ng/mL的系列对照品标准使用液；
步骤4：取黄曲霉毒素标准使用液经0.22μm孔径的有机相滤膜过滤后，采用液相色谱-串联质谱仪检测所述待测液，得图2～图5黄曲霉毒素B1、B2、G1、G2色谱图，具体条件如下：
色谱条件：色谱柱：Unitary C18(100mm×3mm,3μm)；
流动相：以10mmol/L醋酸铵溶液为流动相A，以甲醇为流动相B；按下表1中的规定进行梯度洗脱。
表1 梯度洗脱程序
Figure PCTCN2019121265-appb-000001
流速：0.3mL/min；
柱温：25℃-40℃；
进样量：5μL；
质谱条件：电离方式，电喷雾正离子电离；
检测模式：多反应监测(MRM)；喷雾压力：7.0Bar；加热气流量：1000L/Hr；干燥气流量：1000L/Hr；干燥气温度：500℃；毛细管电压：3.1kV；锥孔电压：68V；锥孔流速：150L/Hr。各化合物监测离子对和碰撞电压(CE)见下表2。
表2 四种黄曲霉毒素的质谱分析参数
Figure PCTCN2019121265-appb-000002
以黄曲霉毒素的质量浓度x为横坐标，对应的峰面积y为纵坐标进行线性回归，黄曲霉毒素B1、B2、G1、G2标准曲线如图6～图9所示，线性方程、线性范围、相关系数、定量限结果如表3，结果显示，四种黄曲霉毒素线性关系良好，r 2不低于0.9901。
表3 四种黄曲霉毒素线性方程、线性范围、相关系数、定量限
Figure PCTCN2019121265-appb-000003
以各黄曲霉毒素的1倍、2倍、4倍定量限为空白样品加标水平，每个添加水平做六组平行实验，按照上述的提取分析方法进行检测，回收率结果及相对标准偏差如表4所示，结果显示，空白样品的加标回收率为81.96％～93.21％，定量限能够符合检测要求。
表4 四种黄曲霉毒素的加标回收率及相对标准偏差(n＝6)
Figure PCTCN2019121265-appb-000004
Figure PCTCN2019121265-appb-000005
以黄曲霉毒素的质量浓度为横坐标，对应的峰面积为纵坐标绘制标准曲线，根据每种黄曲霉毒素的标准曲线及样品中的峰面积，得实施例中黄曲霉毒素B1含量为15.65μg/kg，黄曲霉毒素B2为5.24μg/kg，黄曲霉毒素G1为4.56μg/kg，黄曲霉毒素G2为0.57μg/kg。
其中，黄曲霉毒素B1对应的峰面积是468652，黄曲霉毒素B2对应的峰面积是240081，黄曲霉毒素G1对应的峰面积是351048，黄曲霉毒素G2对应的峰面积是45290。
本实施例中前期提取处理在30分钟左右，耗时较短，操作简单，易上手，涉及的有机溶剂用量较少，因此环境污染少，相比较现有的免疫亲合柱来净化样品(中国药典)，成本较低，更经济适用。
表5 本发明实施例和现有技术的参数对比
Figure PCTCN2019121265-appb-000006
以上所述，仅为本发明的较佳实施例，并非用于限定本发明的保护范围，故凡依照本发明专利范围所做的等效变化或修饰，均属于本发明专利权利要求范围内。

Claims (7)

