experimental material and reagents : sulphachloropyrazine - diaveridine 
 ( spz - dvd ) suspension , 20.0% spz with 4.0% dvd , was jointly developed by nanjing animal 
 husbandry pharmaceutical co. , ltd .
the standard spz of chemical pure was purchased from china institute of veterinary 
 drugs control ( beijing , china ) , and dvd of chemical pure was purchased from augsburg 
 ( frankfurt , germany ) . the acetonitrile , methanol , n - hexane and other chemicals ( potassium 
 dihydrogen phosphate , ortho - phosphoric acid 85% and dichloromethane ) of hplc grade were 
 purchased from poch chemical - company ( gliwice , poland ) at the highest purity available .
the 
 deionized water ( 18.2 m / cm ) used for preparing all the aqueous solutions was obtained by the 
 reverse osmosis method with milli - q - plus 185 system ( millipore , molsheim , france ) .
this work 
 was approved by the ethic committee of college of veterinary medicine , yangzhou 
 university .
apparatus : identification and quantification of analytes were carried out 
 using a liquid chromatography apparatus ( agilent 1200 , chicago , il , u.s.a . ) .
it consisted of 
 a solvent delivery pump ( star9002 ) , a manual injector and a variable wavelength uv - vis 9050 
 detector .
the samples and standards were analyzed using a kromasil c18 ( 250  4.6 mm ) column 
 ( kromasil ; eka chemicals , bohus , sweden ) .
an analytical balance ( sartorius bp 61s ) , vortex 
 ( wh-1 , bio - mix , warsaw , poland ) , a centrifuge ( tgl-16c , anting , shanghai , china ) , a solid 
 phase extraction ( spe ) ( waters mcx , new york , ny , u.s.a . ) and milli q plus 185 system 
 ( milipore - waters ) to produce deionized water were also employed .
animals : all experiments were carried out on 30 healthy broilers of 40 
 days ( d ) of age of both sexes .
broilers were fed with spz - dvd suspension mixed in water for 
 seven successive days , and then , they were divided into 5 experimental groups according to 5 
 different slaughter times ( 4 hr , 1 , 3 , 5 and 7 days , after withdraw time ) .
there were also a 
 group of broilers as a control , without feeding any drug . before commencing the study ,
the 
 broilers were marked with alphabetical code ( broilers1 , 2 , 3 , 4 and 5 ) which shows each 
 term of killing and taking of the samples .
the water was 
 changed two times a day ( at the morning , 1 hr before drug administration and at the 
 evening ) .
all of the broilers 
 ate and drank ad libitum throughout the period of this study .
control 
 groups were given pure water by the same method of administration for 7 days continuously . 

next , 6 broilers from a control group were humanitary slathered , and the marker 
 tissues  muscles ( 100200 g ) , liver ( all ) , kidneys ( all ) and skin with fat ( 1020 g ) were 
 taken for the recovery of spz and dvd .
broilers were adapted to normal temperature ( 25c ) , humidity ( 65% ) and sunlight .
the birds 
 were freely fed standard food and water in metabolic cages for 1 week before the experiment . 

the chickens were not given food for 12 hr before the experiment started , but water was 
 still given .
five chickens ( 3 females and 2 males ) were administered a single dose of 
 spz / dvd suspension in drinking water .
the control chickens were housed and fed in the same 
 way , but they were administered the same volume of 0.9% physiological saline .
the tissue samples were taken after 4 hr , 1 , 3 , 5 and 7 days of last drug application , in 
 broilers .
matrices were cleaned of blood and stored at 20c until the day of analysis by 
 hplc .
chromatographic conditions : the sulfa drug under investigation is 
 separated on a sep column by isocratic elution using a mobile phase that consisted of 
 acetonitrile - kh2po4 ( 13:87 ; v / v ) at a constant flow rate of 1.7 
 ml / min .
the kh2po4 was of ph 7.4 , 0.01 m. the 
 standards of sulphachloropyrazine and the tissue extracts were monitored at a wavelength of 
 270 nm . all analyses were performed at ambient temperature .
the retention time of 
 sulphachloropyrazine and dvd was about 10 min , similar to those in other studies .
extraction procedure : frozen tissue samples were thawed to room 
 temperature prior to extraction .
