CN108226355A - The assay method of aflatoxin in a kind of tealeaves - Google Patents
The assay method of aflatoxin in a kind of tealeaves Download PDFInfo
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- 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
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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
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.
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|---|---|---|---|---|
| CN112034079A (en) * | 2020-09-29 | 2020-12-04 | 中国科学院上海营养与健康研究所 | Novel method for detecting aflatoxin content in tea sample |
Citations (5)
| 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 |
-
2018
- 2018-01-05 CN CN201810012304.8A patent/CN108226355A/en active Pending
Patent Citations (5)
| 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)
| 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》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112034079A (en) * | 2020-09-29 | 2020-12-04 | 中国科学院上海营养与健康研究所 | Novel method for detecting aflatoxin content in tea sample |
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