EuroResidue VIII – 23 -25 May 2016 - Egmond aan Zee, The
Netherlands
Nora Mestorino, Martín Daniele, Andrea Buchamer, Juan Cruz
Chiarizia, María Laura Marchetti
Laboratory of Pharmacological and Toxicological Studies (LEFyT),
Faculty of Veterinary Science, Universidad Nacional de La Plata, 60
and 118, 1900 La Plata,
Argentina. [email protected]
Doxycycline (DOX) is a derivate of tetracycline with similar
properties, but with a longer action period. It is the most active
antibiotic of tetracycline group, so
the minimum inhibitory doses are low. Is an antimicrobial widely
used in swine production against Gram-positive and Gram-negative
bacteria, including some
anaerobes.
Doxycycline is more soluble in lipids than other tetracyclines
and after application penetrates better in body tissues and fluids.
Long persistence of DOX in an
animal’s body could cause unacceptable concentration in animal
tissues. The European Commission had set maximum residue limits
(MRLs) for
oxytetracycline, tetracycline, and chlortetracycline as a sum of
parent compounds with their 4-epimers and for doxycycline, only as
parent compound without
its 4-epimer. The established levels of MRLs are as follows:
muscles = 100 ng/g, liver and skin plus fat = 300 ng/g; and kidneys
= 600 ng/g (EU 37/2010)1.
The purpose of this study was to determine the residues of DOX
in edible tissues (kidney, liver, skin + fat and muscle) of pigs
after 5 days of oral medication
via drinking water of 10 mg DOX 25%/kg body weight (BW) per day.
Based on the tissue residues, a withdrawal time was calculated
according to Guideline N°
EMEA/CVMP/036/95 of the Committee for Veterinary Medicinal
Products2.
C18 column (Luna, 5 µm, 4.6 mm x 150 mm;
Phenomenex, Torrance,CA, USA) was eluted with
water-acetonitrile with 0.02 M oxalic acid and 0.0005M
EDTA (72:28, v/v) at 1.2 mL/ min flow rate
UV-VIS detector (346 nm)
This method performed accurately and reproducibly over a range
of 0.1 to 6 µg/mL for DOX. The linearity was between r= 0.9913 to
0.9975 values in all tissues assayed.
The chromatographic analysis time was short and DOX was
presented in 4 min as a sharp and symmetrical peak with no
interfering peaks (Figure 1).
The LODs were 0.020, 0.020, 0.030 and 0.026µg/g for DOX in
kidney, skin/fat, muscle and liver, respectively; while LOQs were
0.050, 0.136, 0.050 and 0.225 µg/g for
kidney, skin/fat, muscle and liver, respectively.
The method for the analysis of tissue samples was thoroughly
validated (Table I), and was specific for all samples with respect
to the interference from endogenous
compounds. The validated analytical methodology showed
satisfactory results of sensitivity, precision and accuracy that
allow its use for the detection and quantification of
DOX residues in tissue of pigs (Figure 2).
In Figure 3 (A, B, C and D), the mean tissue concentrations and
their SD values of muscle, kidney, liver, skin +fat, respectively
at 1, 3, 7 and 11 days after cessation of
medication are presented. Highest residues were found in liver,
followed by kidney, muscle and skin + fat. The concentrations in
all matrices were near or below the MRL
at 3 days and below the respective LOQ at 11 days after
completion of treatment, respectively. In muscle sample, DOX only
was detected at 24 h and 3 days post
treatment.
In our study, taking into account the MRLs in pigs and
considering that the marker residue is the doxycycline, the
calculated WT was 7.23, 4.87, 4.50 and 4.33 days for
liver, kidney, skin + fat and muscle, respectively.
8-EVAPORATION
1- Grinding the tissue
2- 0.4 g of tissue + Mc
Ilvaine- EDTA (1.4 mL)
3-homogenize
4-Mix
5-be centrifuged
6- S1+ S2 + S3
+ S4
7- SPE extraction
9-HPLC ASSAY
Supernatant S1
The WT for edible tissues of pigs (muscle,
liver, kidney and skin plus fat) were
estimated by linear regression analysis of
the log transformed tissue concentrations
and determined at the time when the upper
one-sided 95% tolerance limit for the
residue was below the MRLs, with a
confidence of 95% (EMEA, 2002).
