CN108226355A - The assay method of aflatoxin in a kind of tealeaves - Google Patents

The assay method of aflatoxin in a kind of tealeaves Download PDF

Info

Publication number
CN108226355A
CN108226355A CN201810012304.8A CN201810012304A CN108226355A CN 108226355 A CN108226355 A CN 108226355A CN 201810012304 A CN201810012304 A CN 201810012304A CN 108226355 A CN108226355 A CN 108226355A
Authority
CN
China
Prior art keywords
aflatoxin
sample
solution
acetonitrile
column
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810012304.8A
Other languages
Chinese (zh)
Inventor
李军明
殷建忠
钟读波
冯月梅
尚乐
卞家亭
胡丽
李向楠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yunnan Cloud Testing Quality Inspection Co Ltd
Kunming Medical University
Original Assignee
Yunnan Cloud Testing Quality Inspection Co Ltd
Kunming Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yunnan Cloud Testing Quality Inspection Co Ltd, Kunming Medical University filed Critical Yunnan Cloud Testing Quality Inspection Co Ltd
Priority to CN201810012304.8A priority Critical patent/CN108226355A/en
Publication of CN108226355A publication Critical patent/CN108226355A/en
Pending legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/04Preparation or injection of sample to be analysed
    • G01N30/06Preparation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Abstract

The present invention relates to a kind of assay methods of aflatoxin in a kind of assay method of aflatoxin more particularly to tealeaves, and including sample extraction, purification, standard solution measures and sample determination step, and sample extraction step uses acetonitrile extracting solution;Purifying step uses solid-phase extraction column and decontaminating column secondary purification;It takes aflatoxin standard reserving solution that standard series is made using liquid in standard solution determination step, is measured after crossing organic phase miillpore filter with liquid chromatography tandem mass spectrometry;Assay method provided by the invention is easy to operate, and quickly, selectivity and high sensitivity are as a result accurate reliable;Pollution is few, safe, reduces the pollution to operating personnel and environment;Testing cost is relatively low, easy to spread.

