DataPDF Available




Levofloxacin is a third generation fluoroquinolone used in food-producing animals in some countries outside of EU. The aim of the study was to investigate the residues of this antibiotic in chicken tissues (meat, skin and giblets). Chicken (n=30) were treated with levofloxacin orally at a dose of 10 mg/kg BW for 5 days. Birds were divided into 5 groups and humanely killed as followed: on day 0 (the day after last administration of levofloxacin), 2, 4, 6 and 8 day. The results of the studies showed the highest residues levels of levofloxacin in liver (1051 µg/kg), followed by breast muscle, gizzard, heart and skin – 428 321 303 and 293 respectively. The rate of reduction of antimicrobial activity was different. Tissue concentrations in heart and gizzard decreased faster than these in muscles and skin. In the liver they decreased from the last day of the treatment to the 2nd day and the levels remained nearly equal up to the 8 th day after the end of drug administration.
Bu lg ar ia n Journal of Veterinary Medicine (201 3) , 16 , Suppl. 1, 216–219
Department of Food Hygiene and Control, Veterinary Legislation and Management,
Department of Veterinary Microbiology, Infectious and Parasitic Diseases, Faculty
of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria;
Kyuchukova, R., V. Urumova, M. Lyutskanov, V. Petrov & A. Pavlov, 2013. Studies on le-
vofloxacin residues in chicken meat and giblets. Bulg. J. Vet. Med., 16, Suppl. 1, 216–219.
Levofloxacin is a third generation fluoroquinolone used in food-producing animals in some countries
outside of EU. The aim of the study was to investigate the residues of this antibiotic in chicken tissues
(meat, skin and giblets). Chicken (n=30) were treated with levofloxacin orally at a dose of 10 mg/kg
BW for 5 days. Birds were divided into 5 groups and humanely killed as followed: on day 0 (the day
after last administration of levofloxacin), 2, 4, 6 and 8 day. The results of the studies showed the
highest residues levels of levofloxacin in liver (1051 µg/kg), followed by breast muscle, gizzard, heart
and skin 428 µg/kg, 321 µg/kg, 303 µg/kg and 293 µg/kg, respectively. The rate of reduction of
antimicrobial activity was different. Tissue concentrations in heart and gizzard decreased faster than
these in muscles and skin. In the liver they decreased from the last day of the treatment to the 2nd
day and the levels remained nearly equal up to the 8
day after the end of drug administration.
Key words: chicken, levofloxacin,residues
Food-producing animals are treated with a
variety of veterinary drugs, including a
large number of different types of com-
pounds which can be administered in the
feed or in the drinking water. They are
applied in animal husbandry for different
reasons and may lead to residues in milk,
eggs and in other edible tissues. These
residues may include the non-altered par-
ent compound as well as metabolites
and/or conjugates, and have direct toxic
effects on consumers, e.g. allergic reac-
tions in hypersensitive individuals, or an-
tibiotics may cause problems indirectly
through selection of resistant strains of
bacteria (Fàbrega et al., 2008). For con-
trolling the residue problem, the EU has
set maximum residue limits (MRLs) for a
variety of veterinary drugs in milk, eggs
and tissues (Reig & Toldra, 2008; Petersa
et al., 2009).
Antibiotics are widely used in all farm
animals species and residues are often
found in meat, and they should not exceed
the maximal residue limits (Okerman et
al., 2000). Fluoroquinolones are a group
of synthetic antimicrobial agents widely
used both in human and veterinary medi-
cine. These agents exert their antibacterial
effect through the inhibition of DNA gy-
rase, interfering with the supercoiling of
bacterial chromosomal material. As a re-
sult, they have a broad spectrum of activ-
ity against Gram-negative and Gram-
R. Kyuchukova, V. Urumova, M. Lyutskanov, V. Petrov & A. Pavlov
BJVM, 16, Suppl. 1 217
positive bacteria, Mycoplasma spp. and
Rickettsia, including those resistant to
beta-lactam antibiotics and sulphonamides
(Brown, 1996; Ramos et al., 2003).
