standards and chemicals : enrofloxacin ( er ) ( baytril 100 , bayer 
 ag , leverkuser , germany ) for injection was used . enrofloxacin and ciprofloxacin standards 
 were purchased from sigma chemical co. ( st . louis , mo , u.s.a . ) . other reagents and chemicals 
 of analytical grade were purchased from sigma chemical co. purified water was produced using 
 the milli - q water purification system from millipore , inc . animals : ten female adult thai swamp buffaloes between 5 and 10 years of 
 age with an average body weight of 500 kg were selected . all ten buffaloes were housed 
 together at the animal unit , faculty of natural resources and agro - industry , chalermphrakiat 
 sakon nakhon province campus , kasetsart university , sakon nakhon province , thailand . all 
 experimental procedures were performed according to the guidelines for animal experiments 
 and approved by the animal ethics research committee of the faculty of veterinary medicine , 
 kasetsart university . prior to the study , each buffalo was assessed to be healthy based on a 
 review of health history , physical examination , complete blood count and serum chemistry 
 analysis . experimental design : to obtain the pharmacokinetic data , the ten thai 
 swamp buffaloes were randomly divided into two groups ( n=5 per group ) . each group was 
 administered er intravenously ( i.v . ) or subcutaneously ( s.c . ) ( baytril , 100 
 mg / ml ) at a dose of 5.0 or 7.5 mg / kg bw . the experiments were performed 
 in a two - phase , two - period , cross - over design with an interval of 3 weeks between the 
 periods and the phases . ten ml of blood was collected from the jugular vein 
 of each animal with heparinized syringes at 0 , 15 and 30 min and at 1 , 2 , 4 , 6 , 8 , 12 , 24 , 
 32 and 48 hr after administration . the plasma was separated by centrifugation ( 1,986  
 g ) for 15 min . the urine and fecal samples were collected from 0 to 48 hr 
 by natural collection and rectal palpation . all of the plasma , urine and fecal samples were 
 frozen at 20c until analysis . sample extraction : the extraction method of er and cp in plasma , urine and 
 feces was performed as described previously [ 19 , 
 26 , 36 ] , 
 after revalidation . 300 l of acetonitrile ( acn ) was added to 200 l plasma in 
 a tube and then shaken for 15 sec . a 150 l sample of the supernatant was transferred to a 
 new tube and mixed with 300 l of water . after passing it through a 
 ministart rc filter ( pore size 0.45 m , sartorius ag , 
 gttingen , germany ) , an aliquot of 200 l of this mixture was injected for 
 high performance liquid chromatography ( hplc ) . one hundred l of urine was diluted with 900 l of 
 distilled water in a microcentrifuge tube . the solution was vortex - mixed and filtered using 
 a ministart rc filter ( pore size 0.45 m , sartorius ag ) ; then , 
 the sample ( 50 l ) was injected onto hplc . fecal samples ( 100 mg ) were homogenized with 400 l of distilled water in 
 a motor driven glass homogenizer for 23 min . to each sample , 2 ml of 
 extraction solution was added , and the samples were mixed . samples were stored at room 
 temperature , in the dark , for 45 min before 1.5 ml of 0.07 m 
 ortho - phosphoric acid were added . samples were then vortexed and allowed 
 to stand for a further 10 min before being centrifuged at 5,800  g at 4c 
 for 10 min . the solution was filtered with a ministart rc filter ( pore size 0.45 
 m , sartorius ag ) ; then , the sample ( 50 l ) was injected 
 onto hplc . hplc conditions : the analytical method was performed in accordance with 
 otero et al . . the hplc analysis 
 was performed using an agilent 1260 series system consisting of a binary pump , an automatic 
 sample injector , a column thermostat and a fluorescence detector ( agilent technologies , 