Description

The assay method of aflatoxin in a kind of tealeaves
Technical field
The present invention relates to technical field of food safety, and in particular to a kind of assay method of aflatoxin more particularly to The assay method of aflatoxin in a kind of tealeaves.
Background technology
Aflatoxin is the secondary metabolite of aspergillus flavus and aspergillus parasiticus toxigenic bacterium strain, is to find toxicity so far Most strong a kind of mycotoxin, it is presently found to have more than 20 kinds, aflatoxin B is common are in food1、B2、G1、G2And M1 Deng.Aflatoxin is mainly hepar damnification to the harm of human body, can induce the diseases such as hepatitis, hepatic sclerosis, liver cancer.Aspergillus flavus poison Element is primarily present in corn, cereal, peanut and nut, in particularly mouldy crops.
The analysis method of aflatoxin mainly has thin layer chromatography, high performance liquid chromatography and enzyme linked immunosorbent assay. Thin layer chromatography equipment is simple, and cost is relatively low, is easy to universal, but the method sample pre-treatments are cumbersome, and extraction, clean-up effect are paid no attention to Think, impurity is more, result is interfered larger.High performance liquid chromatography is current most common analysis method, which automates journey Degree is higher, but needs to perform the derivatization sample processing, there are the shortcomings of complicated for operation, analysis time is long, and it is current before Processing method is based on the immune affinity column method of purification, but the method cannot remove tea polyphenols and Tea Pigment in tealeaves completely, right There are severe jammings for testing result.Enzyme linked immunosorbent assay is used for rapid screening, and the tea polyphenols and Tea Pigment in tealeaves equally can Result is interfered, is susceptible to " false positive ".
《Standard GB/T 5009.22-2016 national food safety standard --- aflatoxin B race and G races in food Measure》Using isotopic dilution Liquid Chromatography-Tandem Mass Spectrometry, high performance liquid chromatography-pre-column derivatization, high-efficient liquid phase color Spectrum-pre-column derivatization, Enzyme-linked Immunosorbent Assay screening method and thin-layered chromatography, but the range that above-mentioned five kinds of methods are applicable in does not have Cover tealeaves.《Import and export the measure of aflatoxin residual quantity in professional standard SN/T 3263-2012 export foods》In, it adopts With high performance liquid chromatography and fluorometry, the scope of application of wherein high performance liquid chromatography has covered tealeaves, but It is that the detection of this method is limited to 0.5 μ g/kg, is quantitatively limited to 1.5 μ g/kg, it is difficult to which meet that aflatoxin in tealeaves detects will It asks.Aflatoxin B in professional standard LS/T 6108-2014 cereal1It is quick measure immunochromatographic method, exempted from using enzyme label Epidemic disease chromatography and colloidal gold immunity chromatography.
Sample and enzyme-linked is purified using immune affinity column currently for the high performance liquid chromatography of aflatoxin The shortcomings that immunoabsorption, can not avoid when analyzing Tea Samples, therefore, be badly in need of studying aspergillus flavus poison in a kind of new tealeaves The assay method of element, to cope with the deficiencies in the prior art.
Invention content
In order to solve the above technical problems, the present invention provides a kind of assay method of aflatoxin in tealeaves, including sample Product extraction, purification, standard solution measures and sample determination step, and sample extraction step uses acetonitrile extracting solution;Purifying step is adopted With solid-phase extraction column and decontaminating column secondary purification;Take aflatoxin standard reserving solution that standard is made in standard solution determination step Series is measured using liquid after crossing organic phase miillpore filter with Liquid Chromatography-Tandem Mass Spectrometry.
For this purpose, present invention employs following technical schemes:
The present invention provides a kind of assay methods of aflatoxin in tealeaves, and the method includes sample extraction, samples The step of aflatoxin content calculates in purification, standard solution measure, sample measure and sample, the purifying step is adopted successively Secondary purification twice is carried out with solid-phase extraction column and decontaminating column;The standard solution is measured in sample determination step, first uses methanol Solution constant volume, then the content using Liquid Chromatography-Tandem Mass Spectrometry detection aflatoxin.
Preferably, it the described method comprises the following steps:
(1) sample extraction:Sample is extracted using acetonitrile, after filtering, obtains extracting solution;
(2) sample purification:Extracting solution crosses solid-phase extraction column, constant volume after elution;It filters, must treat after decontaminating column, elution Survey liquid;
(3) standard solution measures:The aflatoxin standard solution of known concentration is taken, after filtering, with liquid chromatogram-string Connection mass spectrograph is measured;
(4) sample measures:It is measured with liquid chromatography-tandem mass spectrometry instrument;
(5) aflatoxin content in sample is calculated.
