CN103652517B - A kind of irradiation-induced degradation facture of mycotoxin - Google Patents
A kind of irradiation-induced degradation facture of mycotoxin Download PDFInfo
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Abstract
The present invention relates to a kind of irradiation-induced degradation facture of mycotoxin; Comprise the following steps: (1) standard items preparing standard solution to mycotoxin, be the radiation treatment of 0-200kGy through irradiation range to standard solution, measured by the content of liquid chromatography-tandem mass spectrometry to mycotoxin; (2) after the irradiation that irradiation range is 0-9kGy, pre-service is carried out to the malicious sample of band, measured by the degradation effect of liquid chromatography-tandem mass spectrometry to mycotoxin; (3) choose the highest mycotoxin standard solution of concentration as sample, after the radiation treatment that irradiation range is 0-200kGy, carry out detection by liquid chromatography-tandem mass spectrometry analyze catabolite; Solve research and the environmental protection application to lacking irradiation-induced degradation mycotoxin catabolite; Good degrading effect and realize to agricultural product refuse environmental protection detoxification treatment of going mouldy.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of irradiation-induced degradation facture of mycotoxin.
Background technology
Mycotoxin is the toxic metabolic products produced under adapt circumstance condition by Toxigenic fungi, and current research mainly concentrates on large especially to mankind's harm and pollutes the high aflatoxin of frequency, ochratoxin, fumonisin and trichothecene toxin deoxynivalenol, T-2 toxin etc.Mycotoxin is the toxin of natural generation, be prevalent in the agricultural product such as corn, wheat, barley, paddy, oat, Chinese sorghum, flavouring, nut, dairy products and feed, have a strong impact on the output of crops, reduce the quality of agricultural product, cause tremendous economic to lose.According to FAO (Food and Agriculture Organization of the United Nation) (FAO) report, the whole world about has the crops of 25% to suffer the pollution of fungi and toxin every year, about has the crops of 2% to lose nutrition and economic worth because of seriously polluted, and the direct and indirect economic loss caused reaches tens billion of dollar.Strict limit standard is formulated to mycotoxins such as the aflatoxin in Cereals, food and feed, ochratoxin A, deoxynivalenol, Gibberella zeae rare ketone, Zhan Celadon mycins both at home and abroad.
The method of existing mycotoxin detoxification comprises at present: (1) chemical method, mycotoxin can be degraded by many chemical substances such as hydrogen peroxide, sodium hypochlorite, but can produce some poisonous secondary products, therefore Europe and many countries do not allow to process mycotoxin with chemical method simultaneously.(2) biological method, biology detoxification method is screening certain micro-organisms, utilizes its biotransformation, mycotoxin is destroyed or changes the method for low toxicity material into.Current biological method is the hot topic of research, but the kind of the detoxification microorganism be suitable for for different toxin is different, as fruit product may need applicable multiple-microorganism just can reach good effect by multiple endotoxin contamination.(3) physics detoxicity method, mainly contains WATER-WASHING METHOD, scalping method, de-embryo detoxification method, heating detoxification method, solvent extraction, radiation method etc.Major part physical method can not remove mycotoxin completely.Solvent extraction adopts organic solvent by mycotoxin extract and separate, and the method disposal cost is high, and organic solvent has residual contamination, without actual application value.
In Chinese patent 201110000805.2 " immune affinity column purification high performance liquid chromatography detects the method for ochratoxin A in Chinese medicine tincture ", disclose and by tablets by HPLC-MS, positive findings is confirmed; In Chinese patent 201010209190.X " a kind of method detecting nivalenol and deoxynivalenol toxin in Chinese medicine ", disclose and under full scan pattern, confirm toxin by tablets by HPLC-MS; In Chinese patent 201110370420.5 detection method of several mycotoxin levels " in the wheat ", disclose and detected by tablets by HPLC-MS, by the regression equation calculation DON of toxin concentration for peak area, the content of T-2 toxin; In Chinese patent 201210054250.4 method of mycotoxin levels " in the LC-MS/MS Araliaceae ", disclosing can be accurate, measures the multicomponent residual quantity of mycotoxin in Araliaceae delicately; In Chinese patent 201210361145.5 " method with Algae toxins in electron beam irradiation-induced degradation water ", disclose when radiation dose is 5kGy, the generation of Algae toxins can be suppressed; In Chinese patent 2009100787702.0 " method of degrading fumonisins ", disclose irradiation can make fumonisins B1 degradation rate reach 63.47%; In Chinese patent 200910078703.5 " a kind of method of degrading fumonisins ", disclose irradiation can make fumonisins B1 degradation rate reach 59.03%; Irradiation detoxification is disclosed without industrial pollution in Chinese patent 200910078708.8 " method of aflatoxin degradation "; In Chinese patent 200910078707.3 " a kind of method of aflatoxin degradation ", disclose irradiation can make aflatoxin B1 degradation rate reach 71.51%.
