CN103645265A - Irradiation degradation treatment method for fumitremorgin and ochratoxin - Google Patents
Irradiation degradation treatment method for fumitremorgin and ochratoxin Download PDFInfo
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Abstract
The invention relates to an irradiation degradation treatment method for fumitremorgin and ochratoxin. The irradiation degradation treatment method comprises the following steps: (1) preparing standard solutions of standard products of the fumitremorgin FB1 and ochratoxin OTA; carrying out irradiation treatment on the standard solutions in an irradiation range of 0kGy-200kGy; determining the content of the FB1 and the OTA by adopting a liquid chromatogram-tandem mass spectrometry; (2) pre-treating a poisoned sample after irradiating in an irradiation range of 0kGy-9kGy; determining degradation effects on the FB1 and the OTA by adopting the liquid chromatogram-tandem mass spectrometry; and (3) selecting the FB1 and OTA standard solutions with the highest concentration as a sample and carrying out detection and analysis on a degraded product by using the liquid chromatogram-tandem mass spectrometry after irradiating in the irradiation range of 0kGy-200kGy. According to the irradiation degradation treatment method for the fumitremorgin and the ochratoxin, researches and applications of FB1 and OTA degraded products which are lack of irradiation degradation are solved; the degradation effect is good and the environment-friendly detoxification treatment of mildewed agricultural product wastes is realized.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to the irradiation-induced degradation facture of a kind of FT and ochratoxin.
Background technology
FT and ochratoxin belong to mycotoxin, mycotoxin is the toxic metabolite product being produced under adapt circumstance condition by Toxigenic fungi, FT and ochratoxin, to mankind's harm is large especially and pollution frequency is high, have been formulated strict limit standard to the FT in Cereals, food and feed and ochratoxin both at home and abroad.
In Chinese patent 201110000805.2 " method of ochratoxin A in immune affinity column purification high performance liquid chromatography detection Chinese medicine tincture ", disclose by tablets by HPLC-MS positive findings has been confirmed; In Chinese patent 201010209190.X " a kind of detect Chinese medicine in the method for nivalenol and deoxynivalenol toxin ", disclose by tablets by HPLC-MS and confirmed toxin under full scan pattern; In Chinese patent 201110370420.5 " detection method of several mycotoxin levels in wheat ", disclose by tablets by HPLC-MS and detected, by toxin concentration for the regression equation calculation DON of peak area, the content of T-2 toxin; In Chinese patent 201210054250.4 " method of mycotoxin levels in LC-MS/MS Araliaceae ", disclosing can be accurate, measures delicately the multicomponent residual quantity of mycotoxin in Araliaceae; In Chinese patent 201210361145.5 " by the method for algae toxin in electron beam irradiation-induced degradation water ", disclose when radiation dose is 5kGy, can suppress the generation of algae toxin; In Chinese patent 2009100787702.0 " method of degrading fumonisins ", disclose irradiation and can make fumonisins B
1degradation rate reaches 63.47%; In Chinese patent 200910078703.5 " a kind of method of degrading fumonisins ", disclose irradiation and can make fumonisins B
1degradation rate reaches 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 and can make AFB
1degradation rate reaches 71.51%.
Existing correlation technique has: the irradiation dose of the report 3kGy～5kGy such as Zhang Xichun has obvious effect to the degraded of mould and mycotoxin; Poplar is waited research quietly and shows that mass concentration is that aflatoxin B1 solution degradation rate when 4kGy of 0.1mg/L can reach more than 80%, and fumonisins B1 solution degradation rate when 10kGy that during 6kGy, degradation rate reaches 96%, 0.1mg/L 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 has reached 90%; The people such as Chi Lei have delivered the paper of gamma-rays to the irradiation-induced degradation of ochratoxin A, mention the method for catabolite structure elucidation, the possible mechanism of degraded in literary composition, but the product that report is not degraded.
Summary of the invention
The object of this invention is to provide a kind of good degrading effect, realize the FT of agricultural product refuse environmental protection detoxification treatment and the irradiation-induced degradation facture of ochratoxin.