  1. 一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，包括以下步骤：
    a)将酸枣仁粉碎，过二号筛，得酸枣仁粉末并置于具塞离心管中，加入甲醇或乙腈，料液比为1:1～1:100；以1000r/min～3000r/min的转速涡旋振荡1min～10min后，以100W～500W的超声功率超声5min～30min；加入0.1g～20g无水硫酸钠或无水硫酸镁或氯化钠，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心8min～16min，取上清液；
    b)取上清液至离心管中，加入0.5g～10g十八烷基硅烷键合硅胶或N-丙基乙二胺净化，以1000r/min～3000r/min的转速涡旋混合1min～10min后，以1500r/min～4500r/min的转速离心3min～10min，经0.1μm～0.6μm孔径的有机相滤膜过滤，得待测液；
    c)取黄曲霉毒素标准使用液经0.1μm～0.6μm孔径的有机相滤膜过滤后，采用液相色谱-串联质谱仪检测所述待测液，色谱柱为Unitary C18，流动相A为甲酸铵或醋酸铵或甲酸，流动相B为甲醇，流速为0.2mL/min～0.5mL/min，柱温为25℃～40℃，进样体积为1μL-10μL，电离方式为电喷雾正离子电离，检测方式为多离子反应监测。
  2. 根据权利要求1所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述有机相滤膜孔径为0.2μm～0.5μm。
  3. 根据权利要求1所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述色谱柱填料为烷基硅烷键合硅胶，所述色谱柱长度为100mm～250mm，内径为2.1μm～4.6μm，粒径为1.7μm～5μm。
  4. 根据权利要求1所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述步骤a)中以4000r/min的转速涡旋振荡3min后，以500W的超声功率超声20min，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心5min。
  5. 根据权利要求1所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述步骤b)中加入N-丙基乙二胺净化，以4000r/min的转速涡旋混合3min后，以4000r/min的转速离心3min后，经0.22μm孔径的有机相滤膜过滤。
  6. 根据权利要求1所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述步骤c)中黄曲霉毒素标准使用液测定步骤包括：取已知浓度的黄曲霉毒素混合对照品溶液，用50％～90％体积分数的甲醇或乙醇或乙腈溶液稀释、定容后在2℃～8℃下保存1～3个月，使用时用50％～90％体积分数的甲醇配制成不同浓度的黄曲霉毒素标准使用液。
  7. 根据权利要求6所述的一种酸枣仁中黄曲霉毒素的提取检测方法，其特征在于，所述甲醇体积分数为70％～90％。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4285698A (en) * 1980-04-28 1981-08-25 Peanut Research & Testing Laboratories, Inc. Analysis of aflatoxins in peanuts by high pressure liquid chromatograph
CN105651899A (zh) * 2016-04-08 2016-06-08 云南健牛生物科技有限公司 一种高灵敏检测黄曲霉毒素的方法及其应用
CN105823844A (zh) * 2016-04-28 2016-08-03 苏州市天灵中药饮片有限公司 一种中药饮片中黄曲霉毒素的检测方法
CN105866295A (zh) * 2016-06-12 2016-08-17 肇庆学院 一种快速检测中药材中黄曲霉素b1含量的方法
CN106526056A (zh) * 2017-01-04 2017-03-22 浙江国正检测技术有限公司 啤酒及其原辅料中afb1的超高效液相色谱‑串联质谱检测方法
CN108195971A (zh) * 2018-02-07 2018-06-22 吉林出入境检验检疫局检验检疫技术中心 用液相色谱法测定灵芝孢子粉中黄曲霉毒素的检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109932467A (zh) * 2018-08-10 2019-06-25 烟台出入境检验检疫局检验检疫技术中心 超高效液相色谱－四级杆/高分辨质谱法测定花生中黄曲霉毒素和农药残留的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4285698A (en) * 1980-04-28 1981-08-25 Peanut Research & Testing Laboratories, Inc. Analysis of aflatoxins in peanuts by high pressure liquid chromatograph
CN105651899A (zh) * 2016-04-08 2016-06-08 云南健牛生物科技有限公司 一种高灵敏检测黄曲霉毒素的方法及其应用
CN105823844A (zh) * 2016-04-28 2016-08-03 苏州市天灵中药饮片有限公司 一种中药饮片中黄曲霉毒素的检测方法
CN105866295A (zh) * 2016-06-12 2016-08-17 肇庆学院 一种快速检测中药材中黄曲霉素b1含量的方法
CN106526056A (zh) * 2017-01-04 2017-03-22 浙江国正检测技术有限公司 啤酒及其原辅料中afb1的超高效液相色谱‑串联质谱检测方法
CN108195971A (zh) * 2018-02-07 2018-06-22 吉林出入境检验检疫局检验检疫技术中心 用液相色谱法测定灵芝孢子粉中黄曲霉毒素的检测方法

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