In the present invention, sample is extracted using acetonitrile, reduces the dissolution of tea polyphenols and Tea Pigment, and extracting solution color is shallower, Convenient for subsequent purified treatment, sample purification uses solid-phase extraction column and decontaminating column secondary purification, to tea polyphenols and Tea Pigment etc. Magazine, which is dispelled, to work well, and is measured using liquid chromatography-tandem mass spectrometry instrument, specific by optimizing multiple-reaction monitoring (MRM) Parameter, greatlys improve monitoring sensitivity, and quantitative limit can reach 0.1 μ g/kg, while multiple-reaction monitoring (MRM) anti-interference energy Power is strong, and as a result accurately and reliably, specific technical counting sees attached list 1,2.
In the present invention, the aspergillus flavus goes the computational methods of content as follows:Concentration is distinguished with the peak area of standard solution The standard curve of each aflatoxin is drawn, sample determines that each is yellow bent by the comparison with standard chromatogram retention time The peak of mould toxin, various aspergillus flavus poison in the calculated by peak area sample in the standard curve and sample of each aflatoxin Cellulose content:
X=(A × V)/(m × f)
Wherein:X --- each aflatoxin content in sample;
A --- sample presses external standard method corresponding concentration in standard curve;
V --- the volume of extracting solution in sample extraction process;
The sampling amount of m --- sample;
F --- cycles of concentration.
Preferably, the aflatoxin is B races aflatoxin and/or G races aflatoxin.
Preferably, B races aflatoxin is aflatoxin B1And/or aflatoxin B2
Preferably, G races aflatoxin is aflatoxin G1And/or aflatoxin G2
Preferably, the sample extraction step specifically includes:Add in acetonitrile in the sample, through vortex mixing, ultrasonic extraction, It is filtered after centrifugation, obtains extracting solution;Specifically preferred embodiment is:It weighs 5g samples (being accurate to 0.0001g) and is placed in 50mL tool plugs In centrifuge tube, 20mL acetonitriles are added in, vortex 2min, ultrasonic extraction 20min centrifuge 10min, then with glass fiber filter paper mistake Filter.
Preferably, the time of the vortex mixing be 1-10min, such as can be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, preferably 2-7min.
Preferably, the time of the ultrasonic extraction be 16-30min, such as can be 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30min, preferably For 18-24min.
Preferably, the time of the centrifugation be 8-16min, such as can be 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min or 16min, preferably 10-12min.
Preferably, described be filtered into is filtered with glass fiber filter paper.
Preferably, the sample purification step includes:Extracting solution crosses solid-phase extraction column, is eluted with acetonitrile, nitrogen is blown, PBS delays After fliud flushing constant volume, cross decontaminating column, water wash, eluted with acetonitrile, nitrogen is blown, after methanol dissolving, filter to obtain prepare liquid;It is specific preferred Mode is:The sample purification step is specially to take 4mL extracting solutions, crosses solid-phase extraction column, is eluted with 2mL acetonitriles, collects 6mL and washes De- liquid, nitrogen are blown to about 4mL, are settled to 50mL with PBS buffer solution, all cross decontaminating column, discard, with 20mL water wash, discard, most It is eluted in two times with 4mL acetonitriles afterwards, collects eluent, nitrogen, which is blown to, closely to be done, and is dissolved with 1mL methanol solutions, is crossed 0.45 μm of organic phase Filter membrane obtains prepare liquid.
Preferably, it is to elute in two times with acetonitrile elution after the water wash, then collects eluent.
Preferably, in the methanol solution volume fraction of methanol for 40-70%, such as can be 40%, 45%, 50%th, 55%, 60%, 65% or 70%, preferably 50-60%.
Preferably, it is described filter prepare liquid was organic phase filter membrane that aperture is 0.1-0.6 μm, such as can be 0.1 μ M, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm or 0.6 μm, the aperture Preferably 0.2-0.5 μm.
Preferably, the standard solution determination step includes:Aflatoxin standard reserving solution known to concentration is taken, uses first It is preserved after alcoholic solution constant volume, mixing;The aflatoxin standard solution of various concentration, mistake are configured to during use with methanol solution After filter, it is measured with liquid chromatography-tandem mass spectrometry instrument.It is specifically as follows:Take the aflatoxin B of a concentration of 25 μ g/mL1、 B2、G1、G2Standard reserving solution 0.04mL is settled to 10mL with methanol solution, mixing, obtains 100 μ g/L intermediate standard solution, 2-8 DEG C It preserves three months, is configured to 0.5,1.0,2.0,3.0,5.0 μ g/L standard series using liquid with methanol solution during use, crosses 0.45 It is measured after μm organic phase miillpore filter with liquid chromatography-tandem mass spectrometry instrument.
Preferably, in the methanol solution volume fraction of methanol for 40-70%, such as can be 40%, 45%, 50%th, 55%, 60%, 65% or 70%, preferably 50-60%.
Preferably, the temperature of the preservation is 2-8 DEG C, for example, can be 2 DEG C, 2.5 DEG C, 3 DEG C, 3.5 DEG C, 4 DEG C, 4.5 DEG C, 5 DEG C, 5.5 DEG C, 6 DEG C, 6.5 DEG C, 7 DEG C, 7.5 DEG C or 8 DEG C, the holding time is 1-3 months.
Preferably, it is described to be filtered into the organic phase filter membrane that aperture is 0.1-0.6 μm, such as be 0.1 μm, 0.15 μ M, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm or 0.6 μm, the aperture are preferably 0.2-0.5μm。
As the preferred embodiment of the present invention, the condition of chromatography used is:
Chromatographic column:VP-ODS C18 chromatographic columns (150mm × 2.0mm, 5.0 μm);
Mobile phase:- 0.1% aqueous formic acid (ammonium acetate containing 0.01mol/L) of acetonitrile (A);