 waldbronn , germany ) with excitation and emission wavelength of 294 and 500 nm , respectively . separation was achieved using a plrp - s column ( 5 m , 4.6  150 mm ) with an 
 rp18-e guard column ( 5 m , 4  40 mm ) ( polymer laboratories inc . , church 
 stretton , u.k . ) . the column was maintained at a temperature of 35c using a column oven . the 
 mobile phase consisted of 14% acn and 86% 0.07 m ortho - phosphoric acid . the retention times of er and cp were 7.93 and 
 5.45 min , respectively . the limit of quantification ( loq ) of er and cp in plasma was 0.1 and 
 0.05 g / ml , respectively . fortification and method validation : the calibration curves were obtained 
 by spiking the working standard solution into buffalo blank plasma , urine and feces to yield 
 final concentrations of 0.1 , 0.5 , 1 , 5 and 10 g / ml of er 
 and cp . buffalo blank plasma , urine and feces ( for quality control samples ) were obtained 
 some days before the treatment protocol commenced . linearity of the regression curve in the 
 range 0.110 g / ml was assessed on the basis of the 
 residual plot , the lack of fit test and the back calculation ( within 20% of known amount ) . five duplicates of the quality control samples at concentrations of 0.5 , 5 and 10 
 g / ml were prepared and used to determine the 
 recoveries , intra- and inter - day precision , and accuracy of the method . the procedure was 
 repeated five times within the same day to gain intra - day run precision and accuracy and 
 five times for each concentration over five consecutive days to obtain inter - day run 
 precision and accuracy . the extraction recoveries of er from plasma were 85.19  2.52 , 87.12 
  1.95 and 86.24  3.11% for 0.5 , 5 and 10 g / ml , 
 respectively . the extraction recoveries were 84.22  4.52 , 83.26  2.9 and 83.24  3.33% for 
 urine , and 82.63  3.32 , 81.64  3.68 and 82.45  4.31% for feces , respectively . the intra- 
 and inter - day precision values in plasma , urine and feces were lower than 7% and accuracy 
 was more than 95% , respectively . the variation coefficient ( r ) 
 value of the er and cp calibration curves was > 0.9997 . pharmacokinetic calculation and statistical analysis : the concentrations 
 of er and cp in thai swamp buffaloes versus time were pharmacokinetically analyzed by 
 applying a two - compartment and non - compartmental model , respectively , using the pk solution 
 2.0 program ( summit research services , montrose , co , u.s.a . ) . the absolute 
 subcutaneous bioavailability ( f ) was calculated using the following 
 equation:(% ) 
 f(s.c.)=(aucs.c . ) / ( auci.v . )  
 100 the statistical analyses were evaluated using the student - ttest . all the analyses were conducted using graphpad instat ( graphpad 
 software , inc . , la jolla , ca , u.s.a . ) . in all the experiments , differences were considered 
 significant , if the associated probability level ( p ) was lower than 
 0.05 . the determination of the er and cp concentrations showed that er and cp were detectable in 
 the plasma of buffaloes following single i.v . and s.c . administrations . the semi - logarithmic 
 plots of the mean plasma concentration - time curves of er and cp at doses of 5.0 and 7.5 
 mg / kg bw are shown in figs . 1.mean values (  sd ) of enrofloxacin ( er ) (  ) and ciprofloxacin ( cp ) (  ) concentrations 
 in plasma of thai swamp buffaloes after intravenous ( a ) and subcutaneous ( b ) 
 administrations at a dose of 5.0 mgkg bw ( n=5 ) . 2.mean values (  sd ) of enrofloxacin ( er ) (  ) and ciprofloxacin ( cp ) (  ) concentrations 
 in plasma of thai swamp buffaloes after intravenous ( a ) and subcutaneous ( b ) 
 administrations at a dose of 7.5 mgkg bw ( n=5 ) .. er was measurable from 5 min , while cp was detectable from 30 min after s.c . 
