Establishment Of Four Methods Of High Performance Liquid Chromatography For Mycotoxin Determinations And An Investigation Of Contaminant Of Mycotoxins In Feedstuffs From Shandong Province

Mycotoxins are toxic secondary metabolites produced by fungi and mold that are contaminants of various feedstuffs and finished feed. The mycotoxins will cause the toxin poisoning when the animal ingests a feed contaminating with mycotoxin and the influence the animal production and a heath problem of human. It is necessary to increase the study on determine technology and to provide a technical support for the further study on detoxification.F-2 toxin (named Zearalenone) is white crystal and is a mycotoxin produced as a secondary metabolite of various Fusarium fungi with a non-steroidal structure. F-2 toxin has the estrogenic activity and has effects on the reproductive performance of animals.T-2 toxin (T-2) belongs to trichothecenes mycotoxins. Trichothecenes, constituting of kinds of sesquiterpene compounds with the similar chemical structures, are secondary metabolites produced by cephalosporium, Fusarium, Stachybotry and trichoderma. Trichothecenes are constituted of type A, B, C and D due to differences of chemical structure. Type A trichothecenes include T-2 toxin, HT-2 toxin and Diacetoxyscirpenol etc., type B trichothecenes include Deoxynivalenol、Nivalenol etc., type C trichothecenes include crotocin and baccharin etc., type D trichothecenes include satratoxin and bacillosporin etc. T-2 toxin is of enterotoxigenic, hepatotoxicity and other toxic effects.Fumonisins (FB), constituting of types of diester compounds with the similar chemical structures of more alcohol hydrogens and tricarhallylic acids, are the water-solubility metabolites produced by F. moniliforme. So far, there are eleven types of fumonisins are discovered, including FA1,FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, FC4 and FP1 and FB1 is the main one. FB1 is neurotoxicity, pulmonary toxicity, immunotoxicity, carcinogenicity, embryotoxicity, phytotoxicity and other toxic effects.Aflatoxins (AFT) are types of structurally similar secondary metabolites produced by Asergillus flavus and Aspergillus parasiticus. So far, there are eighteen types of aflatoxins and the derivatives are discovered, including AFTB1, AFTB2, AFTG1, AFTG2, AFTM1, AFTM2, AFTB2a, AFTG2a, AFTP,, AFTQ1 and AFTR0 etc. and AFTB1, AFTB2, AFTG, and AFTG2 are naturally occurring aflatoxins and the others are derivatives. The toxicity is depending on the different structure and it is a high toxicity and carcinogenicity of aflatoxin with a double bond structure by the end of furan ring. It is proved that AFB1, AFB2, AFTG1 and AFTM1 can induce hepatoma in various experimental animals and AFB1 is the most toxicity and carcinogenicity one.The aim of this paper is to develop four high performance liquid chromatography technologies for F-2, T-2, FB1 and AFTB1, respectively and determine the mycotoxins in feedstuffs rapidly, efficiently, accurately and economically and then provide technical supports for the research institutions and the large enterprise groups. The study is a support for the further study on detoxification, transformation and toxicity mechanism for mycotoxins in feed aiming to reduce the harm for animal.Test I Establishment of high performance liquid chromatography for F-2 toxinA method has been established and improved for quantitative determination of F-2 toxin in feedstuffs using high-performance liquid chromatography with fluorescence detection (HPLC/FLD). The clean-up procedure for extracts was immunoaffinity columns combining with chromatographic column. Feedstuff samples were extracted with acetonitrile/water (90:10, V/V). Analysis was carried out by using a mobile phase composed of methanol/acetonitrile/water (10:44:46, V/V/V), with a flow speed of 0.6 mL/min, a column temperature of 25 ℃ and fluorescence detection (excitation wavelength 274 nm, emission wavelength 440 nm). The method exhibited good linearity over the relevant working range of 0.025～0.8 μg·mL-1 with a linear equation of y=1416362.2459x+2771.5771 and R2 with 1.0000. Recoveries were 88.74%, with relative standard deviations (RSD) of less than 4.15%. The limit of detection (LOD) was 5.7 ng-mL" 1 and the limit of quantization (LOQ) was 19 μg·kg-1. HPLC/FLD was an effective