Since their discovery in the early
1960s, the quinolone group of antibacteri-
als has generated considerable clinical and
scientific interest. Nalidixic acid, the first
used quinolone was obtained as an impu-
rity during the manufacture of quinine.
Since that time, many derivatives have
been synthesised and evaluated for their
antibacterial potency (Andersson & Mac-
Gowan, 2003). A number of new fluoro-
quinolones have become available for use
worldwide since the initial introduction of
ciprofloxacin in the late 1980s (Appel-
baum & Hunter, 2000).
Levofloxacin is a third generation flu-
oroquinolone, an optical isomer of oflo-
xacin having two-fold higher antimicro-
bial activity than the parent compound.
Currently, it is successfully used in human
medicine in the treatment of infections of
upper and lower respiratory tract, genito-
urinary system, skin and soft tissue. This
compound has been applied in food-
producing animals (Patel et al., 2009).
However, the data about residues after
repeated oral administration of levofloxa-
cin in chickens are lacking. Therefore, the
present study was planned to investigate
the residues of this antibiotic in chicken
tissues (meat, skin and giblets).
The study was conducted on thirty chick-
ens aged two months. Chickens were
treated orally with levofloxacin at a dose
of 10 mg/kg BW via the drinking water
for 5 days. Water was provided ad libi-
tum. Birds were divided into 5 groups and
humanely killed on the day after last ad-
ministration of levofloxacin (day 0), and
on post treatment days 2, 4, 6 and 8.
Breast muscle, liver, gizzard, heart and
skin (with fats) were separated from each
carcass. Samples were weighed and ho-
mogenised with Maximum Recovery
Diluent (MRD, HIMEDIA, India) in an
amount equal to the mass of the sample,
then were centrifuged for 15 min at 2500
(for liver samples 20 min). The su-
pernatant was collected and dropped (100
µL) on a medium with the test microor-
ganism Escherichia coli ATCC 25922. It
was inoculated on plain agar (HIMEDIA,
India), previously sterilized and cooled to
50 °C, with concentration of cells 0.5 of
McFarland standard. Sterile plates (90
mm) were filled with 14 mL E.coli ATCC
25922 infected agar as described by
Okerman et al. (1998; 2007). After
incubation for 24 h at 37° C, the widths
of each inhibition zone were measured
from the edge of the sample to the edge of
the inhibition zone. Results were proc-
essed by GraphPad statistical software.
The results of the studies are presented in
Table 1. The data show that the highest
residues levels of levofloxacin were in the
liver (1051 µg/kg), followed by breast
muscle, gizzard, heart and skin 428
µg/kg, 321 µg/kg, 303 µg/kg and 293
µg/kg, respectively.
Decreasing concentrations of residues
were found in all investigated tissues up
to the 8
day after treatment. On the sec-
ond day the levels of gizzard and heart
were below the MRL for fluoroquinolo-
nes. In breast muscle and skin this was
observed on day 4 and in the liver levels
remained high until the 8
day after the
end of the treatment (rates with no statisti-
cally significant difference vs previous
Studies on levofloxacin residues in chicken meat and giblets
BJVM, 16, Suppl. 1
The rate of reduction of antimicrobial
activity was different for the various tis-
sues. Concentrations of levofloxacin in the
heart and in the gizzard decreased faster
in comparison to the levels in the muscle
and in the skin. After initial decrease in
the liver up to the 2
day after the treat-
ment they remained similar between days
2 and 8. These results were different from
our previous research on tissue concentra-
tion of gatifloxacin (Kyuchukova &
Pavlov, 2012) where the residue levels in
muscles, heart and gizzard were below
MRL on the second day.