2.00 g of test minced tissue ( muscle , liver , kidney and 
 skin with fat ) was transferred into a test - tube and mixed with 15 ml of 
 acetonitrile .
after homogenization and centrifugation for 2 min at 15,000 rpm , the samples 
 were sonicated for 5 min and centrifuged at 5,000 rpm for 10 min .
the upper supernatant 
 layer was transferred into a clean tube , for mixed 30 sec in vortex with 15 
 ml of acetone and sonicated for 5 min .
after third centrifugation for 3 
 min , the upper layer was combined and mixed with 5 ml of n - propanol .
four ml of acetonitrile and 0.017 mol / l 
 kh2po4 ( 32/68 ) were added and next shaken for 5 min .
the same amount of n - hexane was 
 added to wash the liquid by vortexing for 2 min and centrifuging for 10 min at 4,000 rpm . 

the lower liquid layer of 2 ml was 
 added onto the mcx solid phase extraction column , which was balanced with 5 
 ml of methanol and 5 ml of hydrochloric acid in advance . 

after the liquid flow off , the column was washed with 2 ml of 0.1 
 mol / l hydrochloric acid , 2 ml of methanol and 10 
 ml of 10% ammoniation acetonitrile .
the residue was dissolved with 1 ml of acetonitrile and 0.017 
 mol / l kh2po4 ( 32/68 ) .
after filtered with 0.22 
 m filter membrane , the extraction of szp and dvd was injected into the 
 hplc system .
the extraction of szp and dvd from the broiler tissues was performed according 
 to fig .
standards : a stock solution ( 10 mg / ml ) of spz and dvd 
 sodium salt was prepared by dissolving 10 mg of compound in 100 ml of 
 methanol .
working solutions ( 100 , 50 , 10 , 4 , 1 and 0.04 mg / l for spz and 2 , 
 1 , 0.1 and 0.04 mg /
l for dvd ) were prepared by appropriate serial dilution 
 of the stock solution with a mixture : and 0.017 mol / l kh2po4 ( 32/68 ) .
accuracy / recovery : the accuracy of the method was determined , by the 
 recovery of drugs from the all control tissue samples of broilers ( muscle , liver , kidney and 
 skin with fat ) spiked at 0.04 , 1.0 and 10.0 g / g for spz and 0.05 , 1.0 and 
 2.0 g / g for dvd .
calibration , accuracy and recovery : linearity was studied from 
 matrix - matched calibration , spiking blank extracts at the five concentration levels .
table 1table 1.analytical performance of the methodanalytelinearity range ( mg / l)regression equationlod ( mg / l)loq ( mg / l)spz0.04100y=64.597  0.44290.99920.020.04dvd0.044y=28.185
 0.3640.99990.020.04 shows that the good linearity was obtained for the 2 analytes with the 
 correlation coefficient of > 0.998 .
the calibration graph was obtained by plotting peak 
 area against amount and was linear over the range 0.04100 mg /
the equation for the calibration curve is y=64.597  
 0.4429 , and the correlation coefficient equals 0.9992 for spz and y=28.185  0.3644 with 
 correlation coefficient of 0.9999 for dnd .
the linear range experiments provided the 
 necessary information to estimate the lod and the loq limits based on the peak of lowest 
 concentration in the linear range with a signal - to - noise ratio , s / n of 3.3 for limit of 
 detection and 10 for limit of quantification .
the limit of detection ( lod=3sxy / a , where sxy 
 is the standard deviation , and a is the slope of the calibration curve ) is the lowest 
 absolute concentration of analyte in a sample that can be detected but not necessarily 
 quantified .
the limit of quantification ( loq=10sxy / a ) is the lowest concentration of 
 analyte in a sample that can be determined with acceptable precision and accuracy .
. the estimated values of lod and loq were 0.02 mg / kg and 
 0.04 mg / kg for spz and 0.02 mg / kg and 0.04 mg / kg for dvd , respectively ( table 1 ) .
moreover , the accuracy of the procedure was obtained from relative standard deviation 
 ( rsd% ) of areas calculated for three replicate injections of three increasing spz ( 0.04 , 1.0 
 and
the results of the assays 
 presented in table 2table 2.determination of spz and dvd in different broiler edible tissues at three 
 concentration levelsdrugtissueadded 0.04 mg / ladded 1.0 mg / ladded 10.0 mg / lrecovery ( % ) rsd ( % ) recovery ( % ) rsd ( % ) recovery ( % ) rsd ( % ) spzmuscle82.446.9883.916.0187.131.95kidney78.054.5682.611.9385.840.76liver75.122.0478.371.3479.750.72skin with fat80.00 3.1881.520.8986.340.98added 0.05 mg / ladded 1 mg / ladded 2 mg / lrecovery ( % ) rsd ( % ) recovery ( % ) rsd ( % ) recovery ( % ) rsd ( % ) dvdmuscle76.05 3.71 93.02 6.91 94.03 2.36 kidney79.60 4.54 92.56 5.20 92.85 4.54 liver75.63 2.83 74.46 3.30 88.80 1.69 skin with fat80.20 5.44 93.16 3.18 95.64 3.01 a ) n=5 .
suggest that the precision of the method , expressed as rsd , ranged from 0.7 to 
 7.0% .
satisfactory results were obtained , and the recovery 
 values were depending on tested tissues .
the average recoveries were greater than 75.12 with 
 coefficients of variation ( cvs ) between 0.72% and 6.98% for spz and greater than 74.46% with 
 coefficients of variation ( cvs ) between 1.69 and 5.44% for dvd .