Existing correlation technique has: Zhang Xichun etc. report that the degraded of irradiation dose to mould and mycotoxin of 3kGy ~ 5kGy has obvious effect; Poplar waits quietly studying and shows that mass concentration is that aflatoxin B1 solution degradation rate when 4kGy of 0.1mg/L fumonisins B1 solution degradation rate when 10kGy that degradation rate reaches 96%, 0.1mg/L when can reach more than 80%, 6kGy reaches more than 90%; The result of study of Yin Qinggang shows when irradiation dose is 18kGy, in powdered form corn, zearalenone degradation rate can reach 74.9%, when irradiation dose is 10kGy, in whole kernel corn, zearalenone degradation rate can reach 94.1%, when irradiation dose is 5kGy, the degradation rate of 30.0mg/kg zearalenone aqueous solution reaches 90%; The people such as Chi Lei have delivered the paper of gamma-rays to the irradiation-induced degradation of ochratoxin A, mention the possible mechanism of the method for catabolite structure elucidation, degraded, but do not report the product of degraded in literary composition.
Summary of the invention
The object of this invention is to provide a kind of good degrading effect, realize the irradiation-induced degradation facture of the mycotoxin to agricultural product refuse environmental protection detoxification treatment.
The technical scheme realizing the object of the invention is a kind of irradiation-induced degradation facture of mycotoxin; (1) to the standard items preparing standard solution of mycotoxin, be the radiation treatment of 0-200kGy through irradiation range to standard solution, measured by the content of liquid chromatography-tandem mass spectrometry to mycotoxin; (2) after the irradiation that irradiation range is 0-9kGy, pre-service is carried out to the malicious sample of band, measured by the degradation effect of liquid chromatography-tandem mass spectrometry to mycotoxin; (3) choose the highest mycotoxin standard solution of concentration as sample, after the radiation treatment that irradiation range is 0-200kGy, carry out detection by liquid chromatography-tandem mass spectrometry analyze catabolite.
Preferred described mycotoxin is FT FB1, ochratoxin OTA, T-2 toxin and deoxynivalenol DON; Described irradiation bomb is gamma-rays, the chromatographic condition of described liquid chromatography-tandem mass spectrometry: adopt Thermo Finnigan liquid chromatographic system, it is SepaxBR-C18 post 100 × 2.1mm that its liquid-phase condition comprises chromatographic column, 5 μm; When detecting FB1, OTA and T-2, mobile phase is A: methyl alcohol, B: Cu Suan An – aqueous formic acid, under flow velocity 250 μ L/min and sample size 25 μ L condition, carries out gradient elution; Concrete gradient is:
| Time | A% | B% |
| 0.00 | 15 | 85 |
| 2.00 | 15 | 85 |
| 2.10 | 90 | 10 |
| 5.00 | 90 | 10 |
| 5.10 | 15 | 85 |
| 7.00 | 15 | 85 |
When detecting DON, mobile phase is A: methyl alcohol, B: water, under flow velocity 250 μ L/min and sample size 25 μ L condition, carries out gradient elution; Concrete gradient is:
| Time | A% | B% |
| 0.00 | 15 | 85 |
| 2.00 | 15 | 85 |
| 2.10 | 90 | 10 |
| 4.00 | 90 | 10 |
| 4.10 | 15 | 85 |
| 6.00 | 15 | 85 |
The Mass Spectrometry Conditions of described liquid chromatography-tandem mass spectrometry: adopt Thermo TSQ Ultra triple level Four bar mass spectrometer system, its Mass Spectrometry Conditions comprises when detecting FB1, OTA and T-2, Ionization mode is ESI, and spray voltage is 3000V, and polar mode is positive ion, atomization temperature is 400 DEG C, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 DEG C, sweep length is 0.01m/z, sweep time is 0.1s, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; When detecting DON, Ionization mode is ESI, and spray voltage is 2500V, and polar mode is negative ion, atomization temperature is 400 DEG C, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 DEG C, sweep length is 0.01m/z, sweep time is 0.1s, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; Ion pair information, wherein * is quota ion pair:
Standard items in preferred described step (1) are
Wherein the compound method of FB1 standard solution selects the purity of Enzo company to be 98.0%, 5mg*2 bottle, and variable concentrations has concentration to be 0.8mg/mL respectively, draws in 315 μ L to 25mL volumetric flasks, dissolve constant volume with acetonitrile+water (volume ratio 50:50); Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, dissolves constant volume with acetonitrile+water (volume ratio 50:50); Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, dissolves constant volume with acetonitrile+water (volume ratio 50:50); Concentration is 100.0ng/mL, draws in 500 μ L to 10mL volumetric flasks, dissolves constant volume with acetonitrile+water (volume ratio 50:50); Concentration is also had to be 50.0ng/mL; Wherein the compound method of OTA standard solution selects the purity of Enzo company to be 98.0%, 5mg*1 bottle, and variable concentrations has concentration to be 0.7mg/mL respectively, draws in 360 μ L to 25mL volumetric flasks, dissolve constant volume with methyl alcohol; Concentration is 10.0 μ g/mL, draws in 500 μ L to 5mL volumetric flasks, dissolves constant volume with methyl alcohol; Concentration is 1.0 μ g/mL, draws in 500 μ L to 5mL volumetric flasks, dissolves constant volume with methyl alcohol; Concentration is 100.0ng/mL, draws in 500 μ L to 5mL volumetric flasks, also has concentration to be 10.0ng/mL; Wherein the compound method of T-2 standard solution selects the purity of Sigma company to be 99.1%, 5mg*2 bottle, and variable concentrations has concentration to be 0.9mg/mL respectively, draws in 315 μ L to 25mL volumetric flasks, use acetic acid ethyl dissolution constant volume; Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is 100.0ng/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is also had to be 10.0ng/mL; Wherein the compound method of DON standard solution selects the purity of Sigma company to be 99.1%, 5mg*2 bottle, and variable concentrations has concentration to be 0.8mg/mL respectively, draws in 315 μ L to 25mL volumetric flasks, use acetic acid ethyl dissolution constant volume; Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is 100.0ng/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume; Concentration is also had to be 10.0ng/mL; 0.8mg/mL to FB1 concentration, OTA concentration is 0.7mg/mL, T-2 concentration is 0.9mg/mL, DON concentration is that the standard solution of 0.8mg/mL adopts irradiation dose 0,3,5,7,9,20,50,100 and 200kGy process respectively, adopts irradiation dose 0,3,5,7 and 9kGy process respectively to the standard solution of other concentration.
In preferred described step (2), the malicious sample of band has 6, is go mouldy 2, corn seed sample (being numbered A, B), rice sample 2 (being numbered C, D), wheat (being numbered E) and soya bean sample each 1 (being numbered F) respectively; Preprocess method accurately takes sample 5.00g (± 0.05g) in the centrifuge tube of 50mL, adds 10mL86% acetonitrile solution, vortex 30s, ultrasonic 30min, the centrifugal 5min of 4000r/min, transfer supernatant, in test tube, extracts with 10mL86% acetonitrile solution again, merge extracted twice liquid, dry up at 45 DEG C with nitrogen, then use acetonitrile+water (volume ratio 50:50) to be settled to 1mL, vortex 30s, with 0.22 μm of filtering with microporous membrane, in order to sample introduction; Irradiation dose is 0,3,5,7 and 9kGy.
0.8mg/mL to FB1 concentration in preferred described step (3), OTA concentration is 0.7mg/mL, T-2 concentration is 0.9mg/mL, DON concentration is the standard solution of 0.8mg/mL, respectively after the radiation treatment that irradiation dose is 0-200kGy, detect by full scan pattern, observe the formation with or without characteristic peak.
