155 155
Oxytetracycline Tetracycline
( , , )
Tissue Distribution after dipping administration of Oxytetracycline and Tetracycline in Olive flounder (Paralichthys olivaceus), Rock-
fish (Sebastes schlegeli), and Red sea bream (Pagrus major)
Suk Kim, Hee Sik Chung , Seok Joong Kang , Ji Young Ha, Won Chul Jung, Sung Hyek Heo, Yong Woon Shin, Kyoung Won Kim, Dae Geun Kim and Hu Jang Lee
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju, 660-701, Korea
Livestock Division, Gyeongnam Provincial Government, Changwon, 641-702, Korea Division of Marine Bioscience, Gyeongsang National University, Tongyeong, 650-160, Korea
Tissue distribution and residue depletion of oxytetracycline (OTC) and tetracycline (TC) following dip- ping administration were evaluated in olive flounder (Paralichthys olivaceus), rockfish (Sebastes schlegeli), and red sea bream (Pagrus major) under field conditions. Fishes were held in floating cages placed in sea water and fed a commercial diet for 15 days to acclimate to a new surrounding. Fishes were dipped in OTC 50 g/ton water for 30 min and TC 18 g/ton water for 5 hours. Blood and muscle were sampled from fishes on 0th, 1th, 2th, 3th, and 5th day after administration. After solid-phase extraction, OTC and TC analyses were carried out by HPLC. The recovery rate of OTC in serum and muscle samples was 71-77% and 78- 84%, respectively. Also, the recovery rate of TC in serum and muscle samples was 70-79% and 73-78%, respectively. The results of recovery rate were similar to previous studies reported.
At the termination of dipping administration of OTC and TC, residue concentration in muscle samples of rockfish was significantly higher than those of olive flounder and red sea bream. At day 5, residue concen- trations of all samples were believed to decrease to lower than 0.05 mg/kg, the detection limit.
The present study showed that residue concentrations of OTC and TC decreased to below 0.05 mg/kg after treatment 5th day, faster than the established withdrawal period. The tissue reside depletion time of dipping administration of OTC and TC seems to be shorter than those of oral or parenteral administration.
Key words: Oxytetracycline, Tetracycline, Olive flounder, Rockfish, Red sea bream.
, .
, 2004 1999
2 ( 23 )
, 6 4
5 2.5
( , 2005).
Corresponding Author : Hu Jang Lee, Tel : 055-751-6642, Fax : 055- 751-5803, E-mail : [email protected]
,
. ,
.
, oxytetracycline (OTC)
, OTC
,
,
( , 2005)
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,
,
. ,
OTC , 2000
3 florfenicol, flumequine, oxolinic acid ,
.
.
, 30
,
tetracycline OTC
( , 2005). OTC
, ,
. ,
tetracycline (TC) ,
,
( , 1996).
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,
, ,
( , 2004).
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.
( , 1992; , 1994; , 1994a), ( , 2002; , 2005),
( ,
1983; , 2002)
, OTC TC
(Brocklebank et. al., 1997; Luzzana et. al., 1994;
, 1998).
,
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. ,
,
.
OTC TC
( , , )
,
.
OTC TC Sigma (St. Louis, MO, USA)
,
. .
Macllvaine Buffer - 0.1 M Citric acid 1 L 0.1 M phosphate 625 ml
(pH 4.0 0.05).
Macllvaine - 1.625 L Macllvaine buffer
0.1 M EDTA .
- 1 L flask 0.01 M oxalic
acid methanol 1 L
.
MOX - 100 ml flask 1.50 g
oxalic acid , methanol 100 ml .
- 100 ml MOX 100 ml .
350 30 g , P. olivaceus, 450 45 g , Sebastes schlegeli, 430
55 g , Pagrus major 25
,
15 .
OTC (oxytetracycline HCl 500g/kg, ,
) 1 ton 100g , ,
25 30
. , TC (tetracycline HCl 120
g/kg, , ) 1
ton 150 g , ,
25 5
. 25
( : 1,000 L) ,
25
.
, 1, 2, 3
5 5
5 . MS-
222 ,
. (2,000 g, 10 )
serum .
: Methanol, acetonitrile, 0.01 M oxalic acid 1 : 2 : 7 (pH 2.0)
, 0.2 membrane filter (Milli-
pore, USA) .
HPLC : UV-visible
detecteor HPLC system (HP1100 Series;
Hewlett-Packard) . column
-BondaPack C18 (3.9 300 mm, 10 , Cresent Sci. Ltd., India) .
OTC TC
, HPLC 1.0 ml/min
360 nm 0.005 0.01 AUFS
, 50 peak
.
: OTC (Sigma, USA) TC (Sigma, USA)
, OTC TC 1 mg/ml
.
. : OTC TC 0.1, 0.2, 0.5, 1.0 ppm
peak .