Microbiological assays for investiga-
tion of antimicrobial residues are consid-
ered as multi-residue screening tests for
antibiotics in milk, meat or other animal
tissues. Karraouan et al. (2009) used a
microbiological method for the detection
of antibacterial substances in poultry mus-
cles. The method is based on the inhibi-
tion of Escherichia coli growth on agar.
and can be used as a screening method for
the detection of antibiotics in animal tis-
Devada et al. (2012) studied the safety
of gatifloxacin after repeated oral admini-
stration in broiler chickens and deter-
mined tissue concentration of the drug
following oral administration. The liver
concentration of gatifloxacin was 0.75±
0.04 µg/g after the fourth dose and
0.22±0.07 µg/g after the tenth dose, re-
spectively, whereas in skeletal muscles the
concentration of gatifloxacin was below
the limit of quantification after the fourth
dose. Gatifloxacin was not detected after
the tenth dose of.
In conclusion it should be noted that
the proposed withdrawal period of eight
days for levofloxacin is applicable in all
tissues with exception of the liver, where
relatively high values persisted after the
end of the study. Therefore we could state
that chicken meat producers have to keep
in mind withdrawal period of veterinary
drugs used in their farms.
Andersson, M. I. & A. P. MacGowan, 2003.
Dvelopment of the quinolones. Journal of
Antimicrobial Chemotherapy, 51, Suppl.
S1, 1–11.
Appelbaum, P. C. & P. A. Hunter, 2000. The
fluoroquinolone antibacterials: Past, pre-
sent and future perspectives. International
Journal of Antimicrobial Agents, 16, 5–
Brown, S. A., 1996. Fluoroquinolones in ani-
mal health. Journal of Veterinary Phar-
macology and Therapeutics, 19, 1–14.
Devada, S. S., U. D. Walunj, A. J. Patil, J. H.
Patel, S. K. Bhavsar & A. M. Thaker,
2012. Safety and tissue residue determi-
nation of gatifloxacin in broiler chicken.
Table 1. Levofloxacin residue levels (µg/kg) in the meat and in the giblets (mean ± SD; n=6)
Days after the end of the treatment
Tissue samples 0 2 4 6 8
Muscle 428 ± 253 105 ± 52 68 ± 10 68 ± 10 56 ± 15
Liver 1051 ± 648 206 ± 210 121 ± 93 106 ± 78 88 ± 31
Gizzard 321 ± 119 70 ± 11 41 ± 11 53 ± 18 61 ± 17
Heart 303 ± 210 50 ± 12 35 ± 8 30 ± 6 43 ± 10
Skin 293 ± 76 63 ± 24 75 ± 55 25 ± 8 25 ± 7
R. Kyuchukova, V. Urumova, M. Lyutskanov, V. Petrov & A. Pavlov
BJVM, 16, Suppl. 1 219
Journal of Advanced Veterinary Research,
2, 9–14.
Fàbrega, A., J. Sánchez-Céspedes, S. Soto & J.
Vila, 2008. Quinolone resistance in the
food chain. International Journal of Anti-
microbial Agents, 31, 307– 315.
Karraouan, B., B. Bouchrif, N. Ziyate, A.
Talmi, K. I. S, Yahia, N. Cohen & A.
Fassouane, 2009. Evaluation of multi-plate
microbial assay for the screening of anti-
bacterial residues in poultry muscle.
European Journal of Scientific Research,
35, 311–317.
Kyuchukova, R. & A. Pavlov, 2012. Kinetics
of the residue levels of gatifloxacin in
poultry meat at storage. Days of Veterinary
Medicine 2012, 3
International Scientific
Meeting, Books of Proceedings, p.293.
Okerman, L., H. Noppe, V. Cornet & L. Zut-
ter, 2007. Microbiological detection of 10
quinolone antibiotic residues and its appli-
cation to artificially contaminated poultry
samples. Food Additives and Contami-
nants, 24, 252–257.
Okerman, L., K. Wasch & J. V. Hoof, 1998.
