the demand for food of animal origin increases each year . to satisfy this demand , livestock production within the european union ( eu ) in 2011 was approximately 10 million heads of goats , 80 million heads of sheep , 80 million heads of cattle , and 150 million heads of pigs . to produce safe and nutritional food products , the animals need to be in good health .
even though veterinary medicines contribute to improving and maintaining animal health , administration of these medicines by the farmer is carried out under licence via a veterinarian .
the amount of drugs employed in food production estimated by kools et al . was 6051  t , with antibiotics the most frequently used class of drug ( 5393  t ) .
the groups of tetracyclines and -lactams were used in high amounts and antiparasitic agents ( 194  t ) were the second most frequently used class of drug .
intensively produced animals are often fed with concentrated feed , a mixture of various materials ( oats , wheat , barley , rye , cottonseed , and crambe ) and additives .
antibiotic sulphonamides , tetracyclines , and -lactam are the most frequently used antibiotics , and coccidiostats and ivermectin are the most frequently used antiparasitic agents .
coccidiostats and histomonostats are a group of antiparasitic agents that have been shown to be persistent during the manufacture of feed and carry - over of this type of drugs has been demonstrated .
the eu introduced maximum levels for these substances in nontarget feed in 2009 ; this regulation was later modified for some coccidiostats by the regulation ec/574/2011 .
cross contamination between medicated and nonmedicated feed could occur with any type of drug added to the feed , not only to coccidiostats , particularly when the cleaning process between batches is inefficient .
recently , a study conducted by stolker et al . in the netherlands confirmed that nonmedicated feed batches were contaminated with antibiotic residues such as tetracyclines , penicillins , and sulfonamides . from 140 samples
the fact that antibiotics could be present as contaminants in feed without the farmers ' knowledge implies that withdrawal times will not be considered and antibiotic residues could remain in animal products ( meat , eggs , milk , honey , seafood , and fish ) , in addition to the development of antibiotic resistant bacteria . due to the problems related to food safety , the authorities involved regularly monitor the presence of veterinary drugs in food of animal origin ( eggs , milk , muscle , and liver ) .
controls on the water and food consumed by the animals have also been implemented , but the analysis conducted only evaluated the presence of substances such as pesticides [ 7 , 8 ] , nitrofurans , and mycotoxins [ 10 , 11 ] .
methods based on hplc - ms / ms detection are considered confirmatory methods because these types of methods provide full or complementary information , enabling substances to be unequivocally identified and if necessary quantified at the level of interest , according to the european commission decision 2002/657/ec .
therefore , it is recommended to use confirmatory methods to detect the presence of antibiotics in nontarget feed .
van poucke et al . reported a method for the analysis of zinc bacitracin , spiramycin , tylosin , and virginiamycin with quantification limits below 500  g / kg .
published a multiclass method for the analysis of 33 analytes for 14 groups of antibiotics ( including tetracyclines , quinolones , penicillins , ionophore coccidiostats , macrolides , and sulphonamides ) with quantification limits of 3.865.0  g / kg .
of the eight forms of commercially available tetracyclines , four are frequently used in food animal production ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) .
maximum residue limits ( mrl ) for these four tetracyclines and their three epimers have been introduced for various foods of animal origin , including eggs , muscle , and milk . as maximum levels ( ml ) for these substances in feed samples
concentrations of tetracyclines in medicated feed are variable and depend on the target animal , with dosage rates between 25 and 700  mg / kg .
therefore , carry - over should be expected in the batch of feed manufactured after a medicated feed .
reported that 100% of samples from the first batch of feed produced after the manufacture of medicated feed were contaminated with tetracyclines , with concentrations of 0.5154  mg / kg .
based on these results , carry - over contamination is also expected for the other two commonly used tetracyclines ( chlortetracycline and tetracycline ) as they have similar chemical properties [ 16 , 17 ] .
based on the common use of tetracyclines in food animal production and the absence of confirmatory methods for the presence of the four tetracyclines in animal feed , the aim of this work is to present an hplc - ms / ms method for the analysis of tetracyclines in nonmedicated feed samples at levels of g / kg .
disodium hydrogen phosphate dehydrate , anhydrous citric acid , ethylenediaminetetraacetic acid disodium salt ( edta ) , trichloroacetic acid ( tca ) , and formic acid ( purity > 99% by analysis ) were purchased from sigma - aldrich ( mo , usa ) .
tetracycline , chlortetracycline , doxycycline , and oxytetracycline ( purity > 98% ) and demeclocycline , used as the internal standard ( is ) , were supplied by sigma - aldrich ( mo , usa ) .
