恩诺沙星在凡纳滨对虾体内代谢与消除规律研究

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摘要

本论文采用质谱法分析了恩诺沙星在凡纳滨对虾体内的主要代谢产物，建立

了同时检测凡纳滨对虾血淋巴、肌肉以及肝胰腺三组织中恩诺沙星及其主要代谢

产物的高效液相色谱法，研究了单次围心腔注射、口灌以及药饵连续给药下，恩

诺沙星及其主要代谢产物环丙沙星在凡纳滨对虾体内的代谢规律，本研究主要结

果如下：

1、采用 UPLC/Q-TOF MS 法分析恩诺沙星在凡纳滨对虾体内代谢产物

本文采用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术

（UPLC/Q-TOF MS），对凡纳滨对虾口灌恩诺沙星给药后血淋巴中恩诺沙星和代

谢产物进行定性分析。AcquityTM HSS T3 C18 色谱柱（100×2.1mm I.D.，1.8 µm

粒径），柱温为 40℃，流速为 0.4 mL/min，流动相为 0.05%甲酸水和甲醇。使用电

喷雾电离源（ESI），正离子模式下采集数据。结果显示，恩诺沙星在凡纳滨对虾

血淋巴中主要有三种代谢产物，其中峰面积最大的为环丙沙星，其次是哌嗪环开

环代谢物及其同分异构体的一个未知结构。

2、凡纳滨对虾组织中恩诺沙星及环丙沙星含量测定方法建立-反相高效液相色谱法

采用反相高效液相色谱法（RP-HPLC）建立了凡纳滨对虾血淋巴、肌肉和肝

胰腺组织中恩诺沙星及环丙沙星同时检测的方法。高效液相色谱仪采用沃特世公

司Waters2695。色谱柱为 Zorbax SB-C18（5µm ，150×4.6mm，I.D.），流动相为

乙腈：四丁基溴化铵溶液（0.01M，85%磷酸调 pH 至2.8）=5:95（V/V）。流速为

1.1 mL/min，柱温为 40℃，荧光检测器，Ex=280nm、Em=450nm。恩诺沙星和环丙

沙星线性范围为 0.01~10µg/mL。不同浓度水平下对对虾空白组织进行添加回收率

试验，恩诺沙星在血淋巴、肌肉和肝胰腺三种组织中平均回收率为 92.78±1.25%、

88.27±0.60%、85.88±1.27%；相应组织中的环丙沙星的回收率分别为 91.09±

3.30%、87.05±1.13%和84.05±3.64%。恩诺沙星和环丙沙星在血淋巴、肌肉和肝

胰腺内平均日内精密度 RSD 分别为 2.64%、2.22%；3.94%、4.31%；2.35%、4.35%，

相应恩诺沙星和环丙沙星日间精密度 RSD 分别为 4.72%、4.30%；4.06%、3.82%；

3.55%、3.59%，均小于 5%。恩诺沙星和环丙沙星在三种组织中最低检测限分别为

0.01 µg/mL 和0.01 µg/mL；0.01 µg/g 和0.01 µg/g；0.01 µg/g 和0.01 µg/g。由此可

见，该方法快速、准确、可靠、稳定，可以满足凡纳滨对虾血淋巴、肌肉、肝胰

腺生物样品中恩诺沙星和环丙沙星含量分析的要求。

3、围心腔注射和口灌给药恩诺沙星在凡纳滨对虾体内药物代谢动力学

采用高效液相色谱法，研究了盐度 33，温度 28.0±1.0℃的自然海水养殖下恩

诺沙星围心腔注射（剂量 10mg/kg）和口灌（剂量 30mg/kg）给药后，恩诺沙星及

其主要代谢产物环丙沙星在凡纳滨对虾体内的药代动力学。注射组血淋巴恩诺沙

星浓度-时间关系符合三室模型，而口灌组较适合采用二室模型来描述。相比之下，

注射组药物吸收较快，1min 即达到峰浓度，为 149.34±50.78μg/mL，而口灌组 Tmax

和Cmax 分别为 1h 和18.55±3.79μg/mL，恩诺沙星在凡纳滨对虾体内的生物利用度

（F）为 65.77%；尽管口灌给药下凡纳滨对虾对恩诺沙星的吸收较注射组慢，但口

灌给药的利用率较高。口灌给药下对虾肝胰腺中恩诺沙星含量很高，存在“肝首

过效应”现象。围心腔注射给药下，恩诺沙星在对虾血淋巴、肌肉和肝胰腺中的

CLz 分别为 0.038L/h/kg、0.153L/h/kg 和0.019L/h/kg；口灌给药下三组织的 CLz 分

别为 0.058 L/h/kg、0.164 L/h/kg 和0.029 L/h/kg，由此可见，恩诺沙星在肌肉中消

除最快，其次是血淋巴，肝胰腺中最慢。恩诺沙星在对虾体内代谢生成环丙沙星

的量很少，注射和口灌血淋巴 Cmax-CIP/Cmax-ENR 百分比分别为 0.31%和3.66%，