 administration . however , the drug er and its metabolite cp were below the limit of detection 
 of the method at 32 hr after i.v . and s.c . administrations , respectively , at doses of 5.0 
 and 7.5 mg / kg bw . the main pharmacokinetic 
 parameters are reported in table 
 1table 1.mean  sd values of the pharmacokinetic parameters of enrofloxacin following 
 intravenous and subcutaneous administrations at doses of 5.0 and 7.5 mg / kg bw in thai 
 swamp buffaloes ( n=5 per group)pharmacokinetic parameter ( unit)intravenoussubcutaneous5.0 mg / kg7.5 mg / kg5.0 mg / kg7.5 mg / kgcp 
 ( g / ml)16.32  3.1623.56  5.23 - -kel ( hr)0.1  0.0080.08  0.0050.20  0.02 * 0.14  0.01*t1/2(hr)3.35  0.666.12  0.862.21  0.19 * 4.31  0.61*t1/2 ( hr)7.05  1.328.64  1.153.43  1.12 * 4.91  0.91*cl 
 ( l / hr / kg)0.28 0.040.21  0.130.63  0.11 * 0.28  0.04*vd(l / kg)2.82  0.482.67  0.333.12  1.161.97  0.32*mrt ( hr)15.0  2.6321.3  3.128.70  1.19 * 13.3  2.65*auc024 
 ( g*hr / ml)16.93  3.6633.27  4.638.12  1.16 * 24.37  3.28*auc0- 
 ( g*hr / ml)18.1  3.2935.0  5.339.9  2.23 * 27.0  4.23*cmax 
 ( g / ml)--0.66  0.152.26  0.61tmax ( hr)--6.0  0.006.0  0.00f ( % ) --52.36  4.2472.26  5.39cp : plasma concentration at initial time ; 
 kel : elimination rate constant of the beta phase ; 
 t1/2 : distribution half - life ; 
 t1/2 : elimination half - life ; cl : 
 clearance ; auc0- : area under the curve from time 0 to 
 infinity ; auc0 - 24 : area under the curve from time 0 to 24 
 hr ; vd : volume of distribution ; mrt : mean 
 residence time ; cmax : maximum concentration ; 
 tmax : time at maximum concentration . administration , the auc values increased with 
 the dose , but not in a dose proportional fashion . in the s.c . treatment groups , 
 cmax and auc values also increased , but not 
 in a dose proportional fashion . interestingly , kel and 
 t1/2 showed more rapid elimination of er in the s.c . the absolute bioavailabilities of er were 52.36  
 4.24% and 72.26  5.39% after s.c . administration at doses of 5.0 and 7.5 mg / kg bw , 
 respectively ( table 
 1 ) . mean values (  sd ) of enrofloxacin ( er ) (  ) and ciprofloxacin ( cp ) (  ) concentrations 
 in plasma of thai swamp buffaloes after intravenous ( a ) and subcutaneous ( b ) 
 administrations at a dose of 5.0 mgkg bw ( n=5 ) . mean values (  sd ) of enrofloxacin ( er ) (  ) and ciprofloxacin ( cp ) (  ) concentrations 
 in plasma of thai swamp buffaloes after intravenous ( a ) and subcutaneous ( b ) 
 administrations at a dose of 7.5 mgkg bw ( n=5 ) . cp : plasma concentration at initial time ; 
 kel : elimination rate constant of the beta phase ; 
 t1/2 : distribution half - life ; 
 t1/2 : elimination half - life ; cl : 
 clearance ; auc0- : area under the curve from time 0 to 
 infinity ; auc0 - 24 : area under the curve from time 0 to 24 
 hr ; vd : volume of distribution ; mrt : mean 
 residence time ; cmax : maximum concentration ; 
 tmax : time at maximum concentration . the plasma concentrations of cp were lower than the respective concentrations of parental 
 drug ( fig . the curves were analyzed according 
 to a non - compartmental method ( table 
 2table 2.mean  sd values of the pharmacokinetic parameters of ciprofloxacin following 
 intravenous and subcutaneous administrations of enrofloxacin at doses of 5.0 and 7.5 
 mg / kg bw in thai swamp buffaloes ( n=5 per group)pharmacokinetic parameters ( unit)intravenoussubcutaneous5.0 mg / kg7.5 mg / kg5.0 mg / kg7.5 mg / kgz ( hr)0.11  0.0250.11  0.0200.075  0.013 * 0.11  0.01t1/2z ( hr)6.44  1.886.66  1.359.43  1.69 * 6.72  1.28mrt ( hr)9.63  2.238.88  1.1516.44  1.88 * 9.24  1.04auc024 
 ( g*hr / ml)4.13  0.405.97  1.682.25  0.28 * 6.59  1.29auc0- 
 ( g*hr / ml)4.50  0.396.42  1.762.85  0.34 * 7.11  1.22cmax 