Preferably, the gradient elution program is specially:0-1min, -70% mobile phase of 80% mobile phase;1-3min, - 50% mobile phase of 70% mobile phase;3-5min, -30% mobile phase of 50% mobile phase keep 2min;7-8min, 30% flowing - 80% mobile phase of phase keeps 2min;
Flow velocity:0.4mL/min;Column oven temperature:40℃;
Sample size:10μL;
Mass Spectrometry Conditions:Ionization mode:Electron spray positive ion electrospray is from (ESI+);
Detection pattern:Multiple-reaction monitoring (MRM);
Atomization gas flow:3L/min;Heat throughput:10L/min;Dry gas stream amount:10L/min;Interface temperature:250 ℃;DL temperature:250℃;Heat deblocking temperature:250℃;Spray voltage:4.0KV;Collide atmospheric pressure:230KPa.
Preferably, the chromatographic column used in liquid chromatography-tandem mass spectrometry instrument measures is VP-ODSC18Chromatographic column.
Preferably, VP-ODSC18The specification of chromatographic column is 150mm × 2.0mm.
Preferably, VP-ODSC18The grain size of chromatographic column is 5.0 μm.
Preferably, mobile phase used in liquid chromatography-tandem mass spectrometry instrument measure is the acetonitrile solution dissolved with formic acid.
Preferably, in the acetonitrile solution dissolved with formic acid, the volume fraction of formic acid is 0.05-0.2%, such as can be 0.05%th, 0.07%, 0.09%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%.
Preferably, the ammonium acetate of a concentration of 0.005-0.03mol/L, institute are also contained in the acetonitrile solution dissolved with formic acid It can be 0.005mol/L, 0.01mol/L, 0.015mol/L, 0.02mol/L, 0.025mol/L or 0.03mol/L to state concentration.
Preferably, gradient elution program is used during the measure.
Preferably, the gradient elution program is specially:0-1min, -70% mobile phase of 80% mobile phase;1-3min, - 50% mobile phase of 70% mobile phase;3-5min, -30% mobile phase of 50% mobile phase keep 2min;7-8min, 30% flowing - 80% mobile phase of phase keeps 2min.
Preferably, the flow velocity of the gradient elution is 0.2-0.6mL/min, such as can be 0.2mol/L, 0.25mol/ L, 0.3mol/L, 0.35mol/L, 0.4mol/L, 0.45mol/L, 0.5mol/L, 0.55mol/L or 0.6mol/L.
Preferably, the temperature of column oven used in the gradient elution is 35-45 DEG C, for example, can be 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C or 45 DEG C.
Preferably, the sample size of the gradient elution is 5-20 μ L, such as can be 5 μ L, 6 μ L, 7 μ L, 8 μ L, 9 μ L, 10 μ L, 11 μ L, 12 μ L, 13 μ L, 14 μ L, 15 μ L, 16 μ L, 17 μ L, 18 μ L, 19 μ L or 20 μ L.
Preferably, ionization mode used is electron spray positive ion electrospray from (ESI+) in the Liquid Chromatography-Tandem Mass Spectrometry;
Preferably, detection pattern used is multiple-reaction monitoring (MRM);
Preferably, atomization gas flow used be 2-10L/min, such as can be 2L/min, 2.5L/min, 3L/min, 3.5L/min、4L/min、4.5L/min、5L/min、5.5L/min、6L/min、6.5L/min、7L/min、7.5L/min、8L/ Min, 8.5L/min, 9L/min, 9.5L/min or 10L/min;
Preferably, heating throughput used is 5-20L/min, such as can be 5L/min, 6L/min, 7L/min, 8L/ min、9L/min、10L/min、11L/min、12L/min、13L/min、14L/min、15L/min、16L/min、17L/min、 18L/min, 19L/min or 20L/min;
Preferably, dry gas stream amount used is 5-20L/min, such as can be 5L/min, 6L/min, 7L/min, 8L/ min、9L/min、10L/min、11L/min、12L/min、13L/min、14L/min、15L/min、16L/min、17L/min、 18L/min, 19L/min or 20L/min;
Preferably, used interface temperature is 230-280 DEG C, for example, can be 230 DEG C, 235 DEG C, 240 DEG C, 245 DEG C, 250 DEG C, 255 DEG C, 260 DEG C, 265 DEG C, 270 DEG C, 275 DEG C or 280 DEG C;
Preferably, DL temperature used is 230-280 DEG C, for example, can be 230 DEG C, 235 DEG C, 240 DEG C, 245 DEG C, 250 DEG C, 255 DEG C, 260 DEG C, 265 DEG C, 270 DEG C, 275 DEG C or 280 DEG C;
Preferably, heating deblocking temperature used is 230-280 DEG C, for example, can be 230 DEG C, 235 DEG C, 240 DEG C, 245 DEG C, 250 DEG C, 255 DEG C, 260 DEG C, 265 DEG C, 270 DEG C, 275 DEG C or 280 DEG C;
Preferably, spray voltage used be 3.0-6.0KV, such as can be 3.0kV, 3.5kV, 4.0kV, 4.5kV, 5.0kV, 5.5kV or 6.0kV;
Preferably, it is used collision atmospheric pressure be 200-300KPa, such as can be 200KPa, 210KPa, 220KPa, 230KPa, 240KPa, 250KPa, 260KPa, 270KPa, 280KPa, 290KPa or 300KPa.
The correlation coefficient r of the standard curve of the aflatoxin is more than 0.998, preferably 0.9981-0.9999, such as Can be 0.9981,0.9982,0.9983,0.9984,0.9985,0.9986,0.9987,0.9988,0.9989,0.9990, 0.9991st, 0.9992,0.9993,0.9994,0.9995,0.9996,0.9997,0.9998 or 0.9999;For example, with instrument certainly The data processing software of band draws aflatoxin B respectively using the peak area of liquid with standard series to concentration1、B2、G1、G2's Standard curve, the correlation coefficient r > 0.998 of standard curve, according to aflatoxin B1、B2、G1、G2Standard curve and sample Middle aflatoxin B1、B2、G1、G2Calculated by peak area sample in aflatoxin content.
As preferred technical solution, the described method comprises the following steps:
(1) sample extraction:Acetonitrile is added in the sample, through vortex mixing 1-10min, ultrasound extraction 16-30min, centrifugation It is filtered after 8-16min with glass fiber filter paper, obtains extracting solution;
(2) sample purification:Extracting solution crosses solid-phase extraction column, is eluted with acetonitrile, nitrogen is blown, after PBS buffer solution constant volume, purification excessively Column, water wash, eluted in two times with acetonitrile, nitrogen is blown, after methanol solution dissolving, it is 0.1-0.6 μm of organic phase filter membrane to cross aperture It is filtered, obtains prepare liquid;The volume fraction of methanol is 40-70% in the methanol solution;