1.representative hplc chromatograms obtained from broiler edible tissue samples 
 ( muscle , kidney , liver and skin with fat ) spiked with 0.02 g / g of 
 spz and blank tissue samples . and 2fig .
2.representative hplc chromatograms obtained from broiler edible tissue samples 
 ( muscle , kidney , liver and skin with fat ) spiked with 0.02 g / g of 
 dvd and blank tissue samples .
show chromatograms obtained from broiler tissues 
 containing spz and dvd , respectively . in order to verify the selectivity of the method
no interference was detected in the 
 region of interest where the analyte was eluted as is shown in the blank sample 
 chromatograms .
the stability of spz and dvd was determined in two different ways : in solvent 
 ( working solutions ) and in tissue samples containing drugs .
the working solutions were 
 analyzed every week , and the instrumental responses were compared with peak areas obtained 
 on the day of solution preparation . no degradation phenomenon was observed during storage of 
 4 months at 4c .
representative hplc chromatograms obtained from broiler edible tissue samples 
 ( muscle , kidney , liver and skin with fat ) spiked with 0.02 g / g of 
 spz and blank tissue samples .
representative hplc chromatograms obtained from broiler edible tissue samples 
 ( muscle , kidney , liver and skin with fat ) spiked with 0.02 g / g of 
 dvd and blank tissue samples .
tissue residue depletion : residues of spz and dvd in tissue specimens 
 after oral administration of spz - dvd suspension ( 300 mg / l spz water , daily 
 for 7 consecutive days ) were determined .
the tissue concentration time profiles are 
 presented in table 3table 3.precision date on the analysis of spz and dvd standard concentrations in 
 different broiler edible tissues on five different daysdrugtissuemean concentration founded 
 ( g / g  sd)4 hr1 day3 day5 day7 dayspzmuscle0.58  0.380.10  0.050.04  0.02ndndkidney5.62  3.340.36  0.120.04  0.02ndndliver1.35  0.960.10  0.030.02  0.01ndndskin with fat0.95  0.540.34  0.110.15  0.050.07  0.02nddvdmuscle0.45  0.170.20  0.020.05 
0.030.02  0.01ndkidney3.15  0.511.25  0.330.57  0.240.16  0.02ndliver2.22  0.500.45  0.150.13  0.080.03  0.01ndskin with fat0.36  0.140.18  0.070.05  0.020.03  0.01nda ) n=5 , nd : not detectable . for kidney , liver , muscle and skin with fat .
mean tissue concentrations of spz 
 and dvd , ranging from 0.58 g / g to 5.62 g / g and from 0.36 
 g / g to 3.15 g / g , respectively , were measured 4 hr 
 after administration of the final dose of drugs ( table 
 3 ) . during the observation time , the spz concentrations depleted much slower from 
 the kidney and liver tissues than the muscle and skin with fat tissues .
spz was detected in 
 skin with fat ( 0.07g / g ) , but not in other three tissues , 5 days after 
 termination of drug treatment ; spz can not be detected 7 days after cessation of drug in any 
 tissue .
dvd depleted 
 much slower from the kidney and liver tissues than the muscle and skin with fat tissues . 

marker residue concentrations of dvd were below the mrl ( 0.05 g / g ) in 
 three tissue samples except in kidney , 5 days after the end of treatment .
dvd can not be 
 detected 7 days after cessation of drug in any tissue .
withdrawal time estimation : the mean spz and dvd concentrations were below 
 the loq at 7 days after cessation of medication in these four tissues ( table 3 ) .
the mean spz concentrations in muscle , kidney and liver 
 were below the loq at 5 days after cessation of medication and below the lod at 7 days after 
 dosing .
linear regression analysis of the logarithmic transformed data can be considered for 
 the calculation of the withdrawal periods by wt1.4 software . using this approach , the 
 withdrawal time was determined as the time when the one - sided 95% upper tolerance limit of 
 the regression line with 95% confidence level was below the mrl . generally , when the majority of data from one slaughter point are 
 below the lod or loq , the whole time point should be excluded .