 ( g / ml)--0.15  0.0222.29  0.57tmax ( hr)0.14  0.0890.10  0.006.0  2.0 * 0.11 0.00z : elimination constant ; t1/2z : 
 elimination half life ; cl : clearance ; 
 auc0- : area under the curve from time 0 to infinity ; 
 auc0 - 24 : area under the curve from time 0 to 24 hr ; 
 mrt : mean residence time ; cmax : maximum 
 concentration ; tmax : time at maximum concentration . the average cp / er aucs ratio was 24.0  2.1% and 18.3  
 3.1% for the i.v . treatments ( 5.0 and 7.5 mg / kg , respectively ) and 28.3  3.97% and 26.6  
 4.41% for s.c . treatments ( 5.0 and 7.5 mg / kg , respectively ) . z : elimination constant ; t1/2z : 
 elimination half life ; cl : clearance ; 
 auc0- : area under the curve from time 0 to infinity ; 
 auc0 - 24 : area under the curve from time 0 to 24 hr ; 
 mrt : mean residence time ; cmax : maximum 
 concentration ; tmax : time at maximum concentration . administrations at doses of 5.0 and 
 7.5 mg / kg bw in urine and feces , respectively , are shown in tables 3table 3.mean  sd values of enrofloxacin ( er ) and ciprofloxacin ( cp ) in urine following 
 intravenous and subcutaneous administrations at doses of 5.0 and 7.5 mg / kg bw in thai 
 swamp buffaloes ( n=5 per group)time ( hr)5.0 mg / kg7.5 mg / kgintravenoussubcutaneousintravenoussubcutaneouser ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)042.34  1.1816.92  4.930.50  0.31 * 4.92  3.83 * 5.14  2.9421.89  7.490.35  0.18 * 5.36  2.13 * 4120.42  0.3118.09  3.570.70  0.2115.99 4.700.54  0.3119.19  1.480.56  0.2915.61  4.91 * 12240.12  0.025.33  3.860.36  0.233.90  1.510.16  0.023.04  1.320.31  0.157.28  2.66**p<0.05 ( statistically significant value if compared to the 
 corresponding i.v . group ) . and 4table 4.mean  sd values of enrofloxacin ( er ) and ciprofloxacin ( cp ) in feces following 
 intravenous and subcutaneous administrations at doses of 5.0 and 7.5 mg / kg bw in thai 
 swamp buffaloes ( n=5 per group)time ( hr)5.0 mg / kg7.5 mg / kgintravenoussubcutaneousintravenoussubcutaneouser ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)er ( g / ml)cp ( g / ml)04nfnf0.03  0.003 * 0.05  0.04 * 0.24  0.05ndnfnf4123.63  1.123.46  0.720.57  0.33 * 0.20  0.17 * 2.06  0.130.51  0.450.33  0.06 * 0.14  0.0612240.10  0.330.32  0.030.62  0.14 * 0.28  0.080.24  0.100.20  0.080.86  0.53 * 0.53  0.21*nf : no fecal collection ; nd : not detected . group ) .. both er and cp were detectable in urine and feces up to 24 hr . generally , er 
 and cp had higher concentrations in urine than in feces . our study was designed to characterize the disposition of er following i.v . and s.c . administrations at doses of 5.0 and 7.5 mg / kg bw in thai swamp buffaloes . this animal is 
 important for the rural economy of thailand , but is often affected by bacterial infections 
 that can reduce its food - production ability 
 ( http://www.dld.go.th/th/index.php/2010-02-16-08-16 ) . for this reason , it is important to have a clear 
 pharmacokinetic profile of this drug in each target species to formulate a sound therapeutic 
 treatment plan . this is 
 slightly lower than the value reported for donkeys ( 9.54 hr ) , but higher than those reported in buffalo calves ( 2.92 hr ) , sheep ( 3.25 hr ) , goats ( 2.73 hr ) , lactating dairy 
 cows ( 3.69 hr )   and beef steers ( 5.15 hr ) . compared to other ruminant species , the plasma 
 clearance in thai swamp buffaloes was lower than those reported in buffalo calves ( 1.94 
 l / hr / kg ) , sheep ( 0.53 
 l / hr / kg ) , goats ( 0.70 
 l / hr / kg ) , lactating dairy 
 cows ( 1.45 l / hr / kg )   and beef 
 steers ( 0.7 l / hr / kg ) . data from 
 the present study indicated a slower rate of elimination of er in thai swamp buffaloes 
 compared to the aforementioned ruminant species . parameters related to elimination ( such as 