(3) standard solution measures:Aflatoxin standard reserving solution known to concentration is taken, with methanol solution constant volume, mixing It preserves at 2-8 DEG C afterwards 1-3 months;The aflatoxin standard solution of various concentration, mistake are configured to during use with methanol solution Aperture is after 0.1-0.6 μm of organic phase filter membrane is filtered, to be measured with liquid chromatography-tandem mass spectrometry instrument;The methanol The volume fraction of methanol is 40-70% in solution;
(4) sample measures:It is measured with liquid chromatography-tandem mass spectrometry instrument, chromatographic column used is VP-ODS C18Chromatography Column, specification 150mm × 2.0mm, 5.0 μm of grain size, and using gradient elution program;
(5) in sample aflatoxin content calculating:Draw each Huang respectively to concentration with the peak area of standard solution The standard curve of aspertoxin, sample determine each aflatoxin by the comparison with standard chromatogram retention time Peak, various aflatoxin contents in the calculated by peak area sample in the standard curve and sample of each aflatoxin:
X=(A × V)/(m × f).
Compared with prior art, the present invention at least has the advantages that:
The present invention provides a kind of assay method of aflatoxin in tealeaves, including sample extraction, purification, standard solution It measures and sample determination step, sample extraction step uses acetonitrile extracting solution;Purifying step uses solid-phase extraction column and decontaminating column Secondary purification;It takes aflatoxin standard reserving solution that standard series is made using liquid in standard solution determination step, crosses organic phase It is measured after miillpore filter with Liquid Chromatography-Tandem Mass Spectrometry;Assay method provided by the invention is easy to operate, quickly, choosing Selecting property and high sensitivity, it is as a result accurate reliable;Pollution is few, safe, reduces the pollution to operating personnel and environment;It detects into This relatively low, easy to spread, aflatoxin especially aflatoxin B suitable for tealeaves1、B2、G1、G2Detection.
Description of the drawings
Fig. 1 is 1 Plays solution aflatoxin B of embodiment1Chromatogram;
Fig. 2 is 1 Plays solution aflatoxin B of embodiment2Chromatogram;
Fig. 3 is 1 Plays solution aflatoxin G of embodiment1Chromatogram;
Fig. 4 is 1 Plays solution aflatoxin G of embodiment2Chromatogram;
Fig. 5 is aflatoxin B in embodiment 11Canonical plotting;
Fig. 6 is aflatoxin B in embodiment 12Canonical plotting;
Fig. 7 is aflatoxin G in embodiment 11Canonical plotting;
Fig. 8 is aflatoxin G in embodiment 12Canonical plotting;
Fig. 9 is sample aflatoxin B in embodiment 21Chromatogram;
Figure 10 is sample aflatoxin B in embodiment 22Chromatogram;
Figure 11 is sample aflatoxin G in embodiment 21Chromatogram;
Figure 12 is sample aflatoxin G in embodiment 22Chromatogram.
Figure 13 is sample aflatoxin B in embodiment 31Chromatogram;
Figure 14 is sample aflatoxin B in embodiment 32Chromatogram;
Figure 15 is sample aflatoxin G in embodiment 31Chromatogram;
Figure 16 is sample aflatoxin G in embodiment 32Chromatogram.
Specific embodiment
Of the invention for ease of understanding, it is as follows that the present invention enumerates embodiment.Those skilled in the art are it will be clearly understood that the implementation Example is only to aid in understanding the present invention, is not construed as the concrete restriction to the present invention.
Embodiment 1
Standard solution measures:Take the aflatoxin B of a concentration of 25 μ g/mL1、B2、G1、G2Standard reserving solution 0.04mL is used Methanol solution is settled to 10mL, mixing, obtains 100 μ g/L, and 2-8 DEG C preserves three months, and when use is configured to respectively with methanol solution 0.5th, 1.0,2.0,3.0,5.0 μ g/L standard series use liquid, cross 0.45 μm of organic phase miillpore filter examination with computer, obtain Fig. 1- Yellow bent toximycin B in standard solution obtained by Fig. 41、B2、G1、G2Chromatogram, abscissa is the time, and ordinate is response, often There are one kind aflatoxin schemes, similarly hereinafter;
It is mapped according to above-mentioned chromatogram to concentration of standard solution, aflatoxin B1、B2、G1、G2Canonical plotting such as Fig. 5- Shown in 8.
Embodiment 2
It weighs 5.0127g and added standard solution Pu'er tea, have in plug centrifuge tube in 50mL, add in a certain amount of aspergillus flavus poison Plain mixed standard solution, adds in 20mL acetonitriles, and whirlpool mixing 2min, ultrasonic extraction 20min centrifuge 10min, glass fiber filter paper Filtering;
Sample purification:4mL extracting solutions are taken, cross solid-phase extraction column, are eluted with 2mL acetonitriles, collect 6mL eluents, nitrogen is blown to About 4mL is settled to 50mL with PBS buffer solution, all crosses decontaminating column, discard, with 20mL water wash, discard, finally with 4mL acetonitriles It elutes in two times, collects eluent, nitrogen, which is blown to, closely to be done, and is dissolved with 1mL methanol solutions;
Sample measures:Aflatoxin in Pu'er tea is detected using Liquid Chromatography-Tandem Mass Spectrometry, actual conditions are such as Under:
Chromatographic condition:Chromatographic column:VP-ODS C18 chromatographic columns (150mm × 2.0mm, 5.0 μm);
Mobile phase:- 0.1% aqueous formic acid (ammonium acetate containing 0.01mol/L) of acetonitrile (A);
Gradient elution program:0-1min, 80%B-70%B;1-3min, 70%B-50%B;3-5min, 50%B-30% B keeps 2min;7-8min, 30%B-80%B keep 2min;Flow velocity:0.4mL/min;Column oven temperature:40℃;Sample size: 10μL;
Mass Spectrometry Conditions:Ionization mode:Electron spray positive ion electrospray is from (ESI+);Detection pattern:Multiple-reaction monitoring (MRM);Mist Change throughput:3L/min;Heat throughput:10L/min;Dry gas stream amount:10L/min;Interface temperature:250℃;DL temperature: 250℃;Heat deblocking temperature:250℃;Spray voltage:4.0KV;Collide atmospheric pressure:230KPa.