Preferred when irradiation dose is 3-9kGy, detoxification treatment can be carried out to having commercial value agricultural product, when irradiation dose is 20-200kGy, can to without commercial value go mouldy agricultural product and or other material that goes mouldy carry out concentrated detoxification treatment.
Preferred when irradiation dose is 7-9kGy, detoxification treatment can be carried out to having commercial value agricultural product, when irradiation dose is 100-200kGy, can to without commercial value go mouldy agricultural product and or other material that goes mouldy carry out concentrated detoxification treatment.
The present invention has positive effect: the radiation treatment of (1) 3-9kGy has degradation effect to FB1 and DON, during 9kGy, concentration is that the degradation rate of the FB1 of 0.8mg/mL, 10.0ug/mL, 1.0ug/mL and 50ng/mL is respectively 22.5%, 51.0%, 59.0% and 64.8%, and concentration is that the degradation rate of the DON of 0.8mg/mL, 10.0ug/mL, 1.0ug/mL, 100ng/mL and 10ng/mL is respectively 13.75%, 99.48%, 93.2%, 86.2% and 90.4%; The radiation treatment of 3-9kGy can be degraded OTA and T-2, but DeGrain.When irradiation dose is 100kGy, when the degradation rate of FB1, OTA and DON all meets or exceeds 90%, 200kGy, FB1 and OTA almost all degrades.Radiation treatment is to the degradation effect of T-2 toxin not as other 3 kinds of toxin, and degradation rate when irradiation dose is 200kGy is 43.3%.
(2) screening 6 may with poison sample in have the testing result of 4 samples to be positive, be respectively A, B, C and E sample.Detected FB1 in A, B sample, the change that in 2 samples, FB1 content produces with the increase of irradiation dose is substantially identical, and dosage lower than there being the trend raised with the increase of dosage during 9kGy, but significantly reduces again during 9kGy; Detect DON in C, E sample, when in sample, the pollution concentration of DON is lower, its content reduces with the increase of irradiation dose, and degradation rate during 9kGy is close to 90%, but when in sample, the pollution concentration of DON is heavier, degradation effect reduces.
(3) feature catabolite is had no after FB1 radiation treatment, compare entirely sweeping collection of illustrative plates with the collection of illustrative plates of control sample, the collection of illustrative plates of irradiation sample creates change, doubtful have catabolite to be formed, but owing to containing acetonitrile in the solvent that FB1 standard items adopt, so compare with the collection of illustrative plates of solvent blank, find that the peak produced is solvent peak; New characteristic peak is had to be formed after OTA standard solution irradiation, the material of m/z=398.19 is created by analysis after radiation treatment, this material starts when irradiation dose is 50kGy to occur, during 100kGy, response is the highest, close when response during 200kGy and 100kGy; Have new characteristic peak to be formed after T-2 toxin standard solution irradiation, create the material of m/z=656.8 by analysis after radiation treatment, this material starts when irradiation dose is 20kGy to occur, during 50kGy, response is the highest, and response during 100-200kGy reduces gradually; Also new characteristic peak is had to be formed after DON standard solution irradiation, 2 kinds of materials of m/z=371.19 and m/z=339.14 are created by analysis after radiation treatment, these 2 kinds of materials all just start when irradiation dose is 3kGy to occur, and along with their response of increase of irradiation dose also increases in 3-9kGy dosage range, the amplitude of the response increase of the material of m/z=339.14 is higher than another kind of material.
(4) detoxification process can be made pollution-free by irradiation-induced degradation, have good environment protecting.Dosage is that the radiation treatment of 3-200kGy has degradation effect to several mycotoxin, and the irradiation dose for the DON toxin 3-9kGy of low concentration can reach good degradation effect, takes second place to the degradation effect of FB1.The irradiation dose of 3-9kGy can be used for the agricultural product detoxification having commercial value, as: grain, food, feed etc., preferred 7-9kGy; The irradiation dose of 20-200kGy can do the utilization of environmental protection aspect, for no longer having the concentrated detoxification treatment of go mouldy agricultural product or other material that goes mouldy of commercial value, preferred 50-200kGy, most preferably 100-200kGy.