organic solvents , methanol and ethyl acetate , hplc or analytical grade , were purchased from scharlau chemie ( barcelona , spain ) and demineralised water ( resistivity 18 mu cm ) was prepared in - house with a milli - q water system ( millipore , bedford , ma , usa ) . mobile phase a consisted of milli - q water acidified to 0.04% with formic acid and mobile phase b consisted of methanol , acidified to 0.1% with formic acid . to prepare the individual stock solution of tetracycline ,
20  mg of tetracycline was dissolved in 20  ml of methanol and stored at 20c for up to six months .
the intermediate solution , a mixture of tetracyclines , was prepared by diluting the stock solution of each tetracycline to a final concentration of 50  g / ml and stored at 20c for up to one month .
a standard working solution of tetracycline was prepared freshly each day by diluting the intermediate stock solution to a final concentration of 1  g / ml . for the internal standard stock solution ,
mcilvaine buffer was prepared with 10.8  g of citric acid , 10.93  g of disodium hydrogen phosphate , and 33.62  g of ethylenediaminetetraacetic acid disodium salt dihydrate ( c10h14n2na2o8 ) .
once the three reagents were completely dissolved they were mixed and the volume was made up to 1  l and the ph adjusted to ph 4 .
hplc - ms / ms determination of tetracyclines was performed according to a previously reported method .
the hplc - ms / ms consisted of an hplc alliance 2795 and a ms quattro premier xe triple quadrupole ( waters , manchester , uk ) controlled by the software masslynx 4.1 ( waters , manchester , uk ) .
the chromatographic analyses were performed by injecting 25  l of extract into a sunfire c18 column ( 150  2.1  mm i.d .
, 5.0  mm ) ( waters , manchester , uk ) . mobile phases a and b were mixed on a gradient mode and with a flow rate of 0.25  ml / min .
an electrospray ionisation ( esi ) probe was set up on the triple quadrupole ms to evaporate the mobile phase coming from the hplc and to ionise the tetracyclines .
analytes were detected in positive - ion mode and under the following conditions : capillary voltage 3  kv , source temperature 120c , desolvation temperature 350c , cone gas flow 49  l  h , and desolvation gas flow 650  l  h. as indicated in the decision 2002/657/ec , tetracyclines were identified on the basis of their selected reaction monitoring ( srm ) transition and their retention time ( rt ) .
different volumes of the tetracycline working standard solution were added to 2  g feed samples ( exempt of tetracycline ) and shaken in the dark for 30  min .
concentrations of tetracycline in the matrix matched feed samples were 0 , 400 , 800 , 1200 , 1600 , and 1600  g / kg .
to extract the tetracyclines from the feed samples , 2  g of grounded feed , 8  ml of mcilvaine buffer , 300  l of tca , and 0,1  ml of is working solution were added to a 50  ml polypropylene tube . after shaking the sample in the dark for 10  min ,
6  ml of ethyl acetate was added and the samples were shaken in an orbital shaker at 200  rpm for 20  min .
after 15 of centrifugation at 4500  rpm , in a model 5415d centrifuge ( eppendorf , hamburg , germany ) , 2  ml of the supernatant was transferred to a 10  ml amber conical tube and evaporated to dryness , in a turboevaporator model turbo vap ii de zyrmark ( hopkinton , ma , usa ) .
the final residue was dissolved in 0.5  ml of a mixture of mobile phase components ( 90a:10b ) and vortexed . to filtrate the final extract ultrafree - mc centrifugal filter ( millipore , ma , us ) was employed and centrifuged at 9000  rpm for 10  min .
the filtrate was transferred into an hplc vial which contained a 0.3  ml microinsert and stored at 20c ; analyses were conducted within 3 days .
the guidelines used to validate the method and to interpret the results were those established in the commission decision 2002/657/ec .
the decision establishes criteria and procedures for the validation of analytical methods to ensure the quality and comparability of analytical results generated by official laboratories .
aspects such us trueness / recovery , precision ( under repeatability and reproducibility conditions ) , specificity , and applicability / ruggedness / stability of the method were investigated .
trueness and the other validation parameters were assessed through recovery of additions of known amounts of tetracyclines in blank feed samples ( except tetracyclines ) as no certified reference material exists for this type of analysis and following the recommendation included in the decision 2002/657/ec . for validation one batch of matrix - matched samples
was prepared with 21 samples fortified with tetracyclines at six concentrations ( 0 , 400 , 800 , 1200 , 1600 , and 4000  g / kg ) .
for concentrations 400 , 800 , and 1200  g / kg six replicated samples were prepared with only one sample for the remaining concentrations ( 0 , 1600 , and 4000  g / kg ) .