Aflatoxin B in sample1、B2、G1、G2Chromatogram as shown in figs9-12.
According to the standard curve of embodiment 1, sample aflatoxin B is calculated1、B2、G1、G2Concentration, circular It is as follows:
According to formula:X=(c × V)/(m × f)
Aflatoxin B1 (μ g/kg)=(in sample corresponding concentration × volume of extracting solution in sample extraction process)/it (takes Sample amount × 4);So as to calculate aflatoxin B1A concentration of 2.17 μ g/kg;
Similarly, aflatoxin B is obtained2For 2.18 μ g/kg, aflatoxin G1For 2.03 μ g/kg, aflatoxin G2 For 2.22 μ g/kg.
Embodiment 3
Sample extraction:It weighs 5.0057g and added standard solution green tea, have in plug centrifuge tube in 50mL, add in a certain amount of Aflatoxin mixed standard solution, adds in 20mL acetonitriles, and whirlpool mixing 2min, ultrasonic extraction 20min centrifuge 10min, glass Fiber filter paper filters.
Sample purification:4mL extracting solutions are taken, cross solid-phase extraction column, are eluted with 2mL acetonitriles, collect 6mL eluents, nitrogen is blown to About 4mL is settled to 50mL with PBS buffer solution, all crosses decontaminating column, discard, with 20mL water wash, discard, finally with 4mL acetonitriles It elutes in two times, collects eluent, nitrogen, which is blown to, closely to be done, and is dissolved with 1mL methanol solutions, is crossed 0.45 μm of organic phase miillpore filter, treat It surveys.
Sample measures:Aflatoxin B in sample is detected using Liquid Chromatography-Tandem Mass Spectrometry1、B2、G1、G2
Chromatographic condition:Chromatographic column:VP-ODS C18 chromatographic columns (150mm × 2.0mm, 5.0 μm);Mobile phase:Acetonitrile (A)- 0.1% aqueous formic acid (ammonium acetate containing 0.01mol/L);Gradient elution program:0-1min, 80%B-70%B;1-3min, 70%B-50%B;3-5min, 50%B-30%B keep 2min;7-8min, 30%B-80%B keep 2min;Flow velocity: 0.4mL/min;Column oven temperature:40℃;Sample size:10μL;
Mass Spectrometry Conditions:Ionization mode:Electron spray positive ion electrospray is from (ESI+);Detection pattern:Multiple-reaction monitoring (MRM);Mist Change throughput:3L/min;Heat throughput:10L/min;Dry gas stream amount:10L/min;Interface temperature:250℃;DL temperature: 250℃;Heat deblocking temperature:250℃;Spray voltage:4.0KV;Collide atmospheric pressure:230KPa.
Aflatoxin B in sample1、B2、G1、G2Chromatogram as shown in figures 13-16.
Sample aflatoxin B is calculated in formula in 1 standard curve of embodiment and embodiment 21、B2、G1、G2 Respectively:2.19μg/kg、2.29μg/kg、2.08μg/kg、2.19μg/kg.
Embodiment 4
The technical data of embodiment 2 and 3 is counted, statistics sees attached list 1 and table 2.As can be seen that the present invention passes through Optimize multiple-reaction monitoring (MRM) design parameter, greatly improve monitoring sensitivity, quantitative limit can reach 0.1 μ g/kg, simultaneously Multiple-reaction monitoring (MRM) strong antijamming capability, as a result accurately and reliably.
1 aflatoxin B of table1、B2、G1And G2The range of linearity, standard curve, related coefficient and quantitative limit
Analyte The range of linearity/(μ g/L) Curvilinear equation Related coefficient Quantitative limit (S/N >=10)/(μ g/L)
Aflatoxin B1 0.1-5.0 Y=84358X+1143 0.9990 0.1
Aflatoxin B2 0.1-5.0 Y=63099X+859 0.9993 0.1
Aflatoxin G1 0.1-5.0 Y=78709X-1628 0.9987 0.1
Aflatoxin G2 0.1-5.0 Y=25978X+1545 0.9995 0.1
2 aflatoxin B of table1、B2、G1And G2Recovery of standard addition and precision (n=6)
Comparative example 1
It is same as Example 1, it differs only in addition methanol aqueous solution in sample and extracts, obtain extracting solution, other It is same as Example 1.
When detecting the aflatoxin in sample with Liquid Chromatography-Tandem Mass Spectrometry, the aflatoxin rate of recovery is poor, fixed Amount limit it is higher, sensitivity is poor, illustrate this comparative example can not accurate quantification measure tealeaves in aflatoxin content.
Comparative example 2
It is same as Example 1, it differs only in standard solution measure and sample measure is measured using liquid chromatogram, Other are same as Example 1, and impurity interference is larger, and quantitative limit is higher, and sensitivity is poor, illustrates that this comparative example can not be accurate Quantitative determine the content of aflatoxin in tealeaves.
In conclusion the present invention provides a kind of assay method of aflatoxin in tealeaves, including sample extraction, only Change, standard solution measure and sample determination step, sample extraction step use acetonitrile extracting solution;Purifying step uses Solid Phase Extraction Column and decontaminating column secondary purification;Take aflatoxin standard reserving solution that standard series use is made in standard solution determination step Liquid is measured after crossing organic phase miillpore filter with Liquid Chromatography-Tandem Mass Spectrometry;Assay method operation letter provided by the invention Single, quickly, selectivity and high sensitivity are as a result accurate reliable;Pollution is few, safe, reduces to operating personnel and environment Pollution;Testing cost is relatively low, easy to spread, the aflatoxin especially aflatoxin B suitable for tealeaves1、B2、G1、G2's Detection.
Applicant states that the present invention illustrates the detailed process equipment of the present invention and technological process by above-described embodiment, But the invention is not limited in above-mentioned detailed process equipment and technological processes, that is, it is above-mentioned detailed not mean that the present invention has to rely on Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention, The addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, selection of concrete mode etc. all fall within the present invention's Within protection domain and the open scope.

