Studies On Pharmacokinetics Of Amprolium Hydrochloride, Ethopabate, Sulfaquinoxaline Soluble Powder In Chicken

Amprolium hydrochloride, Ethopabate and Sulfaquinoxaline premix is a newbroad-spectrum anticoccidial compound preparation, and now is used as aconventional additive in chicken feed. The anticoccidial mechanism of Amproliumhydrochloride, Ethopabate and Sulfaquinoxaline premix is preventing essentialnutrients of coccidium from uptaking and utilizing. Amprolian hydrochloride,Ethopabate and Sulfaquinoxalinum soluble powder is a new formulation based onpremix. Compared to premix, it has the same pharmacological activity and it is moreconvenient and effective. At the present, the preparation is researched as the third typeof new veterinary drug, so it is not shown in the Chinese drug market yet. In this study,Amprolium hydrochloride, Ethopabate and Sulfaquinoxalinum soluble powder isstudied as tested preparation, while Amprolium hydrochloride, Ethopabate andSulfaquinoxaline premix is used as the referential preparation. With the LC-MS-MSand the internal standard method, we investigated the pharmacokinetics of the solublepowder in chicken and bioequivalence studies of the premix.MS conditions of detection method: ESI ion source, positive ion scan, ion sprayvoltage:-5.5V; TEM:550℃; the pressure of GAS1and GAS2:15psi and30psi; thepressure of collision CAD:6psi; the pressure of CUR:30psi; EP:10V; CXP:10; thequantitative ion: m/z→243.2/150.1（Amprolium hydrochloride）, m/z→238.2/206.1（Ethopabate）, m/z→301.3/155.8（Sulfaquinoxalinum）, m/z→166/137.8（p-aminobenzoic acid ethyl ester）. The chromatographic conditions: ZORBAX SB-C18（2.1×150nm,3.5μm）HPLC column, mobile phase: methanol-0.1%formic acid inwater, gradient elution with0min （10:90）,8min （90:10）,12（90:10）,12.01（10:90）,17min （10:90）, flow rate：0.2μL.min^-1, the column temperature was room temperature,the injection volume was10μL. The crossover experimental design has been used in this study.206-weekbroilers were randomly divided into T, R groups （n=10） to respective oral testedpreparation and referential preparation of the same dose （125mg/kg） in the twocycles. This medication was given to each subject two times with the interval at leastten half-life. Respectively collected blood0.17,0.25,0.5,1,1.5,2,3,4,6,8,12,24,36and48h after the drug administrated, put them into the anticoagulant tube andcentrifuged at the rate of3000rpm for10minutes, saved the plasma into-80℃. Theplasma samples were extracted with acetonitrile. Using the mixed standard method,the APL, EPA and SQ respectively were rendered in2²50ng/mL,0.2¹0ng/mL,2¹000ng/mL concentration within a good linear relationship. The LOD were2ng/mL,0.2ng/mL and0.2ng/mL; the LOQ were6.6ng/mL,0.66ng/mL and0.66ng/mL; therecoveries of different concentrations were90.12⁹3.16%,86.2%⁹1.09%and88.48⁹2.21%respectively. The Intra-day precision and Inter-day variable coefficientwere controlled under15%. The APL, EPA and SQ at room temperature, long-termfreezing and thawing conditions showed that this method was stable and reached thebiological sample analysis requirements.Pharmacokinetics experimental results showed that: the C_maxof the APL in testpreparation and referential preparation were respectively110.34±4.54μg/mL and108.71±4.48μg/mL; the T_maxof two preparation were both0.75h, the AUC_0-twasrespectively643.24±29.94ng/mL·h and649.54±30.48ng/mL·h; the AUC0-∞wasrespectively638.24±32.76ng/mL·h and675.53±30.33ng/mL·h; the C_maxof the EPA intest preparation and referential preparation were respectively17.06±2.53μg/mL and16.57±4.72μg/mL; the T_maxwere respectively2h, the AUC were respectively24.9±2.39ng/mL·h and24.72±6.703ng/mL·h; the AUC0-∞were respectively25.13±1.99ng/mL·h and25.24±8.64ng/mL·h; the C_max of the SQ in test preparation andreferential preparation were respectively1746.57±76.72μg/mL and1648.07±180.12μg/mL; the T_max were respectively4h; the AUC_0-t were respectively25710.72±1251.70ng/mL·h and23832.68±3415.77ng/mL·h; the AUC0-∞wererespectively25839.24±1247.64ng/mL·h and23939.89±3415.24ng/mL·h.The results of analysis of variance of the main pharmacokinetic parametersshowed that the C_max, T_max, AUC_0-tand AUC0-∞of the APL, EPA and SQ between test preparation and referential preparation had no significant difference （P>0.05）; theAUC_0-tof APL, EPA and SQ in the two preparations were analyzed with a statisticanalysis of two one-side t-test, confidence interval of [1-2É‘] was respectivelyduring95.7%⁹9.7%、93.9%¹00.4%and101.7%¹15.5%; the AUC0-∞was analyzed with astatistic analysis of two one-side t-test, confidence interval of [1-2É‘] was respectivelyduring96%¹00%、93.2%¹00.4%and102.5%¹15.7%，the C_maxwas analyzed witha statistic analysis of two one-side t-test, the confidence zone of [1-2É‘] wasrespectively during99.2%¹03.9%、97.2%¹03.2%and100.9%¹11.7%. Theseresults were during the judgement standard range of bioequivalence. The results ofthe statistical analysis showed that the two compound formulations are bioequivalence.This experiment provides scientific guidance for clinical rational drug use ofAmprolium hydrochloride, Ethopabate and Sulfaquinoxalinum soluble powder testedpreparation, also these results provides reference for acceleration of the formulationand research and development for the similar preparation.