 kel and t1/2 ) showed rapid 
 elimination of s.c . it might be due to the saturated 
 elimination of er at highest concentration of drug . in fish species , er has been reported to 
 inhibit cytochrome p450 . on the contrary , 
 cl values ( 5.0 vs. 7.5 mg / kg i.v . treatments ) appeared to be unchanged , 
 despite a large variability has been found in the 7.5 mg / kg group . however , further studies 
 ( with a larger animal sample size ) are warranted to support this hypothesis . on the other 
 hand , the vd of er found in the present study was large after 
 both i.v . and s.c . administrations . these data are in agreement with the results from 
 previous investigations in other ruminant species [ 14 , 18 , 20 , 25 , 32 ] , which indicated that er was able to penetrate well into various tissues . surprisingly , the auc values increased irrespective of the dose ( 2 and 3 
 times after i.v . and s.c . this is in disagreement with other 
 animal species where er showed dose - dependent pharmacokinetic parameters . the reason for this phenomenon is not clear , and a 
 study with larger animal sample size should be used to verify this datum . administration , er given at a dose of 7.5 mg / kg was absorbed more 
 efficiently than when given at a dose of 5.0 mg / kg as indicated by its higher 
 bioavailability ( 72.26% ) . some studies involving er injected via extravascular injective 
 routes , reported that local irritation and soft tissue necrosis can occur in the injection 
 area . although this possibility was not checked 
 at the time of experiment , the higher dose of the drug might have evoked a stronger 
 inflammation response , increasing the portion of drug absorbed by dilated blood vessels . it 
 might be a concern in clinical practice , however , some recent studies reported in turtles 
 have shown that an intramuscular injection of a diluted concentration of er ( 10 
 mg / ml ) [ 10 , 34 ] did not produce irritative effects as the more concentrate drug 
 marketed solution ( 25 mg / ml ) for pets [ 6 , 31 ] . however , the nonlinear 
 auc values increase on increasing dose might suggest elimination 
 saturation and consequently a non - linear pharmacokinetic . under this condition , the 
 f% data should have a negligible value and should be used with caution . cp is the main active metabolite of er , possessing a similar spectrum of antibacterial 
 activity . its concentration has been reported as always being lower than that of parental 
 molecule in mammals , birds and reptiles . cp is known to account for about 2535% of the 
 amount of the parental drug in mammals . these 
 earlier data are in line with the results of the cp / er auc ratio reported 
 in the present study ( range 18.328.3% ) . er and cp were detectable in urine and feces up to 24 hr after i.v . and s.c . administrations , in the present study . however , the concentration of cp in urine was 
 significantly higher than that detected in feces . this indicated that the urine was a major 
 route of excretion of er metabolite . this is in line with a previous study on the er 
 excretion in pigs . it has been reported that er can kill bacteria in a concentration - dependent manner [ 1 , 9 ] . the minimum 
 inhibitory concentration ( mic ) values of er for most veterinary pathogens 
 range from 0.001 to 0.1 g / ml ; however , bovine pathogens are usually susceptible to er at 
 mic  0.06 g / ml 
 ( baytril-100 package insert ) . in addition , er was proven to be one of the most 
 active antimicrobial agents against pasteurella multicida with an 
 mic of 90% of the isolates ( mic90 ) of 0.05 
 g / ml . this 
 organism causes a disease called hemorrhagic septicemia ( hs ) or bovine respiratory disease 