Doxycycline concentrations in function of
time found in edible tissues were plotted
and analysed with the program WT 1.4 in
order to recommend a period of withdrawal
time for this experimental formulation.
Fig 1. HPLC Chromatograms
of DOX, 4 and 6 µg/mL
Table 1. Validation results for the analysis of DOX in pigs
tissues
This analytical method exhibited good linearity and
reproducibility over the calibration range for DOX in edible
tissues of pigs.
Antibiotics are used in pig farms to enhance growth, feed
efficiency and reduce diseases. Additionally, prophylactic
treatment is common during periods of stress. In
Argentina DOX is frequently used in pigs production; therefore,
it is important to control their residues in edible tissues.
Doxycycline given to pigs orally raise possibility
for residues which, remain in edible tissues, particularly when
the animals are slaughtered without the observance of withdrawal
period. Such residues may pose public
health hazards to consumers depending on the type of food and
the amount of residue present3.
Additionally, the use of antibiotics in related food may lead to
resistance in bacterial populations that do not respond to
treatment commonly used for human illnesses4.
Studies on tissue concentrations after different drug
formulation administration are essential for the control of
antibiotic residues in food animal products, in order to
recommend the withdrawal time appropriate.
Our results demonstrate that DOX oral administration at the 10
mg/kg for 5 days together with drinking water requires WT
respecting the MRL fixed for 8 days for human
consumption.
1.- Commission Regulation EU No 37/2010. Off J Eur Commun 2010,
L15, 1–72. 2.- The European Agency for the Evaluation of Medicinal
Products, Note for Guidance: Approach Towards Harmonisation of
Withdrawal Periods, No. EMEA/CVMP/036/95, 1995. 3.- Oka H, Ito
Y, Matsumoto H (2000). Chromatographic analysis of tetracycline
antibiotics in foods. Journal of Chromatography, 882: 109–133. 4.-
Marchetti,
ML; Mestorino N. Effect of 1-(1-naphthylmethyl)-piperazine on
antimicrobial agents susceptibility in mdr isogenic and veterinary
field Escherichia coli strains. J Med Microbiol jmm. (2012), 61,
786–792
Acknowledgements Mr. Daniel Buldain is acknowledged for check
the manuscript.
Intra-day Inter-day (over 3 days)
matrix r µg/g Accuracy
(%), n=6
Precision
(%), n=6
Accuracy
(%)
Precision
(%)
Muscle
0.9952
(0.2-2µg/g)
0. 2
0.5
1
2
97.21
92.25
108.62
97.91
3.01
1.22
11.07
3.05
97.40
91.42
104.87
97.47
2.02
1.74
9.18
2.47
Liver
0.9944
(0.2-2 µg/g)
0.2
0.5
1
2
88.37
93.79
110.93
97.25
4.12
1.82
3.03
2.16
89.46
93.26
108.98
95.42
5.69
1.18
2.40
1.74
Kidney
0.9975
(0.2-2 µg/g)
0.2
0.5
1
2
93.40
100.01
103.30
99.01
2.09
7.05
1.24
3.17
92.80
100.00
102.77
97.00
2.75
6.00
2.47
2.73
Skin/Fat
0.9913
(0.2-2 µg/g)
0.2
0.5
1
2
89.01
97.66
108.95
97.21
2.31
6.17
1.95
2.13
88.00
91.22
106.32
93.57
3.01
7.53
2.85
3.38
Fig 2. Chromatograms of blank
liver and spiked liver samples
with DOX at levels of 0.2; 0.5; 1
and 2 µg/g
Fig 3. Mean tissue concentrations of doxycycline (A: muscle, B:
kidney, C: liver and D: skin + fat) in pigs slaughtered 1, 3, 7 and
11 d
after oral administration of DOX 25% (dose of 10 mg/kg body
weight during 5 days).
A
B
C
D
P114
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