:
0.5 1.0 ppm spiking ,
, HPLC
, peak
, . (%) = TCs
TCs 100
: OTC TC
5.0 g (serum
5ml) 50 mL Macllvaince
buffer 20 ml 2
, (3,000 g, 10 min, 5 )
. 20 ml 10 ml
Macllvaince buffer 2
. (3,000 g,
30 min, 5 )
Sep-Pak C18cartridge (Vac 3 cc, 500 mg, Waters,
USA) OTC TC .
Sep-Pak C18cartridge OTC TC 20
ml methanol (45 ) ,
HPLC 2 ml
0.45 syringe filter (Nalgene)
HPLC .
OTC TC Ongi (1984)
. Sep-pak C18cartridge
10 ml methanol, 20 ml 0.5
ml/mim cartridge
.
Fig. 1 .
Sigma plot student's t-test , p < 0.05
.
Fig. 1. Summary of sample clean-up procedure for oxyte- tracycline and tetracycline for samples.
Weigh sample (5 g or 5 ml) in polypropylene tube
Homogenize for 2 min in 20 ml Macllvaine buffer using Stomacher
Pour the supernatant in a 50 ml centrifuge tube
Centrifuge (3,000 g, 10 min, 5 )
Transfer the upper layer into another 50 ml tube
Add Macllvaine 20 ml into the residue and centrifuge (3,000 g, 10 min, 5 )
Remove the upper layer into the 50 ml tube
Add Macllvaine 20 ml into the residue and centrifuge and remove the upper layer
Add Macllvaine 10 ml into the residue and centrifuge and remove the upper layer
Centrifuge the combined the buffer in 50 ml tube (3,000 g, 10 min, 5 )
Filter the upper layer
Load the filtered extract in a C18 cartridge
Elute with 20 ml methanol
Evaporate the eluate in 45 water bath to dryness
Redissolve the residue with 2 ml mobile phase
Filter with 0.45 syringe filter and inject 50 into HPLC
OTC TC 0.1, 0.2, 0.5
1.0 mg/l HPLC
peak
OTC TC r2
0.9957 0.9951
(r2>0.995) (Fig. 2).
OTC TC
OTC TC , ,
(5 g) (5 ml) 0.5
1.0 ppm spiking
(Table 1). Fig. 3 OTC TC
0.5 mg/kg spiking
, HPLC
. OTC , 0.5 ppm
, 78%
, 71%
,
. , OTC 1.0 ppm ,
80%
, 76%
, 0.5 ppm
. , TC , 0.5 ppm
, 73%
, 70%
, OTC ,
. , TC 1.0
ppm ,
Table 1. Spiked Recovery in Blood and Muscle.
Spiked Recovery (%)
Fishes Sample concentration OTC TC
(mg/kg) (Mean SD, n=3) (Mean SD, n=3)
P. olivaceus Muscle 0.5 78.25 0.23 73.64 0.63
1.0 80.51 1.28 74.52 0.71
Serum 0.5 71.27 0.36 70.85 0.42
1.0 76.72 0.49 72.44 0.35
S. schlegeli Muscle 0.5 83.24 0.33 75.43 0.54
1.0 81.93 0.56 76.24 0.63
Serum 0.5 72.12 0.34 70.52 0.51
1.0 77.27 0.62 71.28 0.49
P. major Muscle 0.5 80.21 0.43 73.96 0.54
1.0 84.35 0.68 78.13 1.22
Serum 0.5 75.54 1.34 73.23 0.72
1.0 77.68 2.31 79.54 1.32
Fig. 2. Standard calibration curves of tetracycline antibi- otics by High-Performance Liquid Chromatography.
HPLC condition; column, -BondaPak C18 (3.9 300 mm, 10 ), mobile phase, 0.01 M oxalic acid-methanol- acetonitrile (7:2:1), flow rate, 1.0 ml/min, injection volume, 50 .
74% ,
71% , 0.5 ppm
.
OTC , (0 day)
, ,
,
. ,
, 5
OTC TC
(0.05 mg/kg) . , TC
(0 day)
OTC , ,
,
. ,
, 3
,
5 , ,
OTC TC
(0.05 mg/kg) (Table 1-2).
OTC Gram-positive Gram-negative
,
, ,
. , TC
, , ,
ND, Not detected; * at the termination of dipping administration of oxytetracycline; ** indicates p<0.05 (Compared to P. oli- vaceus and P. major).
Table 2. Concentration of oxytetracycline in serum and muscle of fishes after dipping of oxytetracycline 50 g/ton water.
Fish Tissue No. of Residue concentration after treatment (mg/kg)
Samples 0 day* 1 day 2 day 3 day 5 day
P. olivaceus Serum 5 0.778 0.023 0.338 0.024 0.304 0.031 0.107 0.042 ND
Muscle 5 1.028 0.047 0.779 0.043 0.428 0.017 0.169 0.038 ND
S. schlegeli Serum 5 0.835 0.034 0.346 0.033 0.310 0.017 0.115 0.023 ND
Muscle 5 1.923 0.041** 0.967 0.076 0.511 0.025 0.196 0.051 ND
P. major Serum 5 0.754 0.055 0.316 0.029 0.279 0.033 0.098 0.028 ND
Muscle 5 1.215 0.039 0.835 0.052 0.324 0.061 0.156 0.047 ND Fig. 3. Chromatograms of a olive flounder muscle spiked to
a level of 0.5 mg/kg with each of oxytetracycline (A) and tetracycline (B).