The technical scheme that realizes the object of the invention is the irradiation-induced degradation facture of a kind of FT and ochratoxin; Comprise the following steps: (1) standard items preparing standard solution to FT FB1 and ochratoxin OTA, to standard solution, through irradiation range, be the radiation treatment of 0-200kGy, by liquid chromatography-tandem mass spectrometry, the content of FB1 and OTA measured; (2) to carrying out pre-service after the irradiation that is 0-9kGy through irradiation range with malicious sample, by liquid chromatography-tandem mass spectrometry, the degradation effect of FB1 and OTA is measured; (3) choose FB1 that concentration is the highest and OTA standard solution as sample, after the radiation treatment that is 0-200kGy through irradiation range, by liquid chromatography-tandem mass spectrometry, detect and analyze catabolite.
Preferred described irradiation bomb is gamma-rays, the chromatographic condition of described liquid chromatography-tandem mass spectrometry: adopt Thermo Finnigan liquid chromatographic system, its liquid-phase condition comprises that chromatographic column is Sepax BR-C18 post 100 * 2.1mm, 5 μ m; Mobile phase is A: methyl alcohol, and B: Cu Suan An – aqueous formic acid, under flow velocity 250 μ L/min and sample size 25 μ L conditions, 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 |
The mass spectrum condition of described liquid chromatography-tandem mass spectrometry: adopt the triple level Four bar of Thermo TSQ Ultra mass spectrometer system, its mass spectrum condition comprises that ionization mode is ESI, and spray voltage is 3000V, and polar mode is positive ion, atomization temperature is 400 ℃, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 ℃, sweep length is 0.01m/z, be 0.1s sweep time, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; Ion pair information, wherein * is quota ion pair:
Standard model in preferred described step (1) is
Wherein the compound method of FB1 standard solution is that to select the purity of Enzo company be 98.0%, 5mg*2 bottle, and it is 0.8mg/mL that variable concentrations has respectively concentration, draws in 315 μ L to 25mL volumetric flasks, with acetonitrile+water (volume ratio 50:50), dissolves constant volume; Concentration is 10.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, with acetonitrile+water (volume ratio 50:50), dissolves constant volume; Concentration is 1.0 μ g/mL, draws in 1.0mL to 10mL volumetric flask, with acetonitrile+water (volume ratio 50:50), dissolves constant volume; Concentration is 100.0ng/mL, draws in 500 μ L to 10mL volumetric flasks, with acetonitrile+water (volume ratio 50:50), dissolves constant volume; Also having concentration is 50.0ng/mL; Wherein the compound method of OTA standard solution is that to select the purity of Enzo company be 98.0%, 5mg*1 bottle, and it is 0.7mg/mL that variable concentrations has respectively concentration, draws in 360 μ L to 25mL volumetric flasks, with methyl alcohol, dissolves constant volume; Concentration is 10.0 μ g/mL, draws in 500 μ L to 5mL volumetric flasks, with methyl alcohol, dissolves constant volume; Concentration is 1.0 μ g/mL, draws in 500 μ L to 5mL volumetric flasks, with methyl alcohol, dissolves constant volume; Concentration is 100.0ng/mL, draws in 500 μ L to 5mL volumetric flasks, and also having concentration is 10.0ng/mL; To FB1 concentration, be 0.8mg/mL, OTA concentration is that the standard solution of 0.7mg/mL adopts respectively irradiation dose 0,3,5,7,9,20,50,100 and 200kGy to process, and to the standard solution of other concentration, adopts respectively irradiation dose 0,3,5,7 and 9kGy to process.
In preferred described step (2), being with malicious sample to have 6, is respectively go mouldy corn seed sample 2 (being numbered A, B), rice sample 2 (being numbered C, D), wheat (being numbered E) and soya bean samples each 1 (being numbered F); Preprocess method is accurately to take 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, shifts supernatant to test tube, again with 10mL86% acetonitrile solution, extracts, merge extracted twice liquid, with nitrogen, at 45 ℃, dry up, then use acetonitrile+water (volume ratio 50:50) to be settled to 1mL, vortex 30s, with 0.22 μ m filtering with microporous membrane, in order to sample introduction; Irradiation dose is 0,3,5,7 and 9kGy.
