species of the genus fusarium are 
 among the most destructive fungal plant pathogens known and are responsible 
 for major yield losses during cultivation of wheat , maize , barley , 
 and soybeans .
many species of fusarium produce mycotoxins as 
 they grow parasitically within the plant , and ingestion of contaminated 
 grain and food products processed from this grain causes acute and 
 chronic health problems for both humans and animals .
some species of fusarium produce trichothecenes , 
 sesquiterpenoid mycotoxins that inhibit eukaryotic protein synthesis 
 and other cellular functions in animals that ingest contaminated feed .
type a trichothecenes ( e.g. , t-2 toxin , ht-2 
 toxin , diacetoxyscirpenol ) are of particular concern because they 
 are considerably more toxic than the type b group ( e.g. , deoxynivalenol 
 and nivalenol ) .
as part of their defense response to xenobiotics , 
 plants can modify the structure of several mycotoxins , including trichothecenes , 
 by conjugation to sugars , organic acids , or sulfates , which reduce 
 their phytotoxicity and may facilitate their sequestration .
whereas conjugation to sugars may protect plants from the ill effects 
 of the toxins , these so - called  masked 
 mycotoxins  present a potential food safety concern because , 
 although toxicological data are scarce , several studies highlight 
 the potential threat to consumer safety from these substances .
in particular , the possible hydrolysis of masked mycotoxins back 
 to their toxic parents during mammalian digestion is of considerable 
 concern .
structurally t-2 toxin , 1 , r = ac ( figure 1 ) is ( 2,3,4,8)-4,15-bis(acetyloxy)-3-hydroxy-12,13-epoxytrichothec-9-en-8-yl 
 3-methylbutanoate , which in animals is known to be predominantly metabolized 
 to the 4-o - deacetylated form known as ht-2 toxin , 1 , r = h ( figure 1 ) .
glucoside conjugates of t-2 toxin have been reported in fusarium - infected grain and fusarium culture 
 material , although to date the anomericity of the 
 3-o - linked glucosyl group is unknown .
t-2 toxin--glucoside , 2 , and t-2 toxin--glucoside , 3 ( figure 1 ) , are anticipated to have very different physical 
 properties .
more importantly , the relative toxicities of the two anomers 
 are as yet unknown , and the ability to test the naturally occurring 
 form needs to be addressed .
structures of t-2 toxin , 1 ( r = 
 ac ) , ht-2 toxin , 1 ( r = h ) , t-2 toxin--glucoside , 2 , and 
 t-2 toxin--glucoside , 3 .
one of the most studied masked trichothecenes is deoxynivalenol-3-glucoside , 
 which has been reported from contaminated cereal crops and in food products derived from these crops .
nuclear magnetic resonance ( nmr ) studies have 
 shown that the naturally occurring glucoside is deoxynivalenol-3--glucoside .
plant glucosyltransferase genes that control the conversion of deoxynivalenol 
 to deoxynivalenol-3--glucoside have been identified , and a barley glucosyltransferase gene has been engineered into wheat 
 to improve resistance to fusarium head scab .
in addition , 
 a deoxynivalenol glucosyltransferase gene has been cloned and expressed 
 in yeast . as a result
, deoxynivalenol-3--glucoside 
 has been prepared using yeast expression and has been available for 
 studies on its stability during food processing and the digestive 
 fate of this masked mycotoxin .
although initial 
 studies reported that deoxynivalenol-3--glucoside 
 is relatively stable to gastric conditions , it was recently reported that masked mycotoxins 
 can be deconjugated by human colon microbiota , thus releasing their 
 parent forms .
because parent toxins may be 
 absorbed in the intestine , this cleavage should be considered of toxicological 
 relevance depending on the colonic absorption of the target compound .
we have recently shown that three species 
 of blastobotrys are able to biotransform 
 t-2 toxin to t-2 toxin--glucoside , 2 ( figure 1 ) , and these yeast 
 species appeared to provide an efficient way to 
 produce the material necessary to study the digestive fate or animal 
 toxicity of t-2 toxin - glucoside and related compounds , as well as develop methods for their detection .
it was first important to determine which anomeric 
 form of t-2 toxin - glucoside was produced in fusarium - contaminated cereals before proceeding with larger scale studies 
 using the yeast biotransformation product .
for this reason , t-2 toxin--glucoside , 3 ( figure 1 ) , was chemically synthesized , 
 and along with t-2 toxin--glucoside , 2 ( figure 1 ) , the yeast biotransformation product , compared 
 to the t-2 toxin - glucoside found in contaminated grain .
we present 
 here a comparison of the chromatographic and spectroscopic properties 
 of the two anomeric forms , their relative reactivities to antibodies 
 prepared with t-2 toxin--glucoside or t-2 toxin , their relative phytotoxicities , the stabilities and 
 bioavailabilities of these masked mycotoxins after ingestion , and 
 the anomeric form of the naturally occurring t-2 toxin - glucoside . 