Accompanying drawing explanation
In order to make content of the present invention more easily be clearly understood, below according to specific embodiment also by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the typical curve of FB1;
Fig. 2 is the typical curve of OTA;
Fig. 3 is the typical curve of T-2;
Fig. 4 is the typical curve of DON;
Fig. 5 be FB1 standard solution control sample entirely sweep collection of illustrative plates;
Fig. 6 be FB1 standard solution irradiation sample entirely sweep collection of illustrative plates (200kGy);
Fig. 7 sweeps collection of illustrative plates (200kGy) after FB1 solvent irradiation entirely;
Fig. 8 be OTA standard solution control sample entirely sweep collection of illustrative plates;
Fig. 9 is that collection of illustrative plates (200kGy) swept entirely by OTA standard solution irradiation sample;
Figure 10 sweeps collection of illustrative plates (200kGy) after OTA solvent irradiation entirely;
Figure 11 is OTA standard solution control sample collection of illustrative plates (m/z=398.19);
Standard solution irradiation master drawing spectrum (m/z=398.19) of Figure 12 to be OTA dosage be 20kGy;
Standard solution irradiation master drawing spectrum (m/z=398.19) of Figure 13 to be OTA dosage be 50kGy;
Standard solution irradiation master drawing spectrum (m/z=398.19) of Figure 14 to be OTA dosage be 100kGy;
Standard solution irradiation master drawing spectrum (m/z=398.19) of Figure 15 to be OTA dosage be 200kGy;
Figure 16 be T-2 standard solution control sample entirely sweep collection of illustrative plates;
Figure 17 is that collection of illustrative plates (200kGy) swept entirely by T-2 standard solution irradiation sample;
Figure 18 is T-2 standard solution control sample collection of illustrative plates (m/z=656.8);
Standard solution irradiation master drawing spectrum (m/z=656.8) of Figure 19 to be T-2 dosage be 20kGy;
Standard solution irradiation master drawing spectrum (m/z=656.8) of Figure 20 to be T-2 dosage be 50kGy;
Standard solution irradiation master drawing spectrum (m/z=656.8) of Figure 21 to be T-2 dosage be 100kGy;
Standard solution irradiation master drawing spectrum (m/z=656.8) of Figure 22 to be T-2 dosage be 200kGy;
Figure 23 be DON standard solution control sample entirely sweep collection of illustrative plates;
Figure 24 be DON standard solution irradiation sample entirely sweep collection of illustrative plates (9kGy);
Figure 25 is DON standard solution control sample collection of illustrative plates (m/z=371.19);
Standard solution irradiation master drawing spectrum (m/z=371.19) of Figure 26 to be DON dosage be 3kGy;
Standard solution irradiation master drawing spectrum (m/z=371.19) of Figure 27 to be DON dosage be 5kGy;
Standard solution irradiation master drawing spectrum (m/z=371.19) of Figure 28 to be DON dosage be 7kGy;
Standard solution irradiation master drawing spectrum (m/z=371.19) of Figure 29 to be DON dosage be 9kGy;
Figure 30 is DON standard solution control sample collection of illustrative plates (m/z=339.14);
Standard solution irradiation master drawing spectrum (m/z=339.14) of Figure 31 to be DON dosage be 3kGy;
Standard solution irradiation master drawing spectrum (m/z=339.14) of Figure 32 to be DON dosage be 5kGy;
Standard solution irradiation master drawing spectrum (m/z=339.14) of Figure 33 to be DON dosage be 7kGy;
Standard solution irradiation master drawing spectrum (m/z=339.14) of Figure 34 to be DON dosage be 9kGy;
Embodiment
To the standard model preparing standard solution of mycotoxin FT FB1, ochratoxin OTA, T-2 toxin and deoxynivalenol DON, detect the typical curve obtaining these four kinds of toxin, see Fig. 1-4:
FB1：Y=-4407.4+35124.6*X、R
2=0.9970，OTA：Y=116009+673499*X、R
2=0.9979，T-2：Y=69665.1+317463*X、R
2=0.9973，DON：Y=11733.1+10933.8*X、R
2=0.9989
The standard solution of the variable concentrations of these four kinds of toxin is all respectively after the gamma-ray irradiation process that dosage is 3-9kGy, and the content of contratoxin detects, the sample in contrast of the standard solution without irradiation that each toxin is corresponding, and the response of toxin is in table 1.The result display of table 3, the radiation treatment of 3-9kGy has degradation effect to FB1 and DON, during 9kGy, concentration is that the degradation rate of the FB1 of 0.8mg/mL, 10.0ug/mL, 1.0ug/mL and 50ng/mL is respectively 22.5%, 51.0%, 59.0% and 64.8%, and concentration is that the degradation rate of the DON of 0.8mg/mL, 10.0ug/mL, 1.0ug/mL, 100ng/mL and 10ng/mL is respectively 13.75%, 99.48%, 93.2%, 86.2% and 90.4%; The radiation treatment of 3-9kGy can be degraded OTA and T-2, but DeGrain.Due in the dosage range of 3-9kGy, the degradation effect of the standard solution of each toxin maximum concentration is remarkable, so the irradiation dose adding them is to 20-200kGy, response is in table 2.The result display of table 3, when irradiation dose is 100kGy, when the degradation rate of FB1, OTA and DON all meets or exceeds 90%, 200kGy, FB1 and OTA almost all degrades.Radiation treatment is to the degradation effect of T-2 toxin not as other 3 kinds of toxin, and degradation rate when irradiation dose is 200kGy is 43.3%.