AUCCIP/AUCENR 百分比分别为 8.06%和3.71%，在凡纳滨对虾体内起药效作用仍是

以恩诺沙星为主。

4、药饵连续投喂给药下恩诺沙星在凡纳滨对虾体内的残留及消除规律

盐度 33，温度 28.0±1.0℃的自然海水养殖下，恩诺沙星以 30 mg/kg 剂量，给

药间隔 12h，连续投喂 6次后，采用 HPLC 测定了凡纳滨对虾血淋巴、肌肉和肝胰

腺组织中的药物含量、消除速率常数和消除半衰期，并依据 WT1.4 软件提出该试

验条件下的休药期。结果表明，随着药饵投喂次数的增加，血淋巴和肝胰腺中恩

诺沙星药物峰浓度逐步上升，但上升幅度不大，最后一个给药周期的峰浓度分别

为14.94±3.82µg/mL 和16.30±4.89µg/g，而肌肉中药物浓度不升反降，最后一个

给药周期峰浓度为 1.80±0.48µg/g，约为第 1个给药周期的峰浓度的 1/2。恩诺沙星

在肝胰腺中的残留浓度高于血淋巴和肌肉，肝胰腺可以视为药残检测的靶器官。

恩诺沙星在肌肉和肝胰腺中消除表现出快消除和慢消除两个阶段，肌肉快消除和

慢消除的 t1/2 分别为 10.34h 和36.37h，肝胰腺分别为 12.38h 和346.50h。恩诺沙星

在水产动物肌肉和肝脏中最高残留量分别为 0.1mg/kg 和0.2mg/kg，以此作为对虾

的MRL 标准，在本试验的条件下，推算对虾肝胰腺和肌肉中恩诺沙星休药期为

249.57h 和94.98h，对于整虾而言，其休药期建议为 11d。

关键词：恩诺沙星代谢产物凡纳滨对虾残留消除药代动力学反

相高效液相色谱法

ABSTRACT

In this paper the major metabolites of enrofloxacin in Litopenaeus vannamei

following an oral gavage were analyzed using UPLC/Q-TOF MS. A reverse-phase

HPLC method was developed for the simultaneous determination of enrofloxacin and

ciprofloxacin in shrimp tissues.Effects of route of administration(pericardial injection,

oral gavage and feed administration) on pharmacokinetics, metabolism and elimination

have been carried out. The main results are as follows:

1. Analysis on the metabolites of enrofloxacin in Litopenaeus vannamei using

UPLC/Q-TOF MS

Enrofloxacin(ENR)and its major metabolites in the hemolymph from Litopenaeus

vannamei, following oral administration,was chromatographed on ultraperformance

liquid chromatography coupled with Q-TOF mass spectrometry (UPLC/Q-TOF MS)

with a AcquityTM HSS T3 C18(100×2.1mm I.D.,1.8 µm)column at 40ºC.The mobile

phase was 0.05% formic acid and methanol at the flow rate of 0.4mL/min. Electrospray

ion source was used and data was collected at positive ion mode. The result showed that

enrofloxacin was biotransformed to three metabolites in the hemolymph,which the peak

area of ciprofloxacin was the biggest among three metabolites，the others were little

piperazidine ring cleavage ciprofloxacin and its isomer.