these results become particularly relevant if international standards 
 are developed for the detection and quantitation of t-2 toxin and 
 t-2 toxin - glucoside in cereal grains and products made from them .
nmr experiments were performed with 
 acetone - d6 as the solvent on a bruker 
 avance amx 500 spectrometer ( bruker biospin corp
. , billerica , ma , 
 usa ) operating at 500.11 mhz using a standard 5 mm z - gradient bbi probe at 27 c .
chemical shifts are reported as 
 parts per million from tetramethylsilane calculated from the lock 
 solvent .
the deuterated solvents used were obtained from cambridge 
 isotope laboratories ( andover , ma , usa ) .
the pulse sequences used 
 were those supplied by bruker , and processing was done with the bruker 
 topspin software package ( v. 1.3 ) .
additional nmr experiments with 
 tetra - o - tips--glucosyl t-2 toxin , 6 , and t-2 toxin--glucoside , 3 , were performed 
 on an agilent 600 mhz nmr spectrometer ( agilent , santa clara , ca , 
 usa ) .
one wheat and 
 one oat sample were extracted for comparison with t-2 toxin - glucoside 
 standards .
samples were finely ground with a tecator cyclotec 1093 
 ( international pbi , milan , italy ) laboratory mill equipped with a 
 500 mm sieve .
ten grams of each ground sample was extracted with 30 
 ml of acetonitrile / water ( 84:16 v / v ) by orbital shaking for 2 h. after 
 filtration through filter paper , 10 ml of extract was cleaned with 
 a mycosep 227 column ( romer laboratories , union , mo , usa ) . purified 
 extract ( 6 ml , equivalent to 2 g of sample ) was dried under an air 
 stream at 50 c .
extracts of naturally contaminated cereals 
 or t-2 toxin - glucoside 
 standards were analyzed by lc coupled to tandem mass spectrometry 
 ( ms / ms ) as previously described .
lc - ms / ms analyses 
 were performed by a qtrap ms / ms system , from applied biosystems ( foster 
 city , ca , usa ) equipped with an electrospray ionization ( esi ) interface 
 and an 1100 series micro - lc system comprising a binary pump and a 
 microautosampler from agilent technologies ( waldbronn , germany ) .
the 
 column used was a 150  3 mm i.d . , 4 m , synergi hydro , 
 with a 4 mm  2 mm i.d . , 10 m , aqua c18 guard column ( phenomenex , 
 torrance , ca , usa ) .
the flow rate of the mobile phase was 200 l / min , 
 whereas the injection volume was 20 l .
eluent a was water and 
 eluent b was methanol , both containing 5 mm ammonium acetate .
eluent b was increased from 20 to 40% in 3 min , then increased 
 to 63% in 35 min , and kept constant for 9 min .
for column re - equilibration , 
 eluent b was decreased to 20% in 1 min and kept constant for 9 min . 
 for ms analyses ,
the esi interface was used with the following settings : 
 temperature , 350 c ; curtain gas , nitrogen , 30 psi ; nebulizer 
 gas , air , 10 psi ; auxiliary gas , air , 30 psi ; ion spray voltage , + 4500 
 v , positive ion mode .
the ms was operated in enhanced product 
 ion ( epi ) mode , applying a collision energy ( ce ) for stimulation of 
 ion fragmentation of 10 , or multiple reaction monitoring ( mrm ) mode 
 to observe several distinctive ions produced upon fragmentation of 
 the toxin with ammonium adduct [ m + nh4 ] ion . 
 for t-2 toxin - glucoside , the parent ion ( m / z 646 ) and fragment ions were monitored to observe elution 
 of the separated t-2 toxin conjugates .
operation of the lc - ms / ms instrument 
 and interpretation of the acquired data were done utilizing the analyst 
 ( absciex ) software provided by the ms instrument manufacturer .
t-2 toxin - glucosides 
 were prepared using t-2 toxin that had been isolated and purified 
 from liquid cultures of fusarium sporotrichioides strain 5493cos9 - 1#11 as described previously .
t-2 toxin--glucoside , 2 ( optical rotation , 
 [ ]d = + 79.74 , c 0.153 , 
 ch3oh ) , was prepared by feeding t-2 toxin to blastobotrys muscicola cultures as described previously .
t-2 toxin--glucoside , 3 , 
 was synthesized as described below ( figure 2 ) .
pentaacetyl--glucopyranose 
 ( 1.0 g , 2.56 mmol ) was dissolved in dichloromethane ( 20 ml ) , and ethanethiol 
 ( 220 l , 3.07 mmol ) was added followed by bf3oet 
 ( 1.6 ml , 12.8 mmol ) at ambient temperature under an argon atmosphere . 