3.plot of the withdrawal time calculation for spz and dvd in broiler kidney at the time 
 when the one - sided 95% upper tolerance limit is below the eu mrl for spz ( a ) and dvd 
 ( b ) ( 500 g kg-1 ) after oral administration of spz - dvd suspension 
 ( 300 mg / l spz water , daily for 7 days ) . illustrated a plot of withdrawal time calculation 
 for spz and dvd in kidney after oral administration ( 300 mg / l spz water , 
 daily for 7 consecutive days ) , respectively . using this approach and considering the marker 
 residue for the mrl , the withdrawal time for spz
could be calculated for muscle , liver , 
 kidney and skin with fat ( 300 mg / l spz water , daily for 7 consecutive 
 days ) : 3.26 , 3.72 , 4.39 and 5.43 days , respectively .
the withdraw time for dvd was 
 calculated for muscle , liver , kidney and skin with fat by using the same approach : 4.77 , 
 4.94 , 6.74 and 4.58 days , respectively .
therefore , by dosing spz - dvd suspension , a final 
 withdrawal time of 7 days was determined .
plot of the withdrawal time calculation for spz and dvd in broiler kidney at the time 
 when the one - sided 95% upper tolerance limit is below the eu mrl for spz ( a ) and dvd 
 ( b ) ( 500 g kg-1 ) after oral administration of spz - dvd suspension 
 ( 300 mg / l spz water , daily for 7 days ) .
diaveridine ( dvd ) is a popular antibacterial synergist that is widely used in combination 
 with spz .
it has been reported to be genotoxic to mammalian cells , but more studies are 
 required to clarify this [ 15 , 18 , 23 ] .
prudent use of highly 
 potent antimicrobials , such as dvd and spz , in veterinary medicine is strongly required to 
 maintain the efficacy and safety of drugs for the future .
therefore , in order to gain a 
 better understanding of the metabolism of dvd , we performed high - performance liquid 
 chromatography ( hplc ) .
a critical aspect of drug residue analysis is the sample extraction purification steps 
 required to isolate spz or dvd residues from broiler biological matrices .
these compounds are not very 
 soluble in non - polar solvents , but have good solubility in polar solvents .
traditionally , 
 the extraction of sulphonamide from meat , milk and eggs has been done with organic solvents , 
 such as : chloroform , methylene chloride , acetone , acetonitrile and ethyl acetate .
clean - up methods reported in the literature use many 
 different techniques : spe cartridges filled with different stationary phases , e.g. , silica 
 gel , cationic exchanger , c18 and clean - up procedure with ultracentrifuge [ 7 , 9 ] .
sample 
 cleanup procedures also include liquid  liquid purification to reduce or eliminate 
 interferences .
based on the extraction method for the determination of sulphonamides in 
 animal tissues , acetonitrile was selected to precipitate proteins , and hexane was employed 
 to extract out lipids [ 3 , 10 ] .
we tried another clean - up step i.e. ethanol , ethyl acetate , 5% 
 solution of trichloroacetic acid and 1 m solution of hydrochloric acid , but this method 
 permitted too many compounds that interfered with the chromatography of the analyte .
results 
 showed that deproteinization of the sample with acetonitrile followed by hexane washing of 
 the aqueous acetonitrile extract could achieve significant purification .
subsequent 
 extraction of spz was continued as a series of liquid  liquid partition clean - up procedures 
 with the aid of dichloromethane and phosphate buffer .
several spectroscopic techniques , such as uv 
 absorption , fld , dad   or ms , are used for 
 detection of different sulphonamides in lc .
uv detection is often carried out at 270280 nm , 
 in some cases at 255 nm and 254 nm .
based on the 
 literature , 270 nm was selected as a wavelength for the separation of scp in poultry tissue 
 samples .
after optimization of the mobile phase 
 parameters , the wavelength of detection and the flow rate , sulphachloropyrazine was detected 
 with satisfactory recovery .
sulphachloropyrazine peak showed a good shape and no 
 interferences with other peaks ( impurities ) , which indicates a high selectivity and 
 sensitivity of this method .
the recovery of spz and dvd was higher than 70% , and the rsd was 
 lower than 8% .
this makes it valuable and adequate in many applications , particularly in 
 veterinary medicine studies .
spz and dvd have a high potency and a broad spectrum of activity against a number of 
 bacterial pathogens including the primary bacterial pathogens involved with chicken 
 infections , such as e. coli , salmonella spp .
