| Mycotoxins are the secondary toxic metabolites of fungi produced under suitable environmental conditions with carcinogenic,teratogenic and mutagenic effects.The intake of mycotoxins may cause toxins and their metabolites residues in livestock and poultry,which will further come into the human food chain through the meat,milk and other animal-derived products and finally bring safety risks to consumes.More than 30 kinds of mycotoxins have been found to be toxic by now,among which the most common ones are aflatoxin(AFB1),ochratoxin A(OTA)and deoxynivalenol(DON).In order to protect consumer safety,legislative limits for mycotoxins in different products have been set by several countries.The mycotoxins contamination in dairy cows feed and milk has attracted widespread attention in recent years.In this article,a sensitive,fast and reliable analytical methods for the simultanously determination of the most common 30 kinds of mycotoxins in feed was established.Meanwhile,the analytical method of deoxynivalenol-3-glucoside(D3G),which is in serious condition and easily overlooked,was focoused and established,and further applied in real feed samples.Exposure conditions of mycotoxins in different feed samples were evaluated.Then,the metabolic kinetics of AFB1 and OTA in dairy cows were studied and the absorption,distribution,metabolism and excretion of these mycotoxins were revealed.These are important for the safe ensurace of milk and the establishment of limited standards in feed.The contents and results were discribed as follows:(1)An analytical method using QuEChERS(Quick,Easy,Cheap,Effective,Rugged,Safe)pretreatment technique coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)for the determination of 30 mycotoxins in different feed samples(premixed feed,formula feed and concentrated feed)were established.Good linear relationships with coefficients of determination(R2)?0.99 were obtained for all targeted mycotoxins.The limit of determination(LOD)and limit of quantitation(LOQ)values were in range of 0.7-20 ?g/L and 2-50 ?g/L,respectively.Satisfactory recoveries at the low,medium and high concentration with mean values in the range of 72.0-118.4% were obtained.The method was simple,rapid and practical,and can be applied to the quantitation of 30 mycotoxins in premixed feed,formula feed and concentrated feed.When 30 mycotoxins in 38 real feed samples were analyzed,DON,the most prevalent mycotoxin,was detected in 31 feed samples at the concentration level ranging from 59.3 to 576.8 μg/kg.15-acetyl-deoxynivalenol(15-ADON)and D3 G were also frequently detected(incidences of 55% and 18%)with the concentration levels ranging from 62.3 to 984.3 μg/kg and from 27.8 to 127.1 μg/kg,respectively.Fumarotoxin B1(FB1),fumonisin B2(FB2),zearalenone(ZEN),b-zearalenone(?-ZOL),patulin(PAT)and AFB1 were also found in different feedstuffs.(2)By optimizing the extraction solvent,the loading solvents,washing and elution solvents for HLB cartridges,a pretreatment method for enriching and purifying of DON,3-acetyl-deoxynivalenol(3-ADON),15-ADON,fusarenon X(FUS-X),and D3 G was first developed.Purified samples were analyzed by the established UHPLC-MS/MS method.The established method was extensively validated by determining the linearities(R2 ≥ 0.99),sensitivity(LOQ in the range of 0.08–4.85 μg/L),recoveries(79.3%–108.1%),and then was successfully applied to determine four type B trichothecenes and D3 G in a total of 31 feed samples.Various mycotoxins in the range of 2.1–864.5 μg/kg were found in 26 samples,and D3 G has also been detected in 17 samples with the concentrations in the range of 2.1–34.8 μg/kg.(3)An analytical method for the determination of AFB1,AFM1,OTA and OT? in milk,urine,plasma,heart,liver,spleen,lung and kidney was established.Samples were first extracted with 1400 ?L of acetone.The supernatant was dried,re-dissolved in 200 ?L of water containing 5 mmol/L ammonium acetate/acetonitrile(80/20,v/v),and analyzed via UHPLC-MS/MS.Satisfactory recoveries at LOQ,low,intermediate and high spiked concentration levels were achieved,with the mean values ranging from 82.8% to 114.1%.Good linearities were obtained(R2?0.99).LOD and LOQ values were ranged from 0.03 to 0.2 μg/L and from 0.1 to 0.5 μg/L,respectively.The stabilities of mycotoxins in various matrix were evaluated in short-term(8h),long-term(20d)and frozen thawing three cycle stability,with the average values ranged from 80.9% to 113.6%.The proposed method was simple,rapid and valuable,and can be a powerful tool for quantitative analysis of four mycotoxins in milk,urine,plasma,heart,liver,spleen,lung and kidney.(4)Substrate reference materials of AFB1 and OTA were mixed with dairy cow feed to obtain feed cotaining certain amounts of mycotoxins(1000 ?g/kg of AFB1 and 750 ?g/kg of OTA).10,35,45,60,120,180,240,360,540,720,1440,2160 and 2880 min after feeding the above mentioned feed,blood of the dairy cows was taken,respectively.6 h after feeding,the cows were slaughtered for the collection of organs including heart,liver,spleen,lung,kidney and brain.The mycotoxins and their typical metabolites in different tissues and organs were analyzed by the method discribed in(3).The result showed that AFB1 could be fast absorbed.It was detected in the blood at 10 min,peaked at 35 min and no longer detected after 48 h.The conversion of AFB1 from feed to milk ranged from 1.1 to 1.6% and AFM1 clearance in fresh milk was 2 days.AFB1 was detected in heart,spleen,lung and kidney with the concentration of 1.6,4.1,3.3 and 5.6 ?g/kg,respectively.AFM1 was detected in spleen and kidney with the concentration of 0.7 and 0.8 ?g/kg,respectively.OTA and its matabolism OTα were not detected in milk,blood and any organs,while little OTA and much OTα were detected in urine with the highest concentration of 1.8 and 324.6 μg/L,respectively,indicating that OTA was difficultly absorbed in the body and most was metabolised to OTα in vivo and then excreted. |
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