after 2 h , the reaction mixture was diluted with dichloromethane and 
 washed with saturated sodium bicarbonate .
the aqueous phase was extracted 
 with dichloromethane , and the combined organic phase was washed with 
 brine and dried over sodium sulfate .
the filtrate was concentrated , 
 and the residue was purified by column chromatography over silica 
 gel ( hexane / ethyl acetate , 1:1 ) , giving 660 mg ( 67% ) of a colorless 
 oil in the form of a 1:1 ,-mixture .
to 
 a solution of 4 ( 660 mg , 1.68 mmol ) in methanol ( 16.5 
 ml ) was added sodium methoxide ( 10 mg , 0.17 mmol ) at ambient temperature . 

after 1 h , the reaction mixture was neutralized with amberlite 120 . 
 the resin was filtered off , and the filtrate was concentrated to give 
 a colorless oil ( 340 mg ) , which was dissolved 
 in 2,6-lutidine ( 15 ml ) ( sigma - aldrich , st .
louis , mo , usa ) and treated 
 with triisopropylsilyl trifluoromethanesulfonate ( 2 ml , 4.9 mmol ) 
 ( sigma - aldrich ) .
the reaction mixture was heated to 130 c , and 
 after 1.5 h , further triisopropylsilyl trifluoromethanesulfonate ( 2 
 ml , 4.9 mmol ) was added .
the reaction mixture was maintained at 130 
 c for 2 h , then further triisopropylsilyl trifluoromethanesulfonate 
 ( 2 ml , 4.9 mmol ) was added .
after 2 h , the reaction mixture was cooled 
 to room temperature , diluted with hexane , and washed with 0.1 m hcl 
 until the water phase remained acidic .
the filtrate was concentrated 
 and purified by column chromatography over silica gel eluting with 
 hexane , a colorless oil ( 680 mg , 53% ) as a 1:1 mixture of ,-isomers .
a solution of 5 ( 110 mg , 0.13 mmol ) and t-2 toxin 
 ( 40 mg , 0.086 mmol ) was dissolved in dry dichloromethane ( 0.4 ml ) 
 with 3  acid - washed molecular sieves ( alfa aesar , ward hill , 
 ma , usa ) .
the reaction mixture was cooled to 78 c under 
 an argon atmosphere , and then n - iodosuccinimide ( 26 
 mg , 0.15 mmol ) ( chem - impex int .
after 0.5 h , trifluoromethanesulfonic acid ( 2.3 l , 0.04 mmol ) 
 ( sigma - aldrich ) was added to the cold solution .
after 3 h , the reaction 
 mixture was quenched with triethylamine ( emd , philadelphia , pa , usa ) , 
 and the molecular sieves were filtered off .
the filtrate was concentrated 
 and purified by column chromatography over silica gel ( eluent , hexane / ethyl 
 acetate 4:1 ) to give a colorless oil ( 100 mg , 95% ) as a 1:5 mixture 
 of ,-isomers .
h nmr ( 600 mhz in cdcl3 ) for -isomer :  5.66 ( d , j = 
 3.3 hz , 1h , h-4 ) , 5.63 ( d , j = 5.9 hz , 1h , h-10 ) , 
 5.27 ( d , j = 5.5 hz , 1h , h-8 ) , 4.91 ( d , j = 5.9 hz , 1h , h-26 ) , 4.31 ( d , j = 12.1 hz , 1h , 
 h-15a ) , 4.27 ( dd , j = 4.8 , 3.67 hz , 1h , h-3 ) , 4.10 
 ( d , j = 5.5 hz , 1h , h-11 ) , 4.08 ( overlapped with 
 h-15b , 1h , h-28 ) , 4.07 ( d , j = 12.5 hz , 1h , h-15b ) , 
 3.95 ( d , j = 2.9 hz , 1h , h-29 ) , 3.90 ( d , j = 5.9 hz , 1h , h-27 ) , 3.89 ( d , j = 5.8 
 hz , 1h , h-30 ) , 3.84 ( d , j = 5.1 hz , 2h , h-31a , b ) , 
 3.73 ( d , j = 4.8 hz , 1h , h-2 ) , 2.92 ( d , j = 4.0 hz , 1h , h-13a ) , 2.75 ( d , j = 4.0 hz , 1h , 
 h-13b ) , 2.29 ( dd , j = 15.0 , 5.9 hz , 1h , h-7a ) , 2.09 
 ( m , 1h , h-18 ) , 2.07 ( s , 1h , h-23 ) , 2.05 ( m , 1h , h-19 ) , 2.00 ( s , h-25 ) , 
 1.99 ( overlapped with h-25 , 1h , h-7b ) , 1.72 ( s , h-16 ) , 0.95 ( d , j = 6.2 or 5.1 hz , 1h , h-20 ) , 0.94 ( d , j = 6.2 or 5.1 hz , 1h , h-21 ) , 0.67 ( s , h-14 ) ; c nmr ( 150 
 mhz in cd2cl3 )  172.4 ( c-17 ) , 170.2 ( c-22 ) , 
 169.8 ( c-24 ) , 135.1 ( c-9 ) , 124.1 ( c-10 ) , 103.4 ( c-26 ) , 83.5 ( c-30 ) , 
 82.8 ( c-3 ) , 80.6 ( c-4 ) , 79.5 ( c-2 ) , 78.0 ( c-28 ) , 77.3 ( c-27 ) , 71.1 
 ( c-29 ) , 68.0 ( c-8 ) , 66.8 ( c-11 ) , 65.7 ( c-31 ) , 64.3 ( c-15 ) , 64.1 ( c-12 ) , 
 48.5 ( c-5 ) , 46.7 ( c-13 ) , 43.4 ( c-18 ) , 43.0 ( c-6 ) , 26.9 ( c-7 ) , 25.7 
 ( c-19 ) , 22.1 ( c-20 ) , 22.1 ( c-21 ) , 20.9 ( c-25 ) , 20.6 ( c-23 ) , 20.1 ( c-16 ) , 
 6.2 ( c-14 ) ; hrms - esi ( m / z ) [ m + 
 na ] calcd for c66h124o14si4na 1275.7966 , found 1275.7947 .