Standard solution to the highest 0.8mg/mL of FB1 concentration and the highest 0.7mg/mL of OTA concentration in preferred described step (3), adopt respectively irradiation dose 0,3,5,7,9,20,50,100 and 200kGy to carry out after radiation treatment, by full scan pattern, detect, observe the formation that has or not characteristic peak.
Preferred when irradiation dose is 3-9kGy, can carry out detoxification treatment to thering are 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 concentrate detoxification treatment.
Preferred when irradiation dose is 7-9kGy, can carry out detoxification treatment to thering are 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 concentrate detoxification treatment.
The present invention has positive effect: the radiation treatment of (1) 3-9kGy has degradation effect to FB1, and 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%; The radiation treatment of the 3-9kGy OTA that can degrade, but DeGrain.When irradiation dose is 100kGy, when the degradation rate of FB1, OTA all meets or exceeds 90%, 200kGy, FB1 and OTA be all degradeds almost.
(2) in 6 of screening may be with malicious sample, there is the testing result of 2 samples to be positive, be respectively A and B sample.In A, B sample, detected FB1, the variation that in 2 samples, FB1 content produces with the increase of irradiation dose is basic identical, the trend that dosage has during lower than 9kGy the increase with dosage to raise, but significantly reduce again during 9kGy.
(3) after FB1 radiation treatment, have no feature catabolite, the collection of illustrative plates of entirely sweeping collection of illustrative plates and control sample is compared, the collection of illustrative plates of irradiation sample has produced variation, doubtful have catabolite to form, but owing to containing acetonitrile in the solvent of FB1 standard items employing, so compare with the collection of illustrative plates of solvent blank, find that the peak producing is solvent peak; After OTA standard solution irradiation, there is new characteristic peak to form, after radiation treatment, produced by analysis the material of m/z=398.19, this material starts appearance when irradiation dose is 50kGy, and during 100kGy, response is the highest, approaching when response during 200kGy and 100kGy.
(4) by irradiation-induced degradation, can, so that detoxification process is pollution-free, there is good environment protecting.Dosage is that the radiation treatment of 3-200kGy has degradation effect to FB1 and OTA, better to the FB1 of low concentration degradation effect under the irradiation dose of 3-9kGy.The irradiation dose of 3-9kGy can be used for having the agricultural product detoxification of commercial value, as: grain, food, feed etc., preferably 7-9kGy; The irradiation dose of 20-200kGy can be done the utilization of environmental protection aspect, for no longer thering is the concentrated detoxification treatment of go mouldy agricultural product or other material that goes mouldy of commercial value, and preferred 50-200kGy, most preferably 100-200kGy.
Accompanying drawing explanation
For content of the present invention is more easily expressly understood, according to specific embodiment also by reference to the accompanying drawings, the present invention is further detailed explanation, wherein below
Fig. 1 is the typical curve of FB1;
Fig. 2 is the typical curve of OTA;
Fig. 3 be FB1 standard solution control sample entirely sweep collection of illustrative plates;
Fig. 4 be FB1 standard solution irradiation sample entirely sweep collection of illustrative plates (200kGy);
Fig. 5 sweeps collection of illustrative plates (200kGy) after FB1 solvent irradiation entirely;
Fig. 6 be OTA standard solution control sample entirely sweep collection of illustrative plates;
Fig. 7 is that OTA standard solution irradiation sample is swept collection of illustrative plates (200kGy) entirely;
Fig. 8 sweeps collection of illustrative plates (200kGy) after OTA solvent irradiation entirely;
Fig. 9 is OTA standard solution control sample collection of illustrative plates (m/z=398.19);
Figure 10 is that OTA dosage is the standard solution irradiation master drawing spectrum (m/z=398.19) of 20kGy;
Figure 11 is that OTA dosage is the standard solution irradiation master drawing spectrum (m/z=398.19) of 50kGy;
Figure 12 is that OTA dosage is the standard solution irradiation master drawing spectrum (m/z=398.19) of 100kGy;
Figure 13 is that OTA dosage is the standard solution irradiation master drawing spectrum (m/z=398.19) of 200kGy;
Embodiment
Standard model preparing standard solution to FT FB1, ochratoxin OTA, detects the typical curve that obtains toxin, sees Fig. 1 and Fig. 2:
FB1：Y＝-4407.4+35124.6*X、R
2＝0.9970，OTA：Y＝116009+673499*X、R
2＝0.9979；
After gamma-ray irradiation that the standard solution of variable concentrations is all 3-9kGy through dosage is respectively processed, the content of contratoxin detects, and the standard solution without irradiation corresponding to each toxin be sample in contrast, and the response of toxin is in Table 1.The result demonstration of table 3, the radiation treatment of 3-9kGy has degradation effect to FB1, and 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%; The radiation treatment of the 3-9kGy OTA that can degrade, but DeGrain.Due in the dosage range of 3-9kGy, the degradation effect of the standard solution of each toxin maximum concentration is not remarkable, thus the irradiation dose that has increased them to 20-200kGy, response is in Table 2.The result demonstration of table 3, when irradiation dose is 100kGy, when the degradation rate of FB1, OTA all meets or exceeds 90%, 200kGy, FB1 and OTA be all degradeds almost.