( , 1996).
(1977) OTC TC
0.1, 0.3 ppm spiking ,
, 0.1 ppm OTC TC
66, 51% , 0.3 ppm OTC TC
51, 52% .
, OTC TC
, OTC
TC 0.1, 0.5, 1.0 ppm spiking
0.1 ppm OTC TC
100% , 0.5 ppm OTC TC
98 97% , 1.0 ppm
OTC TC 97 96%
( , 2005).
OTC TC 0.5 ppm spiking
78 73%, 1.0 ppm 80
74% ,
(1977)
, (2005)
. , OTC
0.1, 0.5 ppm spiking
, 84 70% (Rupp et al.,
2005), Pena (2003) TC 0.05,
0.1, 0.2 ppm spiking
, 83%
. Hiroyuki (1999)
OTC TC 0.1, 0.5 ppm spiking 0.1
ppm OTC TC 78% ,
0.5 ppm 70% ,
.
OTC TC
, . ,
.
, OTC TC
. (1998) OTC 100 mg/kg
B.W. 1 ,
OTC ,
20
. Jose (1996) OTC 75 ppm 14
, ,
10
0.1 ppm . ,
5 0.05 ppm
ND, Not detected; * at the termination of dipping administration of tetracycline; ** indicates p<0.05 (Compared to P. oli- vaceus and P. major).
Table 3. Concentration of tetracycline in serum and muscle of fishes after dipping of tetracycline 18 g/ton water.
Fish Tissue No. of Residue concentration after treatment (mg/kg)
Samples 0 day* 1 day 2 day 3 day 5 day
P. olivaceus Serum 5 0.489 0.014 0.345 0.035 0.173 0.023 0.068 0.015 ND
Muscle 5 0.524 0.078 0.276 0.054 0.156 0.035 0.075 0.024 ND
S. schlegeli Serum 5 0.515 0.035 0.354 0.021 0.150 0.046 0.053 0.012 ND
Muscle 5 0.974 0.102** 0.469 0.034 0.249 0.042 0.103 0.022 ND
P. major Serum 5 0.419 0.048 0.293 0.037 0.127 0.036 ND ND
Muscle 5 0.626 0.086 0.326 0.053 0.168 0.029 0.072 0.017 ND
,
.
Rigis (2004) OTC 50 mg/kg B.W.
, , half-life 21
, 4 0.1
ppm .
OTC 24
, 3
0.1 ppm ,
, ,
.
, , ,
OTC
71-77%, 78-84% , TC
70-79%, 73-75%
, OTC TC
. , OTC TC 100 g/ton,
150 g/ton ,
, ,
5 OTC
(0.2 mg/kg) , TC
0.1 mg/kg . OTC
TC
(30 ) OTC
. ,
OTC TC
( , , )
,
,
, .
Oxytetracycline tetracycline ,
, .
,
15 .
, oxytetracycline 50 g 1
, , 30
, tetracycline 18 g 1
5 .
, 1,
2, 3, 5
. Oxytetracycline tetracycline
. Oxytetracy-
cline 71-77% ,
78-84% . , tetracycline
70-79%, 73-78%
.
, oxytetracycline
tetracycline
, 5 ,
oxytetracycline tetracycline
0.05 mg/kg .
, oxytetracycline tetracy-
cline ,
,
.
( )
(R05-2004-000-10627-0).
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L., Valfre, F. and Polidori, P.: Effect of tem- perature and diet composition on residue depletion of oxytetracycline in cultured channel catfish. Analyst, 119: 2757-2759, 1994.
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mination of tetracycline antibiotics in salmon muscle by liquid chromatography using post-column derivatization with fluo- rescence detection. J. AOAC Int., 86: 925- 929, 2003.
Reimer, G. J. and Young, L. M.: Validation of a method for determination of tetracycline antibiotics in salmon muscle tissue. J. Assoc.
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Rigos, R., Nengas, I., Alexis, M. and Athanas- sopoulou, F.: Bioavailability of oxytetracy- cline in sea bass, Dicentrarchus labrax (L.).
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. , 38: 365-
371, 2005.
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, 102-103, 2002.
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conjugation R plasmid
. , 35: 115-121,
2002.
, , : , pp.171-176,
, , 1996.
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: HPLC
. , 38: 372-378, 2005.
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. , 16:
17-24, 1983.
, :
. , 5: 38-47, 2002.
, , , , , ,
, : Chloramphenicol
, Paralichthys oli- vaceus
. , 17: 113-121, 2004.
, :
. , 11: 77-81, 1998.
, , : MSPD HPLC
. , 12: 117-124, 1997.
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pefloxacin
. , 34:
153-163, 1994.
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Gentamicin .
, 18: 327-338, 1994.
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Manuscript Received : April 7, 2006 Revision Accepted : May 24, 2006 Responsible Editorial Member : Joon-Ki Chung (Pukyong Univ.)