the above ,-mixture 
 of 6 ( 100 mg , 0.08 mmol ) was dissolved in thf ( 1.0 ml ) 
 and treated at 0 c with tetrabutylammonium fluoride ( 1.6 ml , 
 1.6 mmol ) ( acros - thermo fisher scientific inc . ,
the reaction mixture was allowed to warm to room temperature and stirred 
 for 2.5 h , after which it was concentrated and purified by column 
 chromatography over silica gel ( eluent , chloroform / methanol , 8:1 ) 
 to give a colorless oil ( 42 mg , 84% ) of a 1:5 ,-mixture . 

further purification of this mixture by c18 hplc ( water  acetonitrile 
 2540% ) and freeze - drying gave 21 mg ( 42% ) of the pure -isomer , 3 , as a white amorphous powder .
h nmr ( 600 
 mhz in cd3od )  5.96 ( d , j = 3.0 
 hz , 1h , h-4 ) , 5.77 ( d , j = 6.00 hz , 1h , h-10 ) , 5.31 
 ( d , j = 5.6 hz , 1h , h-8 ) , 4.46 ( dd , j = 4.9 , 3.2 hz , 1h , h-3 ) , 4.43 ( d , j = 7.7 hz , 1h , 
 h-26 ) , 4.37 ( d , j = 6.2 hz , 1h , h-11 ) , 4.36 ( d , j = 12.7 hz , 1h , h-15a ) , 4.08 ( d , j = 12.5 
 hz , 1h , h-15b ) , 3.81 ( dd , j = 12.0 , 2.2 hz , 1h , h-31a ) , 
 3.70 ( d , j = 5.0 hz , 1h , h-2 ) , 3.63 ( dd , j = 12.0 , 5.8 hz , 1h , h-31b ) , 3.34 ( t , j = 9.0 , 8.9 hz , 1h , h-28 ) , 3.28 ( t , j = 9.0 , 9.0 
 hz , 1h , h-29 ) , 3.24 ( dd , j = 9.0 , 7.8 hz , 1h , h-27 ) , 
 3.19 ( ddd , j = 9.6 , 5.8 , 2.2 hz , 1h , h-30 ) , 3.03 
 ( d , j = 3.8 hz , 1h , h-13a ) , 2.85 ( d , j = 3.9 hz , 1h , h-13b ) , 2.36 ( dd , j = 15.2 , 6.0 hz , 
 1h , h-7a ) , 2.14 ( dd , j = 7.6 , 2.4 hz , 1h , h-18 ) , 
 2.07 ( s , h-23 ) , 2.05 ( s , 1h , h-25 ) , 2.05 ( m , h-19 ) , 1.92 ( d , j = 15.2 hz , 1h , h-7b ) , 1.73 ( s , h-16 ) , 0.95 ( d , j = 6.6 hz , 1h , h-20 ) , 0.94 ( d , j = 6.6 
 hz , 1h , h-21 ) , 0.72 ( s , 1h , h-14 ) ; c nmr ( 150 mhz in 
 cd2cl3 )  174.1 ( c-17 ) , 170.8 ( c-24 ) , 
 170.7 ( c-22 ) , 137.2 ( c-9 ) , 125.2 ( c-10 ) , 103.8 ( c-26 ) , 83.9 ( c-3 ) , 
 81.3 ( c-4 ) , 80.6 ( c-2 ) , 78.4 ( c-30 ) , 78.2 ( c-28 ) , 74.9 ( c-27 ) , 71.6 
 ( c-29 ) , 69.4 ( c-8 ) , 68.6 ( c-11 ) , 65.9 ( c-15 ) , 65.4 ( c-12 ) , 62.8 ( c-31 ) , 
 50.2 ( c-5 ) , 48.0 ( c-13 ) , 44.6 ( c-18 ) , 44.5 ( c-6 ) , 28.9 ( c-7 ) , 27.0 
 ( c-19 ) , 22.9 ( c-20 ) , 22.8 ( c-21 ) , 21.3 ( c-25 ) , 20.9 ( c-23 ) , 20.6 ( c-16 ) , 
 7.2 ( c-14 ) ; hrms - esi ( m / z ) [ m + 
 na ] calcd for c30h44o14na 651.2629 , found 651.2618 ; optical rotation [ ]d = + 7.51 ( c 0.080 , ch3oh ) .