The response of table 1 toxin standard solution after various dose irradiation (3-9kGy)
The response of table 2 high concentration toxin standard solution after various dose irradiation (20-200kGy)
The degradation rate (%) of the toxin of table 3 standard solution after various dose irradiation
External standard method is adopted to the detection of the recovery of the detection method of toxin in the malicious sample of band; It is FB1:10.0 μ g/kg, T-2:5.0 μ g/kg, OTA:5.0 μ g/kg that sample A-F adds scalar, and in each sample, DON adds scalar difference, is be in 20.0 μ g/kg, D and E samples be 50.0 μ g/kg in 100.0 μ g/kg, C and F samples in A and B sample.The method recovery is respectively FB1:53.7-87.2%, T-2:57.6-94.2%, OTA:43.0-57.8%, DON:41.3-52.8%.
Screening 6 may with poison sample in have the testing result of 4 samples to be positive, be respectively A, B, C and E sample.Detected FB1 in A, B sample, the change that in 2 samples, FB1 content produces with the increase of irradiation dose is substantially identical, and dosage lower than there being the trend raised with the increase of dosage during 9kGy, but significantly reduces again during 9kGy; Detect DON in C, E sample, when in sample, the pollution concentration of DON is lower, its content reduces with the increase of irradiation dose, and degradation rate during 9kGy is close to 90%, but when in sample, the pollution concentration of DON is heavier, degradation effect reduces.
The irradiation-induced degradation effect of toxin in table 4 sample
Using 4 kinds of standard solutions that toxin concentration is the highest as sample, FB1 and DON:0.8mg/mL, OTA:0.7mg/mL, T-2:0.9mg/mL, respectively after the radiation treatment that dosage is 0-200kGy, detect by the pattern of full scan, catabolite is analyzed.
Feature catabolite is had no after FB1 radiation treatment.As can be seen from entirely sweeping collection of illustrative plates, compare with the collection of illustrative plates (Fig. 5) of control sample, the collection of illustrative plates of irradiation sample creates change (Fig. 6), doubtful have catabolite to be formed, but owing to containing acetonitrile in the solvent that FB1 standard items adopt, so compare with the collection of illustrative plates (Fig. 7) of solvent blank, find that the peak produced is solvent peak.
Formed as can be seen from there being new characteristic peak after Fig. 8-10, OTA standard solution irradiation.By analysis (Figure 11-15), create the material of m/z=398.19 after radiation treatment, this material starts when irradiation dose is 50kGy to occur, during 100kGy, response is the highest, close when response during 200kGy and 100kGy.
Formed as can be seen from there being new characteristic peak after Figure 16-17, T-2 toxin standard solution irradiation.By analysis (Figure 18-22), create the material of m/z=656.8 after radiation treatment, this material starts when irradiation dose is 20kGy to occur, during 50kGy, response is the highest, and response during 100-200kGy reduces gradually.
Formed as can be seen from also there being new characteristic peak after Figure 23-24, DON standard solution irradiation.By analysis, create m/z=371.19(Figure 25-29 after radiation treatment) and m/z=339.14(Figure 30-34) 2 kinds of materials, these 2 kinds of materials all just start when irradiation dose is 3kGy to occur, and along with their response of increase of irradiation dose also increases in 3-9kGy dosage range, the amplitude of the response increase of the material of m/z=339.14 is higher than another kind of material.