Detection of antibiotics in muscle tissue
with microbiological inhibition tests:
Effects of the matrix. The Analyst, 123,
Okerman, G., K. D. Wasch, & J. V. Hoof,
2000. An inhibition test intended to detect
and to differentiate between penicillins,
cephalosporins, tetracyclines and qui-
nolones, for use in muscle tissue from dif-
ferent animal species. In: Proceedings of
Euroresidue IV, Veldhoven 7–10/5, pp.
Patel, J. H., R. D. Varia, U. D. Patel, P. D.
Vihol, S. K. Bhavsar & A. M. Thaker,
2009. Safety level of levofloxacin follow-
ing repeated oral adminstration in White
Leghorn layer birds. Veterinary World, 2,
Petersa, R. J. B., Y. J. C. Bolcka, P. Rutgersa,
A. A. M. Stolkera & M. W. F. Nielena,
2009. Multi-residue screening of veteri-
nary drugs in egg, fish and meat using
high-resolution liquid chromatography ac-
curate mass time-of-flight mass spectrome-
try. Journal of Chromatography A, 1216,
Ramos, M., A. Aranda, E. Garcia, T. Reuvers
& H. Hooghuis, 2003. Simple and sensi-
tive determination of five quinolones in
food by liquid chromatography with fluo-
rescence detection. Journal of Chromatog-
raphy B, 789, 373–381.
Reig, M. & F. Toldra, 2008. Veterinary drug
residues in meat: Concerns and rapid
methods for detection. Meat Science, 78,
Dr. Ralitsa Kyuchukova
Department of Food Hygiene and Control,
Veterinary Legislation and Management
Faculty of Veterinary Medicine
6000 Stara Zagora, Bulgaria
Studies on levofloxacin residues in chicken meat and giblets
BJVM, 16, Suppl. 1
R. Kyuchukova, V. Urumova, M. Lyutskanov, V. Petrov & A. Pavlov
BJVM, 16, Suppl. 1 221

File (1)

Content uploaded by Ralitsa Kyuchukova
Author content
... In the same antibiotic family, our study reported a significant influence of beef (O.R. 0.69; CI 0.533 -0.893) and chicken consumption (O.R. 1.965; C.I. 1.3970 -2.820) on the risk of resistance to levofloxacin. This is in line with findings by Kyuchukova et al. [30], who reported a high concentration of levofloxacin (428 µg/kg) in chicken meat samples from the end of antibiotic treatment up to 8 days, although the concentration tends to decrease with time (56 µg/kg). These findings suggest a high risk to public health and add to the hypothesis that these food products could be causing resistance in humans if withdrawal periods are not observed. ...
Full-text available
Background: Antibiotic resistance is a public health concern in Democratic Republic Congo and worldwide. It is usually caused by antibiotic over prescription or dispensing practices. The consumption of animal source food (ASF) could be another source of antibiotic resistance but is rarely studied. The objective of the study was to evaluate the eating habits of ASF by outpatients with antimicrobial resistance through an analysis of (i) the association of their antimicrobial resistance with ASF consumption; (ii) the influence of the types of ASF on their antimicrobial resistance. Methods: This is a retrospective analytical study conducted at three major Hospitals in Bukavu City (D. R. Congo). A total number of 210 patients, whose samples (mainly faeces and urine) had been subjected to bacterial examination, was included in this study. Morphological, biochemical and antibiotic susceptibility (using disc diffusion method) tests were performed on the samples. This served to isolate and identify resistant bacteria. Afterwards, patients responded to questions about the types and quantity of ASF eaten in the last week. We analysed data using descriptive statistics, logistic regression and non-parametric ranking tests. Results: Escherichia coli (37.1%), Klebsiella pneumonae (14.7%), and Staphylococcus aureus (13.8%) were the most prevalent bacteria. E. coli (68.4%) and K. pneumonae (87.5%) were multidrug resistant (MDR), while S. aureus (7.7%) was minor. Low beef (O.R. 0.737, C.I. 0.542-1.002) and pork (O.R. 0.743, C.I. 0.560 - 0.985) consumption led to significantly (p < 0.05) lower risks of resistance to ciprofloxacin. Patients eating three different ASF per week had the highest resistance score (20.67) and high consumption rates of goat meat, pork and milk (41.5%). Conclusion: The findings of this study suggest a contribution of human nutrition to antimicrobial resistance frequency. Our results show the existence of a high prevalence of multi-drug resistant bacteria in patients for which eating beef, pork and drinking milk are major risk factors. Therefore, a stricter control of antibiotic usage in livestock production and of their presence in ASF is recommended.
