| The pacific white shrimp, Litopenaeus vannamei farming has developed rapidly in recent years, and become the main species of shrimp farming in China. But large-scale intensive farming has brought some diseases, especially shrimp vibriosis, which has significant impact on the shrimp farming industry. An antibacterial drug are needed to prevent and control the vibriosis, which should have characteristics of wide antimicrobial spectrum, low residues, harmonious to aquatic environment, and not easy to produce drug resistance. Studies of pharmacodynamics and pharmacokinetics should be conducted to provide a theoretical basis for the effective use of drugs.Qxolinic acid(OA) is one of the national standard drugs of fishery in China, of which the pharmacokinetics mostly concentrated on fish and no data found in Litopenaeus vannamei. In this paper, the method of determination of oxolinic acid residue was first established. Then pharmacokinetics of oxolinic acid in two formulations(oxolinic acid powder and compound oxolinic acid powder) in Litopenaeus vannamei were studiedas well as its distribution and elimination in tissues. Considering the eliminate in muscle and the maximum residue limits standard, withdrawal time of two oxolinic acid formulations in Litopenaeus vannamei were determination. Finally, antimicrobial activity in vitro on vibrios were studied and PK/PD parameters were made to provide technical support for the rational use of oxolinic acid in shrimp farming. 1. RP-HPLC method for determination of oxolinic acid residue in shrimp tissues.Influence of different detection wavelengths and mobile phases on chromatographic behavior oxolinic acid, and influence of different extraction methods on recovery of oxolinic acid were carried out. The method of reversed-phase high performance liquid chromatography(RP-HPLC)was developed to determine oxolinic acid residue in shrimp, Litopenaeus vannamei. The mobile phase was acetonitrile and 0.01 mol·L-1 tetrabutyl ammonium bromide(p H was adjusted to be 2.75 with phosphate solution). Aglient Zorbax SB-C18(4.6×150mm,5μm) was used with a mobile phase flow rate of 1.0 m L·min-1 and column temperature of 40℃.Fluorescence detector was used with excitation wavelength of 265 nm and emission wavelength of 380 nm. Sampling volume was of 10 μL. The method was found to be linear and reproducible in the concentration ranges of 0.002~10μg·m L-1 with the correlation coefficient of 0.9996~0.9999. Oxolinic acid in shrimp tissues were extracted by acetonitrile, and the average recoveries of oxolinic acid from hemolymph, muscle, hepatopancreas and gill were 84.70% to 90.60%, The relative standard deviations for intra-day and inter-day precisions were 1.60%~4.02% and 2.91%~4.73%, respectively. The quantitative detection limits of oxolinic acid of hemolymph, muscle, hepatopancreas and gill were 0.02μg·m L-1, 0.01μg·g-1, 0.02μg·g-1 and 0.02μg·g-1, respectively. The method is simple, reproducible and suitable to determine oxolinic acid in shrimp tissues. 2. Pharmacokinetics, tissue distribution and elimination of oxolinic acid powder in the Pacific white shrimp, Litopenaeus vannamei.The pharmacokinetics, tissue distribution and elimination of oxolinic acid(OA) in the pacific white shrimp, Litopenaeus vannamei, was investigated following oral administration of OA powder via feed at three dosages of 10 mg·kg-1, 30mg·kg-1 and 80mg·kg-1.After oral administration at three dosages, the hemolymph OA concentration-time curve of shrimp werewell fitted to a two-compartment model with first-order absorption. Maximum concentrations(Cmax) of hemolymph in three dose groups were 4.31 mg·L-1, 14.93 mg·L-1, 16.62 mg·L-1, respectively, and 1.62 mg·kg-1, 5.80 mg·kg-1, 7.36 mg·kg-1 in muscle, respectively, and all peak time were 2h. The maximum concentrations in hepatopancreas of three groups were 7.90 mg·kg-1, 27.23 mg·kg-1 and 60.51 mg·kg-1, respectively and the peak time were 0.5 h. The maximum concentrations in gill were 2.87 mg·kg-1, 8.08 mg·kg-1 and 12.12 mg·kg-1, respectively and all peak time were 4h. Although the maximum concentration in hepatopancreas were highest, the area under the curve(AUC) of hepatopancreas was lower than that in hemolymph, which could be explained by fast eliminationin hepatopancreas. With the dosage increased, eliminate time of oxolinic acid in shrimp muscle prolonged, and the withdrawal time extended. According to OA elimination in muscle and the maximum residue limit of 30 μg·kg-1 in shrimp, the theoretical withdrawal time were 74.7h, 90.1h and 135.9h, respectively. 3. Study on pharmacokinetics of compound oxolinic acid powder in Litopenaeus vannamei.The pharmacokinetics and tissue elimination of oxolinic acid(OA) in the pacific white shrimp, Litopenaeus vannamei, was investigated following oral administration via feed and dosage regime and withdrawal time were developed. The haemolymph OA concentration vs. time after oral administration via feed was best described by a two-compartment model with first absorption following a single dose of 30 mg·kg-1. The peak concentration(Cmax), peak time(Tmax), area under curve(AUC0-24) and elimination half-life time(t1/2z)were 14.70 mg·L-1、2 h、244.6 mg·L-1·h and 18.56 h, respectively. Peak concentrations(Cmax)in muscle, hepatopancreas and gill were 4.11 mg·kg-1、17.20 mg·kg-1 and 7.01 mg·kg-1,respectively; elimination half-life time(t1/2z)were 10.71h、12.31 h and 16.75 h, respectively. The pacific white shrimp could well absorb OA and the residual OA was rapidly eliminated from muscle. According to OA elimination in muscle and the maximum residue limit of 30 μg·kg-1 in shrimp, the theoretical withdrawal time of OA were 91.7h. 4. In vitro antimicrobial activity on Vibrios of oxolinic acid and PK/PD parameters.The minimum inhibitory concentrations(MIC) and the minimum bactericidal concentration(MBC) of OA on the pathogenic vibrio were detected and the pharmacokinetics/pharmacodynamics(PK/PD) parameters were calculated. The results showed, the OA MICs and MBCs of 132 Vibrios isolates were almost the same and varied mainly from 0.15 ~ 1.25 μg·m L-1 with MIC50 of 0.62 μg·m L-1 and MIC90 of 1.25 μg·m L-1. Considering the ratios of Cmax/MIC90 and AUC0-24/MIC90, which were two important indices for PK/PD relationship, the results showed that OA dosage of 30 mg·kg-1 in two formulations a day once bait delivery could effectively combat bacterial diseases caused by the vibrios and not easy to produce drug resistance strains of vibrio. According to OA elimination in muscle and the maximum residue limit of 30 μg·kg-1 for OA in shrimp, the withdrawal time of 5 days was recommended. |
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