method to determine of F-2 toxin quantitatively in terms of high sensitivity and accuracy.Test II Establishment of HPLC for T-2 toxinA method has been established and improved for quantitative determination of T-2 toxin in feedstuffs using HPLC/FLD and 2-naphthoyl chloride (2-NC) as labeling reagent and 4-Dimethylamino pyridine as reaction accelerator. The clean-up procedure for extracts was immunoaffinity columns combining with chromatographic column. Feedstuff samples were extracted with methanol/water (80:20, V/V). Analysis was carried out by using a mobile phase composed of acetonitrile/water (75:25, V/V), with a flow speed of 0.6 mL/min, a column temperature of 30 ℃ and fluorescence detection (excitation wavelength 381 nm, emission wavelength 470 nm). Recoveries were 72.24%～83.31%, with relative standard deviations (RSD) of less than 4.25%. LOD was 1.8 ng-mL-1 and LOQ was 6×103 ng·kg-1. The method exhibited good linearity over the relevant working range of 25～800 ng-mL"1 with a linear equation of y=2186.2x -31953 and R2 with 0.9992. The method was an effective method to determine of T-2 toxin in feedstuffs in terms of fast, sensitivity, accuracy and good repeatability.Test Ⅲ Establishment of HPLC for FB1A method has been established and improved for quantitative determination of FB1 using HPLC/FLD and O-phthalaldehyde (OPA) as labeling reagent. The clean-up procedure for extracts was immunoaffinity columns combining with chromatographic column. Feedstuff samples were extracted with acetonitrile/water/ acetic acid (49:50:1, V/V/V). Analysis was carried out by using a mobile phase composed of methanol/0.1 M (pH 3.3) of sodium phosphate dibasic (77:23, V/V), with a flow speed of 0.6 mL/min, a column temperature of 30 ℃ and fluorescence detection (excitation wavelength 333 nm, emission wavelength 440 nm). Recoveries were 95.03%, with relative standard deviations (RSD) of less than 3.9%. LOD was 2.16 ng-mL"1 and LOQ was 7.2 μg·kg-1. The method exhibited good linearity over the relevant working range of 25～1000 ng·mL-1 with a linear equation of y=20257x-104237 and R2 with 0.9996. The method was an effective method to determine of FB1 in feedstuffs in terms of fast, sensitivity, accuracy and good repeatability.Test IV Establishment of HPLC for AFTB1A method has been established and improved for quantitative determination of AFTB1 using HPLC/FLD and trifluoroacetic acid (TFA) as labeling reagent. The clean-up procedure for extracts was immunoaffinity columns combining with chromatographic column. Feedstuff samples were extracted with acetonitrile/water/ acetic acid (84:15:1, V/V/V). Analysis was carried out by using a mobile phase composed of acetonitrile/ water (25:75, V/V), with a flow speed of 0.6 mL/min, a column temperature of 30 ℃ and fluorescence detection (excitation wavelength 360 nm, emission wavelength 440 nm). Recoveries were 93.17%, with relative standard deviations (RSD) of less than 4.4%. LOD was 0.034 ng-mL"1 and LOQ was 0.114 μg·kg-1. The method exhibited good linearity over the relevant working range of 0.5～100 ng·mL-1 with a linear equation ofy=118682x-35105 and R2 with 0.9999. The method was an effective method to determine of AFTB1 in feedstuffs in terms of fast, sensitivity, accuracy and good repeatability.Test V Investigation of contaminant of four mycotoxins in feedstuffs from Shandong ProvinceA total of 420 feedstuff samples were selected from the main regions in Shandong Province, China, such as Ji’nan, Ji’ning, Heze, Liaocheng, Dezhou, Binzhou, Dongying, Zibo, etc., ranging from January to September,2011. The samples were used to investigate the contaminant of mycotoxins in feedstuffs by determining F-2, T-2, FB1 and AFTB1. Of the 420 analyzed feedstuff samples, the incidence of F-2, T-2, FB1 and AFTB1 was 85.24, 79.52,96.19 and 83.81%, respectively and it proved that the contaminant was widely. The mean level of F-2, T-2, FB1 and AFTB1 was 410.16,78.73,2583.85 and 12.75 μg·kg-1 respectively. The contaminative level of FB1 was the most severe and the one of AFTB1 was microamount. The exceeding rate of T-2 was zero and it proved that the contaminant of T-2 was minimal or the lime was wide. The exceeding rate of F-2, FB1 and AFB1 was 37.99, 70.79 and 20.45%, respectively with FBi of the largest exceeding rate, F-2 of the larger one and AFTB1 of the less one.