additionally , two reagent samples were prepared for control , a blank reagent ( containing only the reagents ) and fortified reagent ( containing 1200  g / kg of tetracycline and the reagents ) . to conduct the validation three batches of matrix - matched samples were prepared ; each batch consisted of 21 samples fortified with tetracyclines at 0 , 400 , 800 , 1200 , 1600 , and 4000  g / kg .
while for levels of 400 , 800 , and 1200  g / kg six replicated samples were prepared , only one sample was used for levels of 0 , 1600 , and 4000  g / kg .
after fortification , and prior to extraction , samples were shaken on an orbital shaker at 200  rpm for 10  min .
after the samples extraction procedure explained above was applied . as well as the 21 matrix - matched samples , with each batch of samples two additional  samples
 were prepared with reagents ( no feed ) ; one was spiked with tetracycline at 1200  g / kg ( fortified reagents ) and the other was not spiked with tetracyclines ( blank reagent ) . additionally , 20 feed samples were analysed to determine selectivity / specificity .
ten were spiked with tetracyclines at a validation level ( 800  g / kg ) and with 400  g / kg of three antimicrobial drugs commonly used in food animal production ( sulfadiazine , sulfamethoxazol , and trimethoprim ) .
the other ten samples were analysed without adding any veterinary drugs ( decision 2002/657/ec ) .
the decision limit ( cc ) and detection capability ( cc ) were determined as described by freitas et al .
, following the decision 2002/657/ec requirements and applying the validation level of 800  g / kg .
nonmedicated feed samples were collected from 50 milk farms located in galicia , spain , to investigate the presence of tetracycline residues in feed that are being consumed by cows that are producing milk daily .
additionally , samples were supplied by feed manufacturers to investigate carry - over levels of tetracyclines after making medicated feed .
the sampling procedures in both cases were conducted following the requirement of the regulation 691/2013 .
feed samples were stored at room temperature and in the dark with the objective of using similar store conditions compared to that in the industry and farms .
the main raw materials of the feed samples according to the labels were corn genetically modified ( between 36 and 15% ) , soy flour produced from soya been genetically modified ( present in some samples , between 4 and 38% ) , colza flour ( present in some samples , between 3 and 47% ) , and barley ( present in some samples , between 6 and 18% ) ; each feed sample had a particular composition which normally depends on the manufacture .
on the other proximate composition the feed samples were crude protein ( between 18 and 26% ) , crude fibre ( between 4 and 10% ) , oil and fat crude ( between 2.5 and 6% ) , crude ash ( between 6 and 10% ) , and sodium ( between 0.7 and 4% ) .
tetracyclines have more than three hydroxyl groups that are easily ionisable by esi to enhance their detection .
therefore , esi is commonly used for the ionisation of tetracyclines when analysis is conducted by hplc - ms / ms , independently of the matrix type [ 14 , 2024 ] . to optimise the ms parameters for a high ms signal response , standard solutions of 1 
g / ml of individual tetracyclines were infused directly into the ms . during this procedure ,
one precursor ion and two product ions were selected for each tetracycline to conduct srm analysis ( table 1 ) . with the hplc - ms / ms operating on srm mode , two transitions and the retention time were used to achieve four identification points for each of the analytes , as required in the decision 2002/657/ec .
it should be highlighted that with other detection methods , such as diode array or a fluorescence detector , only one identification point is achieved   and more steps are required .
the use of precursor and product ions identified in this research for tetracyclines was also reported by boscher et al .
2010 for the analysis of these analytes in feed , royal jelly , and muscle [ 14 , 20 ] .
the separation gradient could be run at ambient temperature ; however , it was observed that an increase in mobile pressure can cause the system to collapse ; therefore a temperature of 35c is recommended .
based on previously reported methods for tetracycline analysis in food and feed matrices [ 14 , 2527 ] different extraction protocols were tested .
firstly , simple extractions with ethyl acetate , hexane , acetonitrile , methanol , and dichloromethane were tested .
however , recoveries were low , due to the tendency of tetracyclines to form chelation complexes with different cations .
therefore , initial extraction with mcilvaine / edta buffer was employed and gave satisfactory results , as in previously reported methods [ 7 , 21 , 28 , 29 ] .
tetracyclines dissolved in the mcilvaine / edta solution were then extracted with ethyl acetate , as these two solutions are immiscible , and separation could be easily conducted as the organic phase stays on the top layer .
other authors employ spe cartridges such as oasis   for dispersive spe   instead of ethyl acetate in order to purify the extract .
the use of spe was avoided to reduce time and cost of the analysis as satisfactory results were achieved with the presented method .
animal feed is derived from a multitude of raw materials from plant and animal origin , as well as pharmaceutical and industrial sources .
as feed ingredients vary depending upon the animal , that is , poultry , swine , and cattle , analysis of tetracyclines in different feed types could be more complicated , particularly as the fat content will vary .