the first of these was 
 a competitive indirect enzyme - linked immunosorbent assay ( ci - elisa ) 
 based upon an antibody ( mab 2 - 13 ) developed against t-2 toxin-3--glucoside , 
 conjugated to ovalbumin ( e.g. , t-2g  ova ) , as described previously .
the second immunoassay format was a commercial 
 test kit previously validated to detect t-2 toxin and ht-2 toxin ( veratox 
 for t-2/ht-2 ) ( neogen corp . , lansing , mi , usa ) .
the 
 methanol content indicated in the test kit instructions , the t-2 toxin - glucoside 
 standards were prepared in 35% ( v / v ) methanol / water .
the single - celled alga chlamydomonas reinhardtii was used to assess the 
 relative phytotoxicity of t-2 toxin , t-2 toxin--glucoside , 
 and t-2 toxin--glucoside , as described previously .
triplicate cultures ( 10 ml ) were initiated with 10 cells / ml 
 containing a 100 m concentration of an individual trichothecene 
 and grown for 6 days under fluorescent lights , with agitation of 200 
 rpm .
culture doublings were calculated as follows : ( log of the final 
 density  log of the initial cell density)/log 2 .
briefly , the main digestive juices were prepared by mixing 
 salts and enzymes and were preheated 
 at 37 c before use .
a volume of 200 l of a water / methanol 
 ( 25:75 , v / v ) solution containing the target compounds was transferred 
 in a 4 ml septum vial , and the solvent was evaporated under nitrogen . 
 the in vitro digestion was started by adding 75 l of artificial 
 saliva to 200 l of the water / methanol ( 25:75 v / v ) solution 
 containing the target analyte ( 500 g / l ) .
after 5 min of incubation 
 at 37 c , 150 l of artificial gastric juice was added 
 and the mixture was incubated again for 2 h. at the end of the gastric 
 step , 25 l of 1 m bicarbonate solution with 150 l of 
 artificial duodenal juice and 75 l of artificial bile juice 
 were added , and then a final incubation step of 2 h was performed . 
 finally , 25 l of acetonitrile was added to stop the reactions , 
 and the sample was centrifuged for 10 min at 10000 rpm , prior to direct 
 lc - ms / ms analysis .
human colonic 
 fermentation of t-2 toxin--glucoside , t-2 toxin--glucoside , 
 and t-2 toxin was carried out as described previously .
these samples were immediately placed 
 in an anaerobic environment and then mixed and weighed to obtain a 
 10% fecal solution in a phosphate saline buffer ( pbs ) . for each sample 

1.8 ml of growth medium , prepared as described previously , and 1.8 ml of fecal solution were mixed and 
 added to 0.4 ml of a toxin solution , at a final concentration of 500 
 g / l .
each vial was then treated with a slight nitrogen flow 
 to eliminate oxygen and perform fermentations under anaerobic condition . 

samples were incubated in a water bath at 37 c and shaken at 
 a rate of 200 strokes / min .
fermentations were stopped immediately 
 to have controls and then after 30 min and 24 h , by cooling samples 
 to room temperature and adding 0.4 ml of acetonitrile .
samples were 
 immediately centrifuged at 14000 rpm for 10 min and stored at 20 
 c . at the same time , samples without added mycotoxin were prepared . 

samples were 
 diluted adding acetonitrile ( 1:2 v / v ) and directly analyzed for t-2 
 toxin - glucoside , t-2 toxin , and ht-2 toxin by lc - ms / ms using mrm as 
 described above .
nmr spectra were recorded for t-2 toxin--glucoside , 
 obtained by yeast biotransformation ( figure 3a , c ) and for chemically synthesized t-2 toxin--glucoside ( figure 3b , d ) .
both compounds are characterized by two spin 
 systems due to a single glucopyranosyl ring and the trichothecene 
 aglycone .
the anomeric linkages are characterized by proton signals 
 in the 4.55.5 ppm range and the c signals by 
 proton signals between 95 and 105 ppm .
t-2 toxin--glucoside gave a doublet at 4.98 ppm that integrates 
 to a single proton ( figure 3a ) and correlates 
 to an adjacent c signal at 98.18 ppm ( figure 3c ) .
the chemically 
 synthesized t-2 toxin--glucoside is similarly characterized 
 by a larger h1ax  h2ax bond angle , resulting in a definitive j1,2 coupling constant of 12 hz for the -anomeric 
 proton at 4.46 ppm ( figure 3b ) .
this h-1 proton 
 correlates to the -c1 c nmr anomeric signal at 
 102.36 ppm ( figure 3d ) .
hence , the t-2 toxin - glucoside 
 anomers are defined by the observed h and c chemical shifts and by the characteristic j1,2 coupling constants .
these assignments were confirmed by 
 hsqc and , together with hmbc , dept , cosy , and noesy experiments , enable 
 the complete nmr assignment of both epimers .
proton nmr spectra of 
 ( a ) t-2 toxin--glucoside , ( b ) t-2 
 toxin--glucoside with signals for the h1 anomeric proton indicated 
 and c nmr anomeric carbon signals of ( c ) t-2 toxin--glucoside 
 ( 98.18 ppm ) and ( d ) t-2 toxin--glucoside ( 102.36 ppm ) .
the two epimeric standards , t-2 toxin--glucoside 
 prepared with yeast and the synthetic t-2 toxin--glucoside , 
 could be separated with lc ( figure 4 ) . in epi 

mode , t-2 toxin--glucoside and t-2 toxin--glucoside 
 [ m + nh4 ] ions were subjected to collision - induced 
 dissociation . under these conditions , t-2 toxin--glucoside 
 more readily ionized to yield an [ m + h ] ion than the 
 t-2 toxin -glucoside , as shown by the relative intensities 
 of the residual [ m + nh4 ] and [ m + h ] ions ( figure 5 ) .
other fragment ions were 
 similar to those previously observed from fusarium - infected grain .
lc - ms / ms chromatograms of ( a ) t-2 toxin--glucoside 
 ( t-2glc ) 
 and t-2 toxin--glucoside ( t-2glc ) standards ,
( b ) t-2 
 toxin - glucoside extracted from wheat , and ( c ) t-2 toxin - glucoside 
 extracted from oats .
tandem mass spectrum of the [ m + nh4 ] ion 
 of ( a ) t-2 toxin--glucoside , ( b ) t-2 toxin--glucoside , 
 ( c ) t-2 toxin - glucoside from oats , and ( d ) t-2 toxin - glucoside from 
 wheat . to 
 determine the form of t-2 toxin - glucoside present in contaminated 
 oats and wheat , extracts were analyzed with lc - ms / ms and compared 
 to the standard t-2 toxin--glucoside and t-2 toxin--glucoside 
 ( figure 4 ) .
under the conditions used , t-2 
 toxin--glucoside eluted at 31.4 min and t-2 toxin--glucoside 
 at 32.0 min .
t-2 toxin - glucoside was detected at 31.5 min in oat and 
 wheat samples , consistent with t-2 toxin--glucoside ( figure 4 ) .
in addition , there were differences in the mass 
 spectra of the two isomers , showing the same fragment ions but with 
 different relative intensities .
similar to t-2 toxin--glucoside , 
 the yeast biotransformation product , the t-2 toxin - glucoside from 
 oats and wheat , had the prominent [ m + h ] product ion 
 from the [ m + nh4 ] ion ( figure 5 ) . in this study
, the stability under in vitro 
 gastrointestinal conditions 
 and the catabolic fate of both t-2 toxin--glucoside and t-2 
 toxin--glucoside were tested in a human gut microbioma assay 
 and compared to t-2 toxin .
we found that the t-2 toxin - glucosides were unchanged after 
 incubation with human artificial saliva .
these results are similar 
 to those reported for deoxynivalenol glucoside and zearalenone glucoside and further demonstrate the stability of some 
 - and -glucosidic bonds upon digestion .
in contrast 
 to zearalenone glucoside , which was hydrolyzed in the 
 first 30 min of colonic fermentation , t-2 
 toxin and the t-2 toxin - glucosides were relatively stable for the 
 first 30 min ( figure 6 ) .
t-2 toxin 
 was mainly transformed into its derivative ht-2 toxin ( 83% at t = 24 h ) .
t-2 toxin--glucoside and t-2 toxin--glucoside 
 were both strongly degraded after 24 h , with < 30% of the starting 
 material remaining .
the latter was mainly cleaved to release its precursor 
 t-2 toxin ( 58% ) with a smaller percentage of ht-2 toxin ( 12% ) , whereas 
 the former is converted into t-2 toxin ( 13% ) , ht-2 toxin ( 30% ) , and 
 other metabolites of unknown structure and toxicity ( 33% ) .
degradation 
 upon human colonic microbiota fermentation of ( a ) t-2 
 toxin--glucoside ( t-2glc ) , ( b ) t-2 toxin--glucoside 
 ( t-2glc ) , and ( c ) t-2 toxin ( t-2 ) .
ht-2 toxin ( ht-2 ) . because smaller amounts of t-2 
 toxin remained following colonic 
 fermentation with t-2 toxin--glucoside
, it may pose less of 
 a risk than t-2 toxin--glucoside , which is not known to occur 
 in crops . however , t-2 toxin--glucoside was also converted 
 into other , as yet uncharacterized , compounds ( figure 6 ) .
in addition to t-2 toxin and ht-2 toxin , other possible 
 products of t-2 toxin - glucoside include ht-2 toxin - glucoside , t-2 
 triol , and t-2 tetraol .
it is important to consider the toxicity of 
 all of the products when the risk posed by t-2 toxin--glucoside 
 in grain is assessed . the relative toxicity 
 of t-2 toxin and 
 the t-2 toxin - glucosides was tested with c. reinhardtii cultures .
previous studies have shown that t-2 toxin is quite phytotoxic and t-2 toxin--glucoside is nontoxic . in this study , cultures grown in 100 m t-2 had only 
 a slight increase in the number of cells , with 0.6 doubling after 
 4 days .
in contrast , neither t-2 toxin--glucoside nor t-2 toxin--glucoside 
 was phytotoxic , with 5.4 and 5.5 doublings , respectively , which are 
 similar to the 5.3 doublings observed for cultures treated with acetone 
 alone .
the first immunoassay was a competitive 
 indirect elisa format using mab 2 - 13 , an antibody developed with t-2 
 toxin--glucoside .
mab 2 - 13 recognized 
 t-2 toxin -glucoside slightly better than t-2 toxin ( cross 
 reaction 108% ) , whereas t-2 toxin--glucoside was recognized 
 more poorly , at about 57% relative to t-2 toxin ( table 1 ; figure 7a ) .
this makes this antibody - based 
 test suitable for the detection of both t-2 toxin and the naturally 
 occurring t-2 toxin--glucoside .
( a ) 
 ci - elisa responses of t-2 toxin--glucoside 
 ( open circles ) and t-2 toxin--glucoside ( solid circles ) with 
 mab 2 - 13 .
data shown are the averages from four plates , with six replicates 
 per toxin concentration per plate ( n = 24 ) ,  
 1 standard deviation .
response 
 equation over the indicated concentration range ( 0.1200 ng / ml ) . 

( b ) elisa responses of t-2 toxin ( open triangles ) , t-2 toxin--glucoside 
 ( open circles ) , and t-2 toxin--glucoside ( solid circles ) to 
 neogen veratox t-2/ht-2 antibody .
data shown are the averages from 
 three plates per toxin , with four replicates per toxin concentration 
 per plate ( n = 12 ) ,  1 standard deviation . 

data for t-2 toxin with this immunoassay 
 are from maragos et al . the second immunoassay was a commercially 
 available t-2/ht-2 kit . 

this kit was used to determine if an elisa , developed before the glucosides 
 of t-2 and ht-2 toxins were even discovered , could detect these  masked  
 mycotoxins .
the commercial elisa test kit for t-2 toxin and ht-2 toxin 
 detection performed very well for detecting t-2 toxin with good sensitivity 
 and good reproducibility .
in addition , the kit cross - reacted fairly 
 well with the t-2 toxin--glucoside , but poorly with the t-2 
 toxin--glucoside ( table 1 ; figure 7b ) . however ,
given that t-2 toxin--glucoside 
 is the form found in naturally contaminated samples and that the cross - reaction 
 with this anomer was only about 16% , this test kit would not be recommended 
 for the simultaneous screening of t-2 toxin and t-2 toxin--glucoside .
the ability of plants to form trichothecene glucosides can be seen 
 as a detoxification mechanism that plants use when infected with trichothecene - producing 
 fungi . because trichothecenes can be virulence factors in plant disease , expression of trichothecene ugt genes in plants 
 is a potential way to increase disease resistance to trichothecene - producing 
 fungi .
however , both the trichothecenes and their masked 
 forms need to be considered to accurately assess the risk to human 
 and animal health posed by plant material contaminated with trichothecenes .
sufficient amounts of t-2 toxin - glucoside are needed to determine the risk posed by the masked form of t-2 toxin in cereals , 
 to develop reliable methods for their detection , and to study the 
 stability , digestive fate , and toxicity of t-2 toxin - glucoside formed 
 in plant materials . whereas the synthesis of deoxynivalenol - glucoside 
 is relatively straightforward , this method 
 was not amenable to the preparation of t-2 toxin - glucosides because 
 the isovaleryl and acetyl groups are labile during the synthesis . 
 a synthesis employing a superarmed tetra - o - triisopropylsilyl 
 protected donor offered a way to selectively 
 remove the protecting groups from the sugar portion of the glucoside 
 without disturbing the isovaleryl and acetyl groups of t-2 toxin , 
 thus providing a means to synthesize t-2 toxin--glucoside . 
 microbial t-2 toxin--glucoside was prepared as a single anomer 
 with b. muscicola yeast cultures , on the basis of lc and lc - ms chromatographic comparisons 
 and ms / ms analyses , the t-2 toxin - glucoside occurring in contaminated 
 wheat and oats was found to be identical to the yeast biotransformation 
 product , t-2 toxin--glucoside .
it is notable that naturally 
 occurring t-2 glucoside has an -linked 
 sugar , whereas naturally occurring deoxynivalenol glucoside has a 
 -linked sugar .
although -linked sugars are more commonly 
 found with xenobiotics and natural products including flavonoids , there is a report of fusarium cultures producing an -linked glycoside , 15-monoacetoxyscirpenol-4--glucoside .
the blastobotrys glucosyltransferase 
 that converts t-2 toxin to t-2 toxin--glucoside has not been 
 identified , but there may be substrate preferences between the ugt 
 that lead to trichothecene - or -glucosides . although 
 the focus of this study was the t-2 toxin - glucosides , 
 ht-2 toxin - glucosides have also been reported in contaminated grain and in culture material .
they may be formed either by hydrolysis of t-2 toxin--glucoside 
 or by conversion of t-2 toxin to ht-2 toxin followed by glycosylation 
 at c-3 or c-4 .
it is possible that the latter route may utilize different 
 glucosyltransferases that result in a  orientation . to date
, 
 no plant ugt that converts t-2 toxin to t-2 toxin--glucoside 
 has been identified .
numerous glucosyltransferases were screened to 
 identify one that had good substrate specificity for deoxynivalenol . although the t-2 toxin - glucoside found in naturally 
 contaminated oats and wheat was identical to the t-2 toxin--glucoside 
 prepared with yeast cultures , the anomericity 
 of ht-2 toxin - glucosides has not been determined .
the present 
 study has demonstrated that naturally occurring t-2 
 toxin - glucoside has an -linked sugar and that t-2 toxin--glucoside 
 can be prepared with b. muscicola in sufficient quantity 
 to study its animal toxicity . the u.s .
fda has not provided guidance 
 levels to the agricultural industry for t-2 toxin or related trichothecenes . 

the european food safety authority ( efsa ) established a group tolerable 
 daily intake of 100 ng / kg body weight for t-2 and ht-2 toxins .
however , at present it is not possible to perform 
 a proper risk assessment for masked mycotoxins in food , due to the 
 lack of data on exposure and toxic properties .
masked mycotoxins may 
 elude analysis because of altered physical properties or because of 
 modification of an epitope recognized by antibodies used for the detection , 
 leading to an underestimation of the total mycotoxin load .
possible 
 release during food processing is also a concern , and efsa currently 
 has a working group on the public health risk of masked mycotoxins 
 in food and animal feed .
research on masked mycotoxins is hampered 
 by the nonavailability of analytical standards or calibrants , and 
 only one compound , deoxynivalenol-3--glucoside , is commercially 
 available .
the occurrence of t-2 toxin--glucoside in 
 wheat and oat 
 samples , and its metabolism by microbes during digestion , suggests 
 that there is a risk in underestimation of the total t-2 toxin load 
 and a need to monitor both mycotoxins and their conjugated forms in 
 cereals and food products . to promote research efforts in the area 
 of masked mycotoxins ,
t-2 toxin--glucoside has now been made 
 available to the scientific community by the authors .
 t-2 
 toxin is a trichothecene mycotoxin produced when fusarium fungi infect grains , especially oats and 
 wheat . 
 ingestion of t-2 toxin contaminated grain can cause diarrhea , 
 hemorrhaging , and feed refusal in livestock . 
 cereal crops infected 
 with mycotoxin - producing fungi form toxin glycosides , sometimes called 
 masked mycotoxins , which are a potential food safety concern because 
 they are not detectable by standard approaches and may be converted 
 back to the parent toxin during digestion or food processing . 
 the 
 work reported here addresses four aspects of t-2 toxin - glucosides : 
 phytotoxicity , stability after ingestion , antibody detection , and 
 the anomericity of the naturally occurring t-2 toxin - glucoside found 
 in cereal plants . 
 t-2 toxin--glucoside was chemically synthesized 
 and compared to t-2 toxin--glucoside prepared with blastobotrys muscicola cultures and the t-2 toxin - glucoside 
 found in naturally contaminated oats and wheat . 
 the anomeric forms 
 were separated chromatographically and differ in both nmr and mass 
 spectrometry . 
 both anomers were significantly degraded to t-2 toxin 
 and ht-2 toxin under conditions that mimic human digestion , but with 
 different kinetics and metabolic end products . 
 the naturally occurring 
 t-2 toxin - glucoside from plants was found to be identical to t-2 toxin--glucoside 
 prepared with b. muscicola . 
 an antibody test for 
 the detection of t-2 toxin was not effective for the detection of 
 t-2 toxin--glucoside . 
 this anomer was produced in sufficient 
 quantity to assess its animal toxicity . 
Introduction
Materials and Methods
Results and Discussion