Dosage is that the radiation treatment of 3-200kGy has degradation effect to several mycotoxin, and the irradiation dose for the DON toxin 3-9kGy of low concentration can reach good degradation effect, takes second place to the degradation effect of FB1.The irradiation dose of 3-9kGy can be used for the agricultural product detoxification having commercial value, as: grain, food, feed etc., preferred 7-9kGy; The irradiation dose of 20-200kGy can do the utilization of environmental protection aspect, for no longer having the concentrated detoxification treatment of go mouldy agricultural product or other material that goes mouldy of commercial value, preferred 50-200kGy, most preferably 100-200kGy.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the irradiation-induced degradation facture of a mycotoxin; It is characterized in that: comprise the following steps: (1) standard items preparing standard solution to mycotoxin, be the radiation treatment of 0-200kGy through irradiation range to standard solution, measured by the content of liquid chromatography-tandem mass spectrometry to mycotoxin; (2) after the irradiation that irradiation range is 0-9kGy, pre-service is carried out to the malicious sample of band, measured by the degradation effect of liquid chromatography-tandem mass spectrometry to mycotoxin; (3) choose the highest mycotoxin standard solution of concentration as sample, after the radiation treatment that irradiation range is 0-200kGy, carry out detection by liquid chromatography-tandem mass spectrometry analyze catabolite; To have commercial value agricultural product adopt irradiation dose be that 3-9kGy carries out detoxification treatment, to without commercial value go mouldy agricultural product and or other go mouldy material employing irradiation dose be that 20-200kGy carries out concentrated detoxification treatment; Described mycotoxin is FT FB1, ochratoxin OTA, T-2 toxin and deoxynivalenol DON; The irradiation bomb of described irradiation is gamma-rays, the chromatographic condition of described liquid chromatography-tandem mass spectrometry: adopt Thermo Finnigan liquid chromatographic system, it is SepaxBR-C18 post 100 × 2.1mm that its liquid-phase condition comprises chromatographic column, 5 μm; When detecting FB1, OTA and T-2, mobile phase is A: methyl alcohol, B: Cu Suan An – aqueous formic acid, under flow velocity 250 μ L/min and sample size 25 μ L condition, carries out gradient elution; Concrete gradient is:
When detecting DON, mobile phase is A: methyl alcohol, B: water, under flow velocity 250 μ L/min and sample size 25 μ L condition, carries out gradient elution; Concrete gradient is:
The Mass Spectrometry Conditions of described liquid chromatography-tandem mass spectrometry: adopt Thermo TSQ Ultra triple level Four bar mass spectrometer system, its Mass Spectrometry Conditions comprises when detecting FB1, OTA and T-2, Ionization mode is ESI, and spray voltage is 3000V, and polar mode is positive ion, atomization temperature is 400 DEG C, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 DEG C, sweep length is 0.01m/z, sweep time is 0.1s, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; When detecting DON, Ionization mode is ESI, and spray voltage is 2500V, and polar mode is negative ion, atomization temperature is 400 DEG C, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 DEG C, sweep length is 0.01m/z, sweep time is 0.1s, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; Ion pair information, wherein * is quota ion pair:
2. the irradiation-induced degradation facture of mycotoxin according to claim 1, is characterized in that: the standard solution in described step (1) and irradiation treatment method are:
Wherein the compound method of FB1 standard solution selects the standard model of Enzo company, standard model purity is 98.0%, 5mg*2 bottle, variable concentrations has concentration to be 0.8mg/mL respectively, draw in 315 μ L to 25mL volumetric flasks, with acetonitrile and the water-soluble solution constant volume of volume ratio 50:50, obtain the solution that concentration is 10.0 μ g/mL; Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, with acetonitrile and the water-soluble solution constant volume of volume ratio 50:50, obtains the solution that concentration is 1.0 μ g/mL; Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, with acetonitrile and the water-soluble solution constant