, 
 pasteurella multocida , pasteurella hemolytica , haemophilus paragallinarum , haemophilus 
 gallinarum and others . tissue depletion of spz and dvd after daily oral 
 administration of spz - dvd suspension ( 300 mg / l spz water for 7 consecutive 
 days ) was also determined .
the dosage regimen of 300 mg / l spz water for 7 
 consecutive days was used , because the results presented here suggest from the integration 
 of in vitro pharmacodynamics and in vivo pharmacokinetics 
 that the drug should be administered orally at 20 mg / kg spz every 12 hr ( or alternatively at 
 40 mg / kg every 24 hr ) , not only to guarantee clinical efficacy but also to minimize the 
 selection and spread of resistant pathogens .
the present work is the first to describe the 
 residue tissue depletion of spz - dvd in edible chicken tissues using a validated hplc method . 

spz and dvd concentrations in kidney , liver , muscle and skin with fat tissues were high 
 initially , especially in kidney and liver and decreased over time .
concentrations of spz in 
 tissues , except for skin with fat , were so much smaller than those detected for the parent 
 drug spz - dvd .
at total of 5 days after the last dose , the mean spz concentrations in all 
 tissues were below the mrl ( table 3 ) , and at 
 total of 7 days after the last dose , the spz concentrations were not detectable . in a 
 preliminary tissue distribution study of spz , the mean tissue concentration of spz also 
 declined to mean values below mrl at 5 days after oral administration of 20 mg / kg for 3 
 consecutive days [ 11 , 13 ] .
this indicates that spz is not removed from the body at a slower rate when 
 dosed at the higher level ( 40 mg / kg spz ) .
it can be assumed that there are no dose - dependent 
 differences in tissue drug distribution and elimination rate between those two doses .
the 
 study of dvd concentrations in broiler after 7 consecutive days also revealed a similar 
 result .
there is a strict legislative framework controlling the use of antimicrobial substances , 
 with the aim of minimizing the risk to human health associated with consumption of their 
 residues .
therefore , to ensure human food safety , the european union ( eu ) has set the 
 tolerance level for these compounds as the maximum residue limit ( mrl ) .
the mrl in all food 
 producing species including chicken was fixed for spz or dvd at 0.1g / g , 
 while 0.05 g / g in japan . according to the depletion regimen , a withdraw 
 time of spz and dvd
the withdraw times for spz in muscle , liver , kidney and skin with 
 fat were 3.26 , 3.72 , 4.39 and 5.43 days , respectively ; and 4.77 , 4.94 , 6.74 and 4.58 days , 
 respectively , for dvd .
based on the data , the suggested withdraw time for spz - dvd suspension 
 administration for 7 consecutive days was 7 days .
our study provides data for a more prudent use of spz and dvd in broiler , suggesting a 
 possible rational dosing and a withdrawal time after treatment to guarantee safety in foods 
 for the consumers .
 diaveridine ( dvd ) is used in combination with sulphachloropyrazine ( spz ) as an effective 
 antibacterial agent and antiprotozoal agent , respectively , in humans and animals . to gain 
 
 a better understanding of the metabolism of spz and dvd in the food - producing animals , a 
 high performance liquid chromatography ( hplc ) method to determine and quantify 
 sulphachloropyrazine ( spz ) and diaveridine ( dvd ) suspension residues from broilers is 
 reported . 
 thirty healthy chickens were orally administered with 
 sulphachloropyrazine - diaveridine ( spz - dvd ) suspension in water of 300 
 mg / l ( spz ) per day for seven successive days . 
 six chickens per day were 
 slaughtered at 0 , 1 , 3 , 5 and 7 days after the last administration . 
 this procedure 
 permitted spz and dvd to be separated from muscle tissue , liver , kidneys and skin with fat 
 after extraction with acetonitrile and acetone under slightly acidic conditions . from the 
 detected residuals in different tissues 
 , we found that spz was quickly eliminated in liver 
 and muscle , and slowly eliminated in kidney and skin with fat . 
 dvd was quickly eliminated 
 in liver and slowly eliminated in kidney . 
 the withdrawal period of spz was 3.26 , 3.72 , 
 4.39 and 5.43 days in muscle , liver , kidney and skin with fat , respectively . 
 the 
 withdrawal period of dvd was 4.77 , 4.94 , 6.74 and 4.58 days in muscle , liver , kidney and 
 skin with fat , respectively . 
 therefore , the suggested withdrawal period for spz - dvd 
 suspension should be 7 days after dosing for seven successive days . 
MATERIALS AND METHODS
RESULTS
DISCUSSION