Aeromonas hydrophila is an opportunistic bacteria with an overwhelming impact on fish farming industry especially with upraising of drug resistant mutants. This study aimed to evaluate and compare the therapeutic and side effects of levofloxacin (LEV), chitosan-nanoparticles (CNPs), and fructooligosaccharides (FOS) in control of this infection in tilapia. A total of 160 Nile-tilapia divided into 8-groups; G1: negative-control, G2: infected-control, G3: non-infected-(levofloxacin (LEV) 10 mg/kg bwt), G4: non-infected-(chitosan-nanoparticles (CNPs) 1 g/kg ration), G5: non-infected-(fructooligosaccharides (FOS) 20 g/kg ration), G6: infected-LEV, G7: infected-CNPs and G8: infected-FOS for 7 days. MICs were (0.125 μg/ml and 1.25 mg/ml) for LEV and CNPs respectively. No mortalities or significant adverse effects were recorded in non-infected treated-groups while infected were (20%) LEV, (30%) CNPs, (40%) FOS and (70%) G2. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) decreased by LEV and CNPs than FOS while all increased total protein (TP) and albumin than G2. Malondialdehyde (MDA) significantly decreased and superoxide dismutase (SOD) and reduced glutathione (GSH) increased in all infected-treated groups than G2 in various degrees. Urea and creatinine descending order were FOS, LEV then CNPs decreased significantly than G2. LEV musculature residues, using HPLC, decreased gradually till the 5th day; 621.00 ± 0.66, 270.00 ± 0.48 then 64.00 ± 0.40, and 471.00 ± 0.79, 175.00 ± 0.52 ppb then not detected at 1st, 3rd, and 5th days of withdrawal in non-infected and infected groups respectively. Finally, LEV and CNPs were superior as bactericidal, decreasing mortalities and enzyme activities while CNPs and FOS increased performance, non-specific immunity, and antioxidant biomarkers.
A potent third-generation antimicrobial fluoroquinolone drug, levofloxacin was introduced into human clinical practice in 1993. Levofloxacin is also used in veterinary medicine, however its use is limited: it is completely banned for veterinary use in the EU, and used extralabel in only companion animals in the USA. Since its introduction to clinical practice, many studies have been published on levofloxacin in animal species, including pharmacokinetic studies, tissue drug depletion, efficacy, and animal microbial isolate susceptibility to levofloxacin. This literature overview highlights the most clinically relevant and scientifically important levofloxacin studies linked to the field of veterinary medicine.