volume of volume ratio 50:50, obtains the solution that concentration is 100.0ng/mL; Concentration is 100.0ng/mL, draws in 5mL to 10mL volumetric flask, with acetonitrile and the water-soluble solution constant volume of volume ratio 50:50, obtains the solution that concentration is 50.0ng/mL; Wherein the compound method of OTA standard solution selects the standard model of Enzo company, and standard model purity is 98.0%, 5mg*1 bottle, variable concentrations has concentration to be 0.7mg/mL respectively, draw in 360 μ L to 25mL volumetric flasks, dissolve constant volume with methyl alcohol, obtaining concentration is 10.0 μ g/mL; Concentration is 10.0 μ g/mL, draws in 500 μ L to 5mL volumetric flasks, dissolves constant volume with methyl alcohol, and obtaining concentration is 1.0 μ g/mL; Concentration is 1.0 μ g/mL, and draw in 500 μ L to 5mL volumetric flasks, dissolve constant volume with methyl alcohol, obtaining concentration is 100.0ng/mL; Concentration is 100.0ng/mL, and draw in 500 μ L to 5mL volumetric flasks, obtaining concentration is 10.0ng/mL; Wherein the compound method of T-2 standard solution selects the standard model of Sigma company, and standard model purity is 99.1%, 5mg*2 bottle, variable concentrations has concentration to be 0.9mg/mL respectively, draw in 278 μ L to 25mL volumetric flasks, use acetic acid ethyl dissolution constant volume, obtaining concentration is 10.0 μ g/mL; Concentration is 10.0 μ g/mL, and draw in 1.0mL to 10mL volumetric flask, use acetic acid ethyl dissolution constant volume, obtaining concentration is 1.0 μ g/mL; Concentration is 1.0 μ g/mL, and draw in 1.0mL to 10mL volumetric flask, use acetic acid ethyl dissolution constant volume, obtaining concentration is 100.0ng/mL; Concentration is 100.0ng/mL, and draw in 1.0mL to 10mL volumetric flask, use acetic acid ethyl dissolution constant volume, obtaining concentration is 10.0ng/mL; Wherein the compound method of DON standard solution selects the standard model of Sigma company, standard model purity is 99.1%, 5mg*2 bottle, variable concentrations has concentration to be 0.8mg/mL respectively, draw in 315 μ L to 25mL volumetric flasks, use acetic acid ethyl dissolution constant volume, obtain the solution that concentration is 10.0 μ g/mL; Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume, obtain the solution that concentration is 1.0 μ g/mL; Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume, obtain the solution that concentration is 100.0ng/mL; Concentration is 100.0ng/mL, draws in 1.0mL to 10mL volumetric flask, uses acetic acid ethyl dissolution constant volume, obtain the solution that concentration is 10.0ng/mL; 0.8mg/mL to FB1 concentration, OTA concentration is 0.7mg/mL, T-2 concentration is 0.9mg/mL, DON concentration is that the standard solution of 0.8mg/mL adopts irradiation dose 0,3,5,7,9,20,50,100 and 200kGy process respectively, adopts irradiation dose 0,3,5,7 and 9kGy process respectively to the standard solution of other concentration.
3. the irradiation-induced degradation facture of mycotoxin according to claim 1, it is characterized in that: in described step (2), the malicious sample of band has 6, that 2, the corn seed sample that goes mouldy is numbered A, B respectively, rice sample 2 is numbered C, D, and wheat is numbered E and soya bean sample number into spectrum is each 1 of F; Preprocess method accurately takes sample 5.00g ± 0.05g in the centrifuge tube of 50mL, adds 10mL86% acetonitrile solution, vortex 30s, ultrasonic 30min, the centrifugal 5min of 4000r/min, transfer supernatant, in test tube, extracts with 10mL86% acetonitrile solution again, merge extracted twice liquid, dry up at 45 DEG C with nitrogen, then be settled to 1mL, vortex 30s with the acetonitrile of volume ratio 50:50 and water, with 0.22 μm of filtering with microporous membrane, in order to sample introduction; Irradiation dose is 0,3,5,7 and 9kGy.
4. the irradiation-induced degradation facture of mycotoxin according to claim 1, it is characterized in that: be 0.8mg/mL to FB1 concentration in described step (3), OTA concentration is 0.7mg/mL, T-2 concentration is 0.9mg/mL, DON concentration is the standard solution of 0.8mg/mL, respectively after the radiation treatment that irradiation dose is 0-200kGy, detect by full scan pattern, observe the formation with or without characteristic peak.
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