chemicals and reagents : the analytical standards of the mycotoxins , afb1 , 
 afb2 , afg1 , afg2 , t-2 toxin , don , niv , zea and ota , were purchased from 
 sigma - aldrich ( st . louis , mo , u.s.a . ) . de - ionized distilled water was produced using the milli - q purification 
 system from millipore , inc . hplc grade acetronitrile and acetic acid were obtained from 
 burdick & jackson ( ulsan , korea ) , while ammonium acetate was purchased from ajax finechem pty ltd . ( taren 
 point , nsw , australia ) . stock and working standard preparation : combinations of standard mycotoxin stocks were 
 prepared in methanol at a concentration of 1,000 ng / ml for the high 
 sensitivity group ( afb1 , afb2 , afg1 , ota and t-2 ) and for the low sensitivity 
 group ( afg2 , don , zea and niv).the working standard solutions were prepared by diluting this stock of 
 standard mixture with methanol at different concentration ranges as follows : 1.0250 
 ng / ml for the high sensitivity group and 2.0500 
 ng / ml for the low sensitivity group . sampling and sample preparation : in total , 100 broiler feed samples ( 10 feed samples per each 
 province ) were randomly collected from broiler farms in central thailand from bangkok , pathum thani , nakhon 
 nayok , nakhon pathom , ayutthaya , saraburi , lopburi , supanburi , chainart , uthai thani and nakhonsawan provinces . 
 in this study , the major ingredients 
 in broiler feed samples include corn , fish meal and soybean . the feed samples were blended , and then , a 10 g ground feed sample was homogenized with 40 ml 
 of the organic extraction solvent mixture ( acetronitrile : water : acetic acid , 79:20:1 v / v ) by shaking for 60 min . the crude extract was centrifuged at 1,509 g for 10 min as described by soleimany et 
 al . . then , 0.5 ml of the supernatant was 
 diluted in 4.5 ml of acetronitrile : water : acetic acid , 20:79:1 v / v . the extract was filtered by 
 passing through a 0.22 m syringe filter and then injected into the unit for lc - ms / ms [ 17 , 18 ] . lc - ms / ms equipment and parameters : lc analysis was performed using an agilent 1260 infinity 
 ( agilent technologies , waldbronn , germany ) consisting of a binary pump , a degasser , a column oven and an auto 
 sampler . the chromatographic separation was performed on a zorbax eclipse plus rrht c18 column ( 50  2.1 mm , 1.8 
 m particle size ) . the mobile phase consisted of 5 mm 
 ammonium acetate with 0.2% acetic acid in water ( mobile phase a ) and 0.2% acetic acid in acetonitrile ( mobile 
 phase b ) . the gradient program of the mobile phase was as follows : 10% b ( initial for 1 min ) , 1095% b ( 5 min ) 
 and 95% b ( 10 min ) . the mobile phase solution was 
 filtered through a 0.22 m membrane and ultrasonically degassed prior to application . the flow 
 rate was 0.5 ml / min , while the injection volume was 10 l . the mass spectrometer used was a triple quadrupole mass spectrometer ( 6460 triple , agilent technologies ) 
 equipped with an electrospray ionization source run in both positive and negative ion modes under the multiple 
 reaction monitoring mode ( mrm ) . the ionization source parameters were optimized as follows : capillary voltage , 
 3,500 v ; gas temperature , 320c ; gas flow rate , 8 l / min ; and nebulizer , 50 psi . the parameters 
 ( for the mass spectrometer ) were optimized as shown in table 
 1table 1.ms/ms parameters for the determination of nine mycotoxinsmycotoxinsprecusor ion ( m / z)product ions ( m / z)collision energy ( ev)retention time ( min)fragmentor ( ev)ionization modeafb1313.07285.1215.13150esi313.07241.035150afb2315.09287.1254.99160esi315.09259.029160afg1329.07311.0214.98150esi329.07243.025150afg2331.08313.0214.85170esi331.08245.029170t-2489.40387.3205.6170esi489.40245.226170ota404.00192.9485.78130esi404.00102.180130zea319.16283.055.9480esi319.16187.01780don355.10265.143.590esi355.1059.11090niv371.10281.042.180esi371.1059.11080afb1 , aflatoxin b1 ; afb2 , aflatoxin b2 ; afg1 , 
 aflatoxin g1 ; afg2 , aflatoxin g2 ; ota , ochratoxin a ; zea , zearalenone ; 
 don , deoxynivalenol ; niv , nivalenol ; esi , electrospray ionization in positive mode ; 
 esi , electrospray ionization in negative mode .. afb1 , aflatoxin b1 ; afb2 , aflatoxin b2 ; afg1 , 
 aflatoxin g1 ; afg2 , aflatoxin g2 ; ota , ochratoxin a ; zea , zearalenone ; 
 don , deoxynivalenol ; niv , nivalenol ; esi , electrospray ionization in positive mode ; 
 esi , electrospray ionization in negative mode . method validation : validation of the lc - ms / ms method was performed to assess the efficiency of 
 this analytical method by investigating the selectivity , sensitivity , accuracy and precision in accordance with 
 the european commission regulation for the performance of analytical methods . linearity : the linearity of an analytical procedure is its ability ( within a given range ) to 
 obtain test results that are directly proportional to the concentration ( amount ) of analyte in the sample . 
 linear regression analysis was conducted for the mycotoxin standard mixtures of the afb1 , 
 afb2 , afg1 , afg2 , ota , t-2 , zea , don and niv toxins under the optimized 
 lc - ms / ms conditions . eight - point calibration curves were plotted as the peak area ratio ( y ) of each mycotoxin 
 against its concentration ( y = mx  c ) at the concentration ranges of 1.0250 
 ng / ml for afb1 , afb2 , afg1 , t-2 and ota ; 
 and 2500 ng / ml for afg2 , don , zea and niv . the linearity was 
 assessed using the correlation coefficient ( r ) ( table 2table 2.linearity , limit of detection ( lod ) and limit of quantification ( loq ) of the optimized lc - ms / ms 
 method for simultaneous determination of mycotoxinsmycotoxinslod in feed sample ( ng / g)loq in feed sample ( ng / g)linear range ( ng / g)rafb10.391.01.02500.9993afb20.481.01.02500.9989afg10.201.01.02500.9995afg20.782.02.05000.9986t-20.391.01.02500.9963ota0.391.01.02500.9991zea0.782.02.05000.9970don0.782.02.05000.9989niv0.782.02.05000.9999r , correlation coefficient . ) . limit of detection and limit of quantification : the limit of detection ( lod ) corresponds to 
 the concentration that will give a signal - to - noise ( s / n ) ratio of 3:1 . the limit of quantification ( loq ) is 
 defined as the concentration of related substance in the sample that will give an s / n ratio of 10:1 ( table 2 ) . recovery and precision : recovery and precision ( repeatability , expressed as relative standard 
 deviation ( rsd ) in% ) were determined within - day by analyzing seven replicates containing nine mycotoxins at 
 three different qc levels ( table 3table 3.accuracy and precision for mycotoxin determination in optimal lc - ms / ms conditions for spiked broiler 
 feed samplesmycotoxinsspiking level ( ng / g)recovery ( % ) intra - day precision ( % rsd)inter - day precision ( % rsd)afb12965.466.3710983.375.43501013.423.89afb22976.387.9810954.434.6450982.213.17afg12923.613.7710954.864.22501032.703.45afg210912.592.9650967.287.67200885.767.11t-22926.416.1110875.535.7950953.153.48ota2895.134.3510854.643.8750863.793.82zea10933.634.0550945.024.69200921.781.98don10825.464.8650843.373.51200853.422.89niv10822.513.1550811.872.03200842.271.89 ) . the inter - day precisions were determined by analyzing qc samples on five different days ( one 
 batch per day ) ( table 3 ) . optimization of lc - ms / ms conditions : for the multi - mycotoxin method , it was decided to perform 
 simultaneous determination within a single run with detection in positive and negative modes . in both ionization 
 modes , the best sensitivity for all investigated compounds was achieved using 5 mm ammonium acetate in 0.2% 
 acetic acid ( mobile phase a ) and 0.2% acetic acid in acetronitrile ( mobile phase b ) as the mobile phase . in 
 addition , nearly all compounds could be separated , except afg1 and afb2 , which eluted at 
 the same retention time ( fig . 1.lc-ms/ms extracted ion chromatogram of the spiked broiler feed sample for mycotoxins under optimized 
 conditions of aflatoxin b1 , aflatoxin b2 , aflatoxin g1 , aflatoxin 
 g2 , t-2 toxin , ochratoxin a , zearalenone , deoxynivalenol and nivalenol . upper chromatogram for each toxin 
 represents the quantifier transition ion , whereas lower chromatogram represents the qualifier transition 
 ion . ) . the differentiation of afg1 and afb2 , the [ m+h ] mass numbers of which 
 are 329.07 and 315.09 , respectively , could be achieved by the difference of characteristic parent ions in the 
 multiple reaction monitor ( mrm ) mode . furthermore , when considering that the selected product ions were 329.07 
 > 311 , 329.07 > 243 and 315.09 > 287.1 , 315.09 > 259 for afg1 and afb2 , 
 respectively , the selectivity of daughter ions could not disturb each other . lc - ms / ms extracted ion chromatogram of the spiked broiler feed sample for mycotoxins under optimized 
 conditions of aflatoxin b1 , aflatoxin b2 , aflatoxin g1 , aflatoxin 
 g2 , t-2 toxin , ochratoxin a , zearalenone , deoxynivalenol and nivalenol . upper chromatogram for each toxin 
 represents the quantifier transition ion , whereas lower chromatogram represents the qualifier transition 
 ion . lc - ms / ms validation method : the linearity and sensitivity results are reported in table 2 . the method exhibited good linearity over the relevant working 
 range , with r values between 0.9963 ( for t-2 ) and 0.9999 ( for niv ) . the lod and loq 
 values in matrices ranged from 0.20 to 0.78 ng / g and from 0.78 to 1.56 ng / g , 
 respectively ( table 2 ) . the recoveries were obtained within the 
 range of 81103% for all nine mycotoxins , as summarized in table 
 3 . occurrence of the mycotoxins in broiler feeds : the 100 broiler feed samples were quantified 
 for the nine mycotoxins studied by simultaneous determination using lc - ms / ms . in summary , 93% of the samples 
 contained afb1 at concentrations ranging from 0.47 to 8.52 ng / g . the second most 
 prevalent mycotoxin was zea found in 63% of the samples at concentrations ranging from 2.22 to 263.51 the other mycotoxins found to be contaminating feed samples were afb2 , don , 
 afg1 , niv and t-2 in 20% , 9% , 7% , 5% and 1% , respectively , of samples . in the present study , 
 afg2 and ota were not detectable in broiler feed samples ( table 4table 4.occurrence of mycotoxins in broiler feeds ( total of 100 samples)mycotoxinspercentage of positive samplesrange of concentration level of contaminations 
 ( ng / g)mean of concentration ( ng / g)afb1930.478.522.02afb2200.793.301.87afg1 70.661.891.30afg2ndndndt-2 11.151.15otandndndzea63 2.22263.5184.27don 933.5860.8145.05niv 512.7535.8327.26mean of concentration ( ng / g ) is the mean of the positive samples . the co - occurrence of mycotoxins was also detected in broiler feeds , with afb1 and zea 
 in 46 of 100 feed samples , afb1 and don in 9 of 100 feed samples , afb1 and niv in 4 of 100 
 feed samples , afb1 , afb2 and zea in 13 of the 100 feed samples and aflatoxins 
 ( afb1 , afb2 and afg1 ) in 4 of the 100 feed samples . mean of concentration ( ng / g ) is the mean of the positive samples . the results illustrated that afb1 and zea were the most common of the contaminants in broiler feed . 
 on the other hand , detection ratios of don and niv , which are fusarium toxins , are very low due 
 to the compositions of such feed samples . accordingly , processing and other ingredients , such as corn , soybean 
 and fish meal , can also play a role regarding afb1 and zea contaminations in broiler feed samples . the levels of mycotoxin contamination in the samples were below the regulatory limits , which corresponded to the 
 u.s . these results suggest that risk of the exposure to 
 mycotoxins in completed feeds obtained from broiler farms in central thailand is very low . on the other hand , 
 low concentrations of mycotoxin contaminants , such as aflatoxins , trichothecenes and zearalenone , moreover , the 
 co - contamination of mycotoxins usually occurs in food or feeds , which can increase the potency of toxicity to 
 the animals . poor management of farm animals , especially with regard to farm feed storage and farm feeding , may 
 have a major influence on the level of mycotoxin contamination in broiler feeds and relate to increased 
 mycotoxin production . the result of the present study corresponded 
 well with the study of streit et al . , since they 
 also surveyed the contamination of mycotoxins in finished feed samples , with 2,743 samples analyzed over a 
 period of 8 years ( 20042011 ) . they found that fumonisins , don , zea , af and ota were the most prevalent in 
 finished feed samples in south - east asia . khatoon et al .   also reported that the contamination of niv and don was found with relatively low level in broiler 
 feeds in pakistan . however , an uhplc - ms / ms - based method for the determination of 191 mycotoxins and other fungal 
 metabolites was developed . they reported that uhplc - ms / ms allowed 
 better separation of the analytes from the matrix . regarding the method validation , the recovery levels were 
 slightly better than those reported by ren et al . the intra- and 
 inter - day precision values were in the range of 1.87.3% and 1.97.7% , respectively . all the recovery and 
 precision results were in agreement with performance criteria recommended by european commission regulation no . an analytical method was developed for the simultaneous determination of nine mycotoxins in broiler 
 feeds by using lc - ms / ms . the analytical method was 
 demonstrated to be an excellent tool for unambiguous identification of mycotoxins using the retention time , 
 accurate mass of precursor and product ions . furthermore , this is the first study to present data on multi - toxin 
 occurrence in completed feeds obtained from broiler farms in thailand using lc - esi - ms / ms . the results of 
 mycotoxin contamination in broiler feeds showed that aflatoxins are the most common present mycotoxin ( mainly 
 afb1 ) and can co - occur with other mycotoxins ( afb1 and zea ) . this evidence highlighted 
 that the production of mycotoxins differs according to the climatic conditions especially as thailand is located 
 in the tropical zone and the climatic conditions which are suitable for mycotoxin production . nevertheless , the 
 levels of mycotoxin contamination in the feeds were below the u.s . these results also suggest that the risk level of exposure to mycotoxins in completed feeds obtained from 
 brolier farms in central thailand is very low due to the low level contamination .
mycotoxins are secondary fungal metabolites that are typically present in grain and feed ingredients used for 
 animal feeds . 
 an analytical method using lc - esi - ms / ms was developed to quantify nine mycotoxins , consisting of 
 aflatoxin b1 ( afb1 ) , afb2 , afg1 , afg2 , t-2 toxin , 
 deoxynivalenol ( don ) , nivalenol ( niv ) , zearalenone ( zea ) and ochratoxin a ( ota ) in broiler feeds . in total , 
 100 samples of broiler feeds were collected from poultry farms in central thailand . 
 the survey found that 
 afb1 and zea were the most prevalent mycotoxins in the feed samples at percentages of 93% and 
 63% , respectively . 
 the limit of detections ( lods ) of investigated mycotoxins was 0.200.78 
 ng / g . 
 afb2 , don , afg1 , niv and t-2 toxin were also detectable at low 
 contamination levels with percentages of 20% , 9% , 7% , 5% and 1% , respectively , whereas ota and afg2 
 were not detected in any of the feed samples . 
 these results suggest that there is a very low level of risk of 
 the exposure to mycotoxins in feeds obtained from broiler farms in central thailand .