Claims (10)

1. the assay method of aflatoxin in a kind of tealeaves, the method includes sample extraction, sample purification, standard solution to survey The step of aflatoxin content calculates in fixed, sample measure and sample, which is characterized in that the purifying step is successively using solid Phase extraction column and decontaminating column carry out secondary purification;The standard solution is measured in sample determination step, is first determined with methanol solution Hold, then the content using Liquid Chromatography-Tandem Mass Spectrometry detection aflatoxin.
2. the method as described in claim 1, which is characterized in that the described method comprises the following steps:
(1) sample extraction:Sample is extracted using acetonitrile, after filtering, obtains extracting solution;
(2) sample purification:Extracting solution crosses solid-phase extraction column, constant volume after elution;It is filtered after decontaminating column, elution, obtains prepare liquid;
(3) standard solution measures:The aflatoxin standard solution of known concentration is taken, after filtering, with liquid chromatography-tandem matter Spectrometer is measured;
(4) sample measures:It is measured with liquid chromatography-tandem mass spectrometry instrument;
(5) content of aflatoxin in sample is calculated.
3. method according to claim 1 or 2, which is characterized in that the aflatoxin for B races aflatoxin and/ Or G races aflatoxin;
Preferably, B races aflatoxin is aflatoxin B1And/or aflatoxin B2
Preferably, G races aflatoxin is aflatoxin G1And/or aflatoxin G2
4. method according to any one of claim 1-3, which is characterized in that the sample extraction step specifically includes: Acetonitrile is added in the sample, is filtered after vortex mixing, ultrasonic extraction, centrifugation, is obtained extracting solution;
Preferably, the time of the vortex mixing is 1-10min, preferably 2-7min;
Preferably, the time of the ultrasonic extraction is 16-30min, preferably 18-24min;
Preferably, the time of the centrifugation is 8-16min, preferably 10-12min;
Preferably, described be filtered into is filtered with glass fiber filter paper.
5. according to the described method of any one of claim 1-4, which is characterized in that the sample purification step includes:Extraction Liquid crosses solid-phase extraction column, is eluted with acetonitrile, nitrogen is blown, after PBS buffer solution constant volume, crosses decontaminating column, water wash, is eluted with acetonitrile, nitrogen It blows, after methanol solution dissolving, filters to obtain prepare liquid;
Preferably, it is to elute in two times with acetonitrile elution after the water wash, then collects eluent;
Preferably, the volume fraction of methanol is 40-70%, preferably 50-60% in the methanol solution;
Preferably, it is described filter prepare liquid was organic phase filter membrane that aperture is 0.1-0.6 μm, the aperture is preferably 0.2- 0.5μm。
6. method according to any one of claims 1-5, which is characterized in that the standard solution determination step includes: Aflatoxin standard reserving solution known to concentration is taken, with being preserved after methanol solution constant volume, mixing;Matched during use with methanol solution The aflatoxin standard solution of various concentration is made, after filtering, is measured with liquid chromatography-tandem mass spectrometry instrument;
Preferably, the volume fraction of methanol is 40-70%, preferably 50-60% in the methanol solution;
Preferably, the temperature of the preservation is 2-8 DEG C, and the holding time is 1-3 months;
Preferably, described to be filtered into the organic phase filter membrane that aperture is 0.1-0.6 μm, the aperture is preferably 0.2-0.5 μm.
7. according to the method described in any one of claim 1-6, which is characterized in that described to use liquid chromatography-tandem mass spectrometry instrument Chromatographic column used in measure is VP-ODS C18Chromatographic column;
Preferably, VP-ODS C18The specification of chromatographic column is 150mm × 2.0mm;
Preferably, VP-ODS C18The grain size of chromatographic column is 5.0 μm;
Preferably, mobile phase used in liquid chromatography-tandem mass spectrometry instrument measure is the acetonitrile solution dissolved with formic acid;
Preferably, in the acetonitrile solution dissolved with formic acid, the volume fraction of formic acid is 0.05-0.2%;
Preferably, the ammonium acetate of a concentration of 0.005-0.03mol/L is also contained in the acetonitrile solution dissolved with formic acid;
Preferably, gradient elution program is used during the measure;
Preferably, the gradient elution program is specially:0-1min, -70% mobile phase of 80% mobile phase;1-3min, 70% stream - 50% mobile phase of dynamic phase;3-5min, -30% mobile phase of 50% mobile phase keep 2min;7-8min, 30% mobile phase -80% Mobile phase keeps 2min;
Preferably, the flow velocity of the gradient elution is 0.2-0.6mL/min;
Preferably, the temperature of column oven used in the gradient elution is 35-45 DEG C;
Preferably, the sample size of the gradient elution is 5-20 μ L.
8. according to claim according to the described method of any one of claim 1-7, which is characterized in that the liquid chromatogram- Ionization mode used is electron spray positive ion electrospray from (ESI+) in tandem mass spectrometry;
Preferably, detection pattern used is multiple-reaction monitoring (MRM);
Preferably, atomization gas flow used is 2-10L/min;
Preferably, heating throughput used is 5-20L/min;
Preferably, dry gas stream amount used is 5-20L/min.
9. according to claim according to the described method of any one of claim 1-7, which is characterized in that used interface temperature is 230-280℃;
Preferably, DL temperature used is 230-280 DEG C;
Preferably, heating deblocking temperature used is 230-280 DEG C;
Preferably, spray voltage used is 3.0-6.0KV;
Preferably, collision atmospheric pressure used is 200-300KPa.
10. according to claim according to the method described in any one of claim 1-9, which is characterized in that the method includes Following steps:
(1) sample extraction:Acetonitrile is added in the sample, through vortex mixing 1-10min, ultrasonic extraction 16-30min, centrifugation 8- It is filtered after 16min with glass fiber filter paper, obtains extracting solution;
(2) sample purification:Extracting solution crosses solid-phase extraction column, is eluted with acetonitrile, nitrogen is blown, after PBS buffer solution constant volume, cross decontaminating column, Water wash, eluted in two times with acetonitrile, nitrogen is blown, methanol solution dissolving after, cross aperture be 0.1-0.6 μm organic phase filter membrane carry out Filtering, obtains prepare liquid;The volume fraction of methanol is 40-70% in the methanol solution;
(3) standard solution measures:Aflatoxin standard reserving solution known to concentration is taken, at 2-8 DEG C after methanol constant volume, mixing It preserves 1-3 months;The aflatoxin standard solution of various concentration is configured to during use with methanol solution, it is 0.1- to cross aperture After 0.6 μm of organic phase filter membrane is filtered, it is measured with liquid chromatography-tandem mass spectrometry instrument;Methanol in the methanol solution Volume fraction be 40-70%;
(4) sample measures:It is measured with liquid chromatography-tandem mass spectrometry instrument, chromatographic column used is VP-ODS C18Chromatographic column, rule Lattice 150mm × 2.0mm, 5.0 μm of grain size, and using gradient elution program;
(5) aflatoxin content in sample is calculated.
CN201810012304.8A 2018-01-05 2018-01-05 The assay method of aflatoxin in a kind of tealeaves Pending CN108226355A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810012304.8A CN108226355A (en) 2018-01-05 2018-01-05 The assay method of aflatoxin in a kind of tealeaves