The response (3-9kGy) of table 1 toxin standard solution after various dose irradiation
The response (20-200kGy) of table 2 high concentration toxin standard solution after various dose irradiation
The degradation rate (%) of the toxin of table 3 standard solution after various dose irradiation
Detection with the recovery of the detection method of toxin in malicious sample is adopted to external standard method; It is FB1:10.0 μ g/kg, OTA:5.0 μ g/kg that sample A-F adds scalar; The method recovery is respectively FB1:53.7-87.2%, OTA:43.0-57.8%.
In 6 of screening may be with malicious sample, there is the FB1 testing result of 2 samples to be positive, be respectively A and B sample.The variation that in 2 samples, FB1 content produces with the increase of irradiation dose is basic identical, the trend that dosage has during lower than 9kGy the increase with dosage to raise, but significantly reduce again during 9kGy.
The irradiation-induced degradation effect of toxin in table 4 sample
Using the highest standard solution of FB1 and OTA concentration as sample, FB1:0.8mg/mL, OTA:0.7mg/mL, after the radiation treatment of irradiation dose 0,3,5,7,9,20,50,100 and 200kGy, by the pattern of full scan, detect respectively, catabolite is analyzed.
After FB1 radiation treatment, have no feature catabolite.By entirely sweeping collection of illustrative plates, can find out, compare with the collection of illustrative plates (Fig. 3) of control sample, the collection of illustrative plates of irradiation sample has produced variation (Fig. 4), doubtful have catabolite to form, but owing to containing acetonitrile in the solvent of FB1 standard items employing, so compare with the collection of illustrative plates (Fig. 5) of solvent blank, find that the peak producing is solvent peak.
By Fig. 6-8, can be found out after OTA standard solution irradiation, have new characteristic peak to form.(Fig. 9-13) by analysis, have produced the material of m/z=398.19 after radiation treatment, this material starts to occur when irradiation dose is 50kGy, and during 100kGy, response is the highest, approaching when response during 200kGy and 100kGy.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; institute is 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 modification of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (7)
1. the irradiation-induced degradation facture of a FT and ochratoxin; It is characterized in that: comprise the following steps: (1) standard items preparing standard solution to FT FB1 and ochratoxin OTA, to standard solution, through irradiation range, be the radiation treatment of 0-200kGy, by liquid chromatography-tandem mass spectrometry, the content of FB1 and OTA measured; (2) to carrying out pre-service after the irradiation that is 0-9kGy through irradiation range with malicious sample, by liquid chromatography-tandem mass spectrometry, the degradation effect of FB1 and OTA is measured; (3) choose FB1 that concentration is the highest and OTA standard solution as sample, after the radiation treatment that is 0-200kGy through irradiation range, by liquid chromatography-tandem mass spectrometry, detect and analyze catabolite.