2. Development of RP-HPLC for simultaneous determination of enrofloxacin and

cipmfloxacin in tissues of Litopenaeus vannamei

The method of reverse-phase high performance liquid chromatography(RP-HPLC)

were developed to simultaneously determine concentrations of ENR and

cipmfloxacin(CIP) in the hemolymph,muscle and hepatopancreas of Litopenaeus

vannamei.RP-HPLC was performed with waters 2695 consisted of Zorbax

SB-C18(5µm,150×4.6mm,I.D.)column at 40℃.The mobile phase consisted 95% 0.01M

tetrabutylammonium bromide,which was adjusted pH to 2.8 with 85%phosphoric acid,

and 5% acetonitrile at the flow rate of 1.1 mL/min.The excitation and emission

wavelengths of the fluorescence detector were set at 280nm and 450nm,

respectively.The good linear relationships were obtained between the peak area and

concentrations of ENR and CIP over the range of 0.01 to 10µg/ml.The recoveries of

ENR in hemolymph,muscle and hepatopancreas were 92.78±1.25%,88.27±0.60% and

85.88±1.27%,respectively; the corresponding values of CIP were

91.09±3.30%,87.05±1.13% and 84.05±3.64%, respectively.The intra-day and inter-day

precision RSD was between 2.22% and 4.72% in all tissues, which were less than

5%.The detection limit of ENR and CIP in hemolymph,muscle and hepatopancreas were

0.01 µg/ml and 0.01 µg/ml;0.01µg/g and 0.01 µg/g;0.01µg/g and 0.01 µg/g,

respectively.Obviously,this method was accurate,repeatable and stable,which could be

used for the analysis of ENR and CIP in the tissues of Litopenaeus vannamei.

3. Pharmacokinetics of Enrofloxacin in Litopenaeus vannamei following pericardial

injection and oral gavage

The pharmacokinetics of ENR and CIP were investigated in Litopenaeus vannamei

following a single pericardial injection at a dose of 10mg/kg and a single oral gavage at

a dose of 30mg/kg. The seawater temperature was 28.0±1.0℃ and the salinity was

33‰.The hemolymph concentration-time curve following pericardial injection was

described by a three-conpartment model,whereas for oral administration it was fitted to

two-compartment model. Enrofloxacin absorption was faster following pericardial

injection than oral gavage. The peak time and peak concentrations(Cmax) were 1min and

149.34±50.78μg/mL, respectively; the corresponding values for oral gavage were 1h

and 18.55±3.79μg/mL, respectively. The biaoavailability for enrofloxacin following oral

gavage was 65.77%. Enrofloxacin levels in hepatopancreas was very high following

oral gavage, which was described as ―first pass effect in liver‖. The body clearances

(CLz) of enrofloxacin in hemolymph, muscle and hepatopancreas for pericardial

injection were 0.038L/h/kg, 0.153L/h/kg and 0.019L/h/kg, respectively;the

corresponding values for oral gavage were 0.058 L/h/kg, 0.164 L/h/kg and 0.029 L/h/kg，

respectively. That showed that enrofloxacin eliminated fastest in muscle, following by

hemolymp, and the slowest was in hepatopancreas. Only a small amount of

enrofloxacin was biotransformed into ciprofloxacin, which Cmax-CIP/Cmax-ENR were 0.31%

and 3.66%,and AUCCIP/AUCENR were 8.06% and 3.71% in hemolymph of pericardial

injection and oral administration. ENR played main role in the efficacy in Litopenaeus

vannamei.

4. Residues and elimination regularity of ENR in Litopenaeus vannamei following

feed administration

The shrimps were continuously fed with enrofloxacin-medicated feed for 6 times at

a seawater salinity of 33 and temperature of 28.0±1.0℃, with dosing interval of 12h

and each dose of 30 mg/kg. The enrofloxacin and ciprofloxacin concentrations in

hemolymph, muscle and hepatopancreas were determined by HPLC. The elimination

rate and half-life time were calculated, and the withdrawal time was calculated by

WT1.4 software. With the increase in feeding times, peak concentrations of

enrofloxacin in hemolymph and hepatopancreas increased gradually but not

significantly. In the last administration cycle, the peak concentrations of enrofloxacin in

hemolymph and hepatopancreas were 14.94 ±3.82µg/ml and 16.30 ±4.89µg/g,

respectively. But the drug concentrations in the muscle did not increase but decrease,

with the last Cmax of 1.80±0.48µg/g, about a half of the first Cmax. Enrofloxacin residues

in the hepatopancreas were higher than that in hemolymph and muscle, so

hepatopancreas could be thought as a target organ for detection of drug residue.

