中华绒螯蟹血淋巴内盐酸环丙沙星的反相高效液相色谱法的建立

The method of RP-HPLC determination of ciprofloxacinum in hemolymph of Eriocheir sinenses

应用反相高效液相色谱法 (RP HPLC)建立了盐酸环丙沙星在中华绒螯蟹血淋巴内含量的测定方法。本法采用肌肉注射的给药方法 ,给药剂量为 8.17± 0 .5 6mg·kg-1和 6 .2 5± 0 .85mg·kg-1。血淋巴从心区抽取。经各种抗凝方法的比较 ,认为等量的ACD与等量的血淋巴混合 ,可得到持久的抗凝效果。血淋巴样经甲醇去蛋白 ,30 0 0r/min离心 10min后 ,取上清液上HPLC。色谱柱 ,RP ODS 5μ15 0× 4.6mm ;流动相 ,甲醇 磷酸缓冲液 四丁基溴化铵 =2 5 0∶75 0∶2 0 (v /v /v) ;流速 ,1mL·min-1;进样量 ,2μL ;检测波长 ,2 80nm。研究表明 ,本法线性关系与重复性良好 ;雌、雄蟹平均回收率分别为 10 0 .0 8± 3 .43%和 99.11± 8.2 0 % ;日间精密度、平均提取精密度分别为 1.19± 0 .92 %、1.5 5± 0 .0 1%。用该方法对肌肉注射给药后 48h内中华绒螯蟹雄蟹血淋巴中的盐酸环丙沙星的血药浓度进行检测 ,揭示出由 37.13± 0 .0 8μg·mL-1降至 5 .96± 0 .18μg·mL-1的代谢规律。

A method of determination of ciprofloxacinum in the hemolymph of Chinese mitten-handed crab, Eirocheir sinensis, was established by RP-HPLC. The drug was administered by intramuscular at a dose of 8.17±0.56 and 6.25±0.85 mg·kg -1 to male and female crab,repectively. The hemolymph was extracted from the cardiac region. By comparison among various anti-coagulant methods, the permanent result of anticoagulation was obtained through ACD mixing with equal volume hemolymph. Hemolymph samples got rid of protein by methanol, centrifuged at 3000 r·min -1 for 10 minutes and then the supernatant was used to HPLC. The RP-ODS-5 μL 50×4.6mm ID column was used, with a mobile phase of methanol-phosphate buffer-tertrabutylammonium bromide=250∶750∶20 ( v/v/v ); The flow rate was 1 mL·min -1; the auto-injected volume, 2 μL and the uv detection wavelength, 280nm. The study showed that the linearity and repeatability were good; the average recovery of both male and female crab were : 100.08±3.43% and 99.11±8.20%; the intra-day precision, the average extracted-precision were 1.19±0.92%, 1.55±0.01% and 2.31±1.03%,respectively. The law of the metabolism of blood-drug concentration was disclosed dropping from 37.12±0.08 μg·mL -1 to 5.96±0.18 μg·mL -1, by using RP-HPLC method to determine the ciprofloxacinum in hemolymph within 48 hours in male crab after administration of the drug through intramuscular.