2. the irradiation-induced degradation facture of FT according to claim 1 and ochratoxin, it is characterized in that: described irradiation bomb is gamma-rays, the chromatographic condition of described liquid chromatography-tandem mass spectrometry: adopt Thermo Finnigan liquid chromatographic system, its liquid-phase condition comprises that chromatographic column is Sepax BR-C18 post 100 * 2.1mm, 5 μ m; Mobile phase is A: methyl alcohol, and B: ammonium acetate-aqueous formic acid, under flow velocity 250 μ L/min and sample size 25 μ L conditions, carries out gradient elution; Concrete gradient is:
The mass spectrum condition of described liquid chromatography-tandem mass spectrometry: adopt the triple level Four bar of Thermo TSQ Ultra mass spectrometer system, its mass spectrum condition comprises that ionization mode is ESI, and spray voltage is 3000V, and polar mode is positive ion, atomization temperature is 400 ℃, sheath gas is 48arb, and assisted gas is 10arb, and ion transfer tube temperature is 400 ℃, sweep length is 0.01m/z, be 0.1s sweep time, and resolution is Q1=0.7FWHM, Q3=0.7FWHM; Ion pair information, wherein * is quota ion pair:
3. the irradiation-induced degradation facture of FT according to claim 2 and ochratoxin, is characterized in that: the standard model in described step (1) is
4. the irradiation-induced degradation facture of FT according to claim 2 and ochratoxin, it is characterized in that: in described step (2), being with malicious sample to have 6, is respectively go mouldy corn seed sample 2 (being numbered A, B), rice sample 2 (being numbered C, D), wheat (being numbered E) and soya bean samples each 1 (being numbered F); Preprocess method is accurately to take 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, shifts supernatant to test tube, again with 10mL86% acetonitrile solution, extracts, merge extracted twice liquid, with nitrogen, at 45 ℃, dry up, then use acetonitrile+water (volume ratio 50:50) to be settled to 1mL, vortex 30s, with 0.22 μ m filtering with microporous membrane, in order to sample introduction; Irradiation dose is 0,3,5,7 and 9kGy.
5. the irradiation-induced degradation facture of FT according to claim 2 and ochratoxin, it is characterized in that: the standard solution to the highest 0.8mg/mL of FB1 concentration and the highest 0.7mg/mL of OTA concentration in described step (3), adopt respectively irradiation dose 0,3,5,7,9,20,50,100 and 200kGy to carry out after radiation treatment, by full scan pattern, detect, observe the formation that has or not characteristic peak.
6. the irradiation-induced degradation facture of FT according to claim 2 and ochratoxin, it is characterized in that: when irradiation dose is 3-9kGy, can carry out detoxification treatment to thering are 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 concentrate detoxification treatment.
7. the irradiation-induced degradation facture of FT according to claim 6 and ochratoxin, it is characterized in that: when irradiation dose is 7-9kGy, can carry out detoxification treatment to thering are 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 concentrate detoxification treatment.
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| CN103931970A (en) * | 2014-05-14 | 2014-07-23 | 河南工业大学 | Method for reducing deoxynivalenol in wheat with gibberellic disease |
| CN104017679A (en) * | 2014-05-18 | 2014-09-03 | 中国食品发酵工业研究院 | Method for reducing OTA (ochratixin A) in beer malting process |
| CN104900295A (en) * | 2015-05-28 | 2015-09-09 | 官爱平 | Irradiation apparatus and method for fluid processing |
| CN106721926A (en) * | 2016-11-09 | 2017-05-31 | 中国农业科学院农产品加工研究所 | The method of irradiation-induced degradation ochratoxin |
| CN108225884A (en) * | 2017-12-15 | 2018-06-29 | 江南大学 | A kind of combined degradation method of zearalenone and ochratoxin A in solution |
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| CN104900295A (en) * | 2015-05-28 | 2015-09-09 | 官爱平 | Irradiation apparatus and method for fluid processing |
| CN106721926A (en) * | 2016-11-09 | 2017-05-31 | 中国农业科学院农产品加工研究所 | The method of irradiation-induced degradation ochratoxin |
| CN108225884A (en) * | 2017-12-15 | 2018-06-29 | 江南大学 | A kind of combined degradation method of zearalenone and ochratoxin A in solution |
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