Enrofloxacin elimination in muscle and liver was found to be two phases of rapid

elimination and slow elimination. The half-life times in muscle for rapid and slow

elimination were 10.34h and 36.37h, respectively, while 12.38h and 346.50h for

hepatopancreas, respectively. MRL for muscle and liver in aquatic animals were

0.1mg/kg and 0.2mg/kg, respectively, which was MRL standard for shrimp in this study.

The withdrawal times for enrofloxacin in hepatopancreas and muscle from shrimp were

calculated to be 249.57h and 94.98h, respectively. For the whole shrimp, the MRL for

enrofloxacin was proposed to be 11 days.

Key Words：enrofloxacin,metabolite,Litopenaeus vannamei, residue,

elimination, pharmacokinetics, RT-HPLC

中文摘要

ABSTRACT

第一章绪论 .................................................................................................................... 1

§1.1 药物代谢动力学概述 ...................................................................................... 1

§1.1.1 药物代谢动力学概念 ........................................................................ 1

§1.1.2 药代动力学主要研究内容以及各参数意义 .................................... 2

§1.1.3 药物代谢动力学模型 ........................................................................ 2

§1.1.3.1 房室模型 ................................................................................. 2

§1.1.3.2 非房室模型的统计矩方法 ..................................................... 3

§1.1.3.3 生理学药物动力学模型 ......................................................... 3

§1.2 常用药物检测方法 .......................................................................................... 4

§1.2.1 气相色谱法（gas chromatography，GC） ...................................... 4

§1.2.2 高效液相色谱法（high performance liquid chromatography，HPLC）

.......................................................................................................................... 4

§1.2.3 色谱-质谱联用（chromatography-mass） ....................................... 5

§1.2.4 免疫分析法（immunoassay） .......................................................... 5

§1.2.5 微生物法（microbiological method） .............................................. 6

§1.3 水产药物代谢动力学研究概况 ...................................................................... 6

§1.3.1 研究意义 ............................................................................................ 6

§1.3.2 研究的药物 ........................................................................................ 7

§1.3. 2.1 原形药物 ................................................................................ 7

§1.3. 2.2 代谢产物 ................................................................................ 8

§1.3.3 研究的动物 ...................................................................................... 10

§1.4 喹诺酮类药物在水生动物体内药动学和休药期的研究 ............................ 11

§1.4.1 喹诺酮类药物简介 ........................................................................... 11

§1.4.2 喹诺酮类药物在水生动物体内药动学研究概况 .......................... 12

§1.4.2.1 噁喹酸 ................................................................................... 12

§1.4.2.2 氟甲喹 ................................................................................... 12

§1.4.2.3 恩诺沙星 ............................................................................... 13

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摘要：

摘要本论文采用质谱法分析了恩诺沙星在凡纳滨对虾体内的主要代谢产物，建立了同时检测凡纳滨对虾血淋巴、肌肉以及肝胰腺三组织中恩诺沙星及其主要代谢产物的高效液相色谱法，研究了单次围心腔注射、口灌以及药饵连续给药下，恩诺沙星及其主要代谢产物环丙沙星在凡纳滨对虾体内的代谢规律，本研究主要结果如下：1、采用UPLC/Q-TOFMS法分析恩诺沙星在凡纳滨对虾体内代谢产物本文采用超高效液相色谱与串联四级杆飞行时间质谱仪联用技术（UPLC/Q-TOFMS），对凡纳滨对虾口灌恩诺沙星给药后血淋巴中恩诺沙星和代谢产物进行定性分析。AcquityTMHSST3C18色谱柱（100×2.1mmI.D.，1.8µm粒径）...

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