1. The aim of this study was to assess the pharmacokinetics of levofloxacin, a third-generation fluoro-quinolone antimicrobial drug, in geese (n = 26) after either single intravenous or oral administration, and to evaluate the depletion profile in goose muscle, heart, liver, kidney and lung after a single oral dose. 2. The pharmacokinetic study involved 16 geese which were randomly divided into two groups (n = 8/group), the first received levofloxacin (2 mg/kg) intravenously while the second was treated with orally (5 mg/kg). The tissue depletion study involved 10 geese which were dosed orally (5 mg/kg) and two animals were killed at different time-points in order to collect the selected tissues. Levofloxacin was quantified in all the matrices tested by a validated high-performance liquid chromatography (HPLC) method, using a spectrofluorimetric detector. The pharmacokinetics were analysed using a non-compartmental model. 3. Plasma concentrations were quantified after up to 24 h in animals administered intravenously and up to 48 h after oral treatment. Levofloxacin was rapidly absorbed after oral administration (Tmax = 0.38 h) showing high bioavailability (95.57 ± 20.61%). The drug showed a moderate volume of distribution (1.40 ± 0.28 ml/g) and rapid clearance (0.28 ± 0.06 ml/g/h). No statistical differences in estimates were found between the two different administration methods (P > 0.05). Drug residues were highest at 6 h and decreased constantly up to 48 h in all the selected tissues. Liver and kidney had the highest levofloxacin concentrations. 4. According to the pharmacokinetic/pharmacodynamic surrogate index (AUC/MIC) the levofloxacin dose regimen (after oral administration) used in the present study could be active against bacteria at a minimum inhibitory concentration (MIC) > 0.24 μg/ml in geese. In addition, drug accumulation in the liver might be controlled using an estimated preliminary withdrawal time of 90 h.
Full-text available
The present paper describes a microbiological method for the detection of antibacterial substances in poulry muscle. The method is based on the growth inhibition of Bacillus subtilis on an agar medium at a pH of 6.0, 7.2, and 8.0, Bacillus cereus on an agar medium at a pH of 6.0, M. luteus on agar medium at a pH of 6.0 and 8.0, and Escherichia coli on agar medium at a pH of 7.2, and on the use of confirmatory solutions (Pase, Paba, MgSO4) for the identification. The proposed method detects the main antibiotic groups: β-lactams, tetracyclines, aminoglycosides, macrolides, and quinolones at low levels. The results are obtained in 18-24 h. This technique can be used as a screening method for the detection of antibiotics in animal tissue, but a more specific method would be necessary for full identification of antimicrobials in screening positive samples.
Full-text available
Levofloxacin is a fluorinated quinolone which has broad-spectrum antibacterial activity at low plasma/tissue concentration. The present study was designed to investigate safety of levofloxacin (10 mg/kg) after repeated oral administration at 12 hours interval for 14 days in layer birds (30-35 weeks old and weighing between 1.5-2.0 kg) and to determine tissue concentration of the drug following oral administration (10 mg/kg) for 5 days. Drug concentration in tissue was determined using High Performance Liquid Chromatography (HPLC). Repeated oral administration of levofloxacin in layer birds was found safe based on evaluation of haematological (Hb, PCV, TLC and DLC), blood biochemical (AST, ALT, AKP, ACP, LDH, BUN, Serum total protein, Serum albumin, Serum Creatinine, Blood glucose and Total bilirubin) and histopathology of liver, kidney and joint cartilage. Levofloxacin could not be detected in body tissues (liver and skeletal muscle) at 12 hours after the last administration. [Vet. World 2009; 2(4.000): 137-139]
Full-text available
Since their discovery in the early 1960s, the quinolone group of antibacterials has generated considerable clinical and scientific interest. Nalidixic acid, the first quinolone to be developed, was obtained as an impurity during the manufacture of quinine. Since this time, many derivatives have been synthesized and evaluated for their antibacterial potency. Two major groups of compounds have been developed from the basic molecule: quinolones and naphthyridones. Manipulations of the basic molecule, including replacing hydrogen with fluorine at position 6, substituting a diamine residue at position 7 and adding new residues at position 1 of the quinolone ring, have led to enhanced antibacterial efficacy. In general these compounds are well tolerated. However, some of these structural changes have been found to correlate with specific adverse events: the addition of fluorine or chlorine at position 8 being associated with photo-reactivity, e.g. Bay y 3118 and sparfloxacin; and the substitution of an amine or a methyl group at position 5 having a potential role in QTc prolongation, e.g. sparfloxacin and grepafloxacin. Progressive modifications in molecular configuration have resulted in improved breadth and potency of in vitro activity and pharmacokinetics. One of the most significant developments has been the improved anti-Gram-positive activity of the newer compounds, such as moxifloxacin and garenoxacin. In the current millennium, these new agents may