 ( brd ) , which is considered to be a livestock disease of major economic importance in 
 southeast asia . the maximum serum 
 concentration / minimum inhibitory concentration ( cmax / mic ) ratio 
 and the area under the serum concentration - time curve 
 ( auc024/mic ) ratio are considered as the major 
 pharmacokinetic - pharmacodynamic ( pk - pd ) indices for antimicrobial activity and optimal dose 
 determination . the criteria of cmax / mic ratios of 810 or 
 auc / mic ratios > 100125 have been suggested for maximum therapeutic 
 efficacy and clinical effectiveness [ 15 , 22 , 23 , 38 ] . using an mic breakpoint of 0.06 
 g / ml , the values of the 
 cmax / mic ratio and the auc024/mic 
 ratio obtained from s.c . administration were 11.0 and 135.3 hr , respectively , whereas they 
 were 37.67 and 406.2 hr at a dose of 5.0 and 7.5 mg / kg bw , respectively . administration of er at a dose of 5.0 
 and 7.5 mg / kg bw might provide effective plasma concentration for the treatment of 
 susceptible bacterial diseases ( mic  0.06 
 g / ml ) in thai swamp buffaloes . however , the 
 cmax / mic ( 6.6 ) and auc024/mic 
 ( 81.2 hr ) values were below the recommended ratios for an mic value of 0.1 
 g / ml after s.c . it is noteworthy that in this theoretical calculation , the efficacy due to cp is not 
 included . cp is reported to have similar efficacy to its parental drug , and an increase of 
 around 20% ( equivalent to the fraction of metabolite formed ) should be expected in the 
 in vivo activity of the drug . regarding our study , the drug plasma level 
 was maintained at a higher level than the mic ( 0.06 
 g / ml ) for 12 and 24 hr after er s.c . the side effects of er , such as vomiting , anorexia , diarrhea 
 and nervous signs , were not observed after s.c . the plasma concentrations of er and its metabolite cp were measured in thai 
 swamp buffaloes , b. bubalis , after s.c . the metabolism of er to cp was identified also in this species , as occurs in 
 other ruminants . er and cp were excreted via the urine . based on the pharmacokinetic data 
 derived from our study and pk - pd indices , s.c . administration of er at a dose of 5.0 and 7.5 
 mg / kg bw would be appropriate for the treatment of susceptible bacterial diseases in thai 
 swamp buffaloes . however , the dose rate of 7.5 mg / kg is recommended to treat infectious 
 diseases caused by other veterinary pathogens having mic  0.1 
 g / ml .
given the limited information available in this species , the aim of this study was to 
 investigate the pharmacokinetic characteristics of enrofloxacin ( er ) and its major 
 metabolite ciprofloxacin ( cp ) in buffaloes , bubalus bubalis . 
 er was 
 administered intravenously ( i.v . ) or subcutaneously ( s.c . ) to buffaloes at doses of 5.0 
 and 7.5 mg / kg bw , and plasma , urine and fecal samples were collected until 48 hr 
 post - administration . 
 the concentrations of er and cp in the plasma , urine and feces were 
 analyzed using high - performance liquid chromatography equipped with a fluorescence 
 detector . 
 the plasma concentrations of er and cp could be determined up to 24 hr and 
 32 hr after i.v . and s.c . 
 administrations at doses of 5.0 and 7.5 mg / kg bw , 
 respectively . 
 cp concentrations were always lower than those of parental drug . 
 the s.c . 
 bioavailability of er was 52.36  4.24% and 72.12  5.39% at doses of 5.0 and 7.5 mg / kg 
 bw , respectively . both er and cp were detectable in urine and feces up to 24 hr . 
 er and cp 
 were mainly excreted via the urine . 
 based on the pharmacokinetic data and pk - pd indices , 
 s.c . 
 administration of er at doses of 5.0 and 7.5 mg / kg bw might be appropriate for the 
 treatment of susceptible bacterial diseases in thai swamp buffaloes .