the method used in this paper has been tested in feed for cattle , laying birds and chickens , rabbits , and dairy cows , and satisfactory results were obtained in all cases .
these matrices were tested by preparing matrix - matched calibration curves with each type of feed , depending on the animal that was going to consume the feed .
calibration curves to quantify concentrations of tetracyclines were obtained by spiking feed samples with the analytes at different concentrations . if linear regression coefficients ( r ) were below 0.98 the extraction procedure was repeated . even if a signal to noise ratio ( s / n ) higher than 10 was achieved at 300  g / kg , validation was conducted at 800  g / kg to provide acceptable results at 0.5 , 1 , and 1.5 times the validation level ( 800  g / kg ) recommended by the decision 2002/657/ec .  
figure 1 shows chromatograms of a blank sample , figure 2 shows a blank sample spiked with all the tetracyclines at 400  g / kg , and figure 3 shows the srm transition employed for each tetracycline in one of the samples spiked at 400  g / kg .
reference materials were not available ; therefore trueness of the method was calculated in terms of recoveries .
however , the advantage of the presented extraction protocol is that it does not require solid phase extraction and the four main tetracyclines can be identified and quantified simultaneously .
results for repeatability , calculated as the mean rds of the rsd ( n = 6 ) for each concentration on each day of the validation , were below 17% for chlortetracycline , doxycycline , oxytetracycline , and tetracycline ( table 2 ) .
results for reproducibility , calculated as the rds of 21 samples at the same concentration , were below 23% for all tetracyclines
( table 2 ) . to determine selectivity / specificity , 10 blank samples and the same samples spiked with the four tetracyclines at 800  g / kg were analysed .
the successful quantification of tetracyclines and the absence of interfering peaks at the retention times of each analyte demonstrated the selectivity / specificity of the method .
the limit of detection ( lod ) and limit of quantification ( loq ) of the method were calculated and verified with feed samples spiked with the tetracyclines at different concentrations .
based on s / n above 3 for lod and above 10 for loq in matrix - matched samples , the lod and loq of the method were set at 35 and 47  g / kg for chlortetracycline , 40 and 60  g / kg for oxytetracycline , 24 and 40  g / kg for tetracycline , and 100 and 150  g / kg for doxycycline .
cc and cc were determined using the conditions for substances for which no permitted limit has been established .
cc and cc were higher than lod and loq for all the compounds , meaning that tetracyclines detected at a higher level than the cc will be positive and levels of tetracyclines were quantifiable , without doubt .
cc and cc for chlortetracycline , doxycycline , oxytetracycline , and tetracycline were below 400  g / kg ( table 2 ) .
validation results already published have shown , in some cases , higher repeatability , reproducibility , and lower loq , such as the work conducted by boscher et al . who reported rsd lower than 12% and loq of 20  g / kg .
similarly , the method reported by stolker et al . achieved a loq of 0.1  g / kg for doxycycline and oxytetracycline .
however , it should be highlighted that none of the methods reported , based on the authors knowledge , have been validated according to the decision 2002/657/ec . from 75 feed samples investigated
oxytetracycline was the tetracycline more frequently detected present in 8% of the samples ( n = 6 ) ; its concentration range was between 90 and 400  mg / kg .
each tetracycline was detected in individual samples and their concentrations were 150 and 110  mg / kg .
chlortetracycline was the pharmaceutical detected at the highest concentration in this study , 15.14  mg / kg in feed samples for calves .
it is important to highlight that the consumption of contaminated feed at level such as 15.14  mg / kg could cause food safety problems giving positive food samples .
carry - over during feed manufacture has been proved for veterinary drugs such as coccidiostats and a similar case can be considered for tetracyclines , a group of antimicrobial agents commonly used in food animal production , due to its low cost .
the research work presents a simple and fast method for the analysis of the four tetracyclines regulated in the production of food of animal origin ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) .
the method was validated according to the european guideline and successfully applied to 75 nonmedicated feed samples .
results showed the presence of tetracyclines in 15% of the samples , indicating that cross contamination occurs and maximum levels for tetracyclines may be required in the future .
 the commission regulation 574/2011/ec set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry - over during manufacturing . 
 carry - over takes place from medicated to nonmedicated feed during feed production . 
 similar contamination could also occur for other pharmaceuticals such as tetracyclines , a group of antibiotics commonly employed in food production animal . 
 the objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) in nontarget feed at a g / kg level . 
 validation of the method was performed according to the guideline included in the commission decision 2002/657/ec for official method . 
 the validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers . 
 while oxytetracycline was the tetracycline most frequently detected , chlortetracycline was the analyte measured at the highest concentration 15.14  mg / kg . from 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline . 