| In this paper, a determination method of sarafloxacin hydrochloride in Litopenaeus vannamei tissues(haemolymph, hepatopancreas and muscle) by reverse-phase high performance liquid chromatography(RP-HPLC) was established. Moreover, protein binding rate of sarafloxacin hydrochloride in haemolymph, pharmacokinetics of sarafloxacin hydrochloride in Litopenaeus vannamei following an intra-sinus injection and oral administration, and the residue and elimination regularity by multiple-dose oral administration were also analyzed. The main results are as follows:1. Development of RP-HPLC for determination of sarafloxacin hydrochloride in haemolymph, hepatopancreas and muscle from Litopenaeus vannameiThe method of RP-HPLC was established to determine sarafloxacin hydrochloride in haemolymph, hepatopancreas and muscle from’Litopenaeus vannamei. The drug in tissues was extracted by acidified acetonitrile, and the tissues were degreased by n-hexane. RP-HPLC was performed by Waters2695with chromatographic column KromasiL Eternity5-C18(250×4.6mm) at40℃, the mobile phase was acetonitrile-trifluoroacetic acid(0.1%)(25:75, by vol) at a flow rate of1.2ml/min, the sample temperature was10℃. The excitation and emission wavelengths of the fluorescence detector were set at280nm and450nm, respectively. The results showed that the average recoveries of sarafloxacin hydrochloride in haemolymph, hepatopancreas and muscle were95.71±0..53%,85.44±4.40%and88.72±2.58%, respectively, The intra-day precision (RSD,%) were1.38%,3.86%and2.77%and inter-day precision (RSD,%) were2.53%,4.32%and3.94%, respectively. The lowest detectable limits (LOD) of sarafloxacin hydrochloride in the three different tissues were0.001mg/L,0.002mg/kg and0.001mg/kg and the limit of qunatiation (LOQ) were0.005mg/L,0.01mg/kg and0.005mg/kg, respectively. The results showed that the developed method was accurate, reliable and stable, which could be applied to analyze sarafloxacin hydrochloride in haemolymph, hepatopancreas and muscle of Liiopenaeus vannamei.2. Protein binding rate of sarafloxacin hydrochloride with haemolymph from Litopenaeus vannameiProtein binding rate of sarafloxacin hydrochloride with haemolymph was determined by ultrafiltration and RP-HPLC in vivo and in vitro. The results indicated that similar regularity was presented in vivo assay and in vitro assay, and when the drug concentration was high, the protein binding rate was low, Conversely, when drug level was low, protein binding rate was high. The protein binding rate of sarafloxacin hydrochloride with haemolymph from Litopenaeus vannamei at about60%-75%, the results suggested that protein binding rate of sarafloxacin hydrochloride with haemolymph was higher.3. Pharmacokinetics and bioavailability of sarafloxacin hydrochloride in Litopenaeus vannameiThe pharmacokinetics and bioavailability of sarafloxacin hydrochloride in Litopenaeus vannamei were investigated following an intra-sinus injection(10mg/kg) and oral administration(30mg/kg) using the RP-HPLC. The shrimp were kept in seawater with salinity of3.3%and temperature of28.0±1.0℃. The curve of sarafloxacin hydrochloride concentration vs. time by intra-sinus was best described by a three-compartment model, whereas two-compartment model was suitable for the oral treatment. Pharmacokinetics parameters of sarafloxacin hydrochloride in haemolymph. hepatopancreas and muscle were calculated by non-compartment model statistical moment principle, as follows that:the peak time (Tmax) were0.08h,1h and0.25h. respectively;The peak concentration (Cmax) were25.824±2.64mg/L,69.844±10.73mg/kg and6.941±0.99mg/kg, respectively:Area Under Curve (AUCo-∞) were73.93mg·h/L793.834mg·h/L and18.447mg·h/L, respectively; elimination half-life (t1/2Z) were19.4h.64.871h and20.211h, respectively; The body clearances (CLz) were0.135L/h/kg,0.013L/h/kg and0.542L/h/kg, respectively. The same method was applied to analyze the pharmacokinetics parameters of sarafloxacin hydrochloride in haemolymph, hepatopancreas and muscle following oral administration, as follows that:Tmax were2h. 2h and2h, respectively; Cmax were12.793±2.36mg/L,312.649±45.96mg/kg and6.019±0.73mg/kg, respectively; AUC0-∞were133.146mg·h/L,2516.513mg·h/L and40.138mg·h/L, respectively; t1/2z were16.706h,16.363h and30.278h, respectively; CLz were0.225L/h/kg,0.012L/h/kg and0.747L/h/kg, respectively. The results suggested that sarafloxacin hydrochloride was quickly absorbed, and mainly distributed to hepatopancreas in Litopenaeus vannamei. However, the drug was less distributed to haemolymph and muscle and quickly eliminated in the two tissues. The bioavailability of sarafloxacin hydrochloride was60.05%。4. Residue and elimination regularity of sarafloxacin hydrochloride in Litopenaeus annametThe residue and elimination regularity of Sarafloxacin hydrochloride in hemolymph, hepatopancreas and muscle of Litopenaeus vannamei, after multiple feed administration, were firstly studied. The shrimp were kept in tanks with recirculated seawater at a salinity of3.3%. The water temperature was maintained at28.0±1.0℃. The drug was administered orally every12h during consecutive5-day periods at doses of30mg/kg. Hemolymph, hepatopancreas and muscle were sampled after administration of sarafloxacin hydrochloride and the residue concentration of sarafloxacin hydrochloride analyses was performed by RP-HPLC. The results showed that, the residue concentration of sarafloxacin hydrochloride in hepatopancreas was higher than hemolymph and muscle. The residue concentration of hemolymph and muscle were very low and the former is higher than the latter. The elimination half-life of sarafloxacin hydrochloride from hemolymph, hepatopancreas and muscle were14.74h.12.16h and14.14h respectively. The concentration-time data of sarafloxacin hydrochloride were analyzed by WT1.4Foftware to get the withdrawl time. Under this experimental condition, withdraw times were393.4h and62.6h when the minimum residue is0.01mg/kg for hepatopancreas and muscle after multiple feed administration of sarafloxacin hydrochloride in Litopenaeus (?)namel. |
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