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810012304.8A CN108226355A (en) 2018-01-05 2018-01-05 The assay method of aflatoxin in a kind of tealeaves

Publications (1)

Publication Number Publication Date
CN108226355A true CN108226355A (en) 2018-06-29

Family

ID=62643173

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810012304.8A Pending CN108226355A (en) 2018-01-05 2018-01-05 The assay method of aflatoxin in a kind of tealeaves

Country Status (1)

Country Link
CN (1) CN108226355A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100948589B1 (en) * 2008-03-05 2010-03-23 한국과학기술원 Method for assaying mycotoxin
CN102507788A (en) * 2011-11-15 2012-06-20 江苏出入境检验检疫局动植物与食品检测中心 Method for measuring six pyrethroid medicaments in breeding or slaughtering environment
CN104535664A (en) * 2014-03-31 2015-04-22 中华人民共和国北京出入境检验检疫局 Method for simultaneously detecting a plurality of mycotoxins in sesame paste
CN104678039A (en) * 2015-01-30 2015-06-03 湖南中烟工业有限责任公司 Method for simultaneously measuring contents of four aflatoxins in tobacco and tobacco products by liquid chromatography-tandem mass spectrometry combination
CN106706635A (en) * 2016-05-21 2017-05-24 广州今典精方药业有限公司 Quality standard of dogbane leaf qualitative and quantitative traditional Chinese medicine decoction piece and manufacturing process thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100948589B1 (en) * 2008-03-05 2010-03-23 한국과학기술원 Method for assaying mycotoxin
CN102507788A (en) * 2011-11-15 2012-06-20 江苏出入境检验检疫局动植物与食品检测中心 Method for measuring six pyrethroid medicaments in breeding or slaughtering environment
CN104535664A (en) * 2014-03-31 2015-04-22 中华人民共和国北京出入境检验检疫局 Method for simultaneously detecting a plurality of mycotoxins in sesame paste
CN104678039A (en) * 2015-01-30 2015-06-03 湖南中烟工业有限责任公司 Method for simultaneously measuring contents of four aflatoxins in tobacco and tobacco products by liquid chromatography-tandem mass spectrometry combination
CN106706635A (en) * 2016-05-21 2017-05-24 广州今典精方药业有限公司 Quality standard of dogbane leaf qualitative and quantitative traditional Chinese medicine decoction piece and manufacturing process thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUKIHIRO GODA 等: "Application and improvement of aflatoxin analysis in foods using a multifunctional column and HPLC", 《JOURNAL OF THE FOOD HYGIENIC SOCIETY OF JAPAN》 *
王可 等,: "茶叶中黄曲霉毒素的高效液相色谱串联质谱法", 《环境与健康杂志》 *

Similar Documents

Publication Publication Date Title
Dong et al. Modified QuEChERS combined with ultra high performance liquid chromatography tandem mass spectrometry to determine seven biogenic amines in Chinese traditional condiment soy sauce
Aguilera-Luiz et al. Multi-residue determination of veterinary drugs in milk by ultra-high-pressure liquid chromatography–tandem mass spectrometry
Van De Steene et al. Comparison of matrix effects in HPLC-MS/MS and UPLC-MS/MS analysis of nine basic pharmaceuticals in surface waters
Chen et al. A rapid method for simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and ultra-performance liquid chromatography
Fan et al. Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS
Han et al. An ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines
Eom et al. Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins
Ballesteros et al. Sensitive gas chromatographic–mass spectrometric method for the determination of phthalate esters, alkylphenols, bisphenol A and their chlorinated derivatives in wastewater samples
Campone et al. Dispersive liquid–liquid microextraction combined with high-performance liquid chromatography–tandem mass spectrometry for the identification and the accurate quantification by isotope dilution assay of Ochratoxin A in wine samples
van Vyncht et al. Multiresidue determination of (fluoro) quinolone antibiotics in swine kidney using liquid chromatography–tandem mass spectrometry
Visconti et al. Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography
Li et al. Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution
CN105548412B (en) Method that is a kind of while measuring 5 kinds of aminoglycoside medicaments residual quantities in food
Peña-Gallego et al. Biogenic amine determination in wines using solid-phase extraction: A comparative study
Wang et al. A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of four tetracycline antibiotics
Godula et al. Pulsed splitless injection and the extent of matrix effects in the analysis of pesticides
Quinto et al. Determination of aflatoxins in cereal flours by solid-phase microextraction coupled with liquid chromatography and post-column photochemical derivatization-fluorescence detection
Han et al. Analysis of ochratoxin A and ochratoxin B in traditional Chinese medicines by ultra-high-performance liquid chromatography–tandem mass spectrometry using [13C20]-ochratoxin A as an internal standard
Lu et al. Multi-walled carbon nanotubes as solid-phase extraction adsorbent for the ultra-fast determination of chloramphenicol in egg, honey, and milk by fused-core C18-based high-performance liquid chromatography–tandem mass spectrometry
Ngwa et al. Simultaneous analysis of 14 benzodiazepines in oral fluid by solid-phase extraction and LC-MS-MS
CN106053638B (en) The detection method of aminoglycoside antibiotics residual quantity in a kind of animal-derived food
Tang et al. Development of a quantitative multi-mycotoxin method in rice, maize, wheat and peanut using UPLC-MS/MS
Leça et al. Rapid and sensitive methodology for determination of ethyl carbamate in fortified wines using microextraction by packed sorbent and gas chromatography with mass spectrometric detection
Saito et al. Determination of ochratoxins in nuts and grain samples by in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry
CN104614466B (en) The assay method of antiseptic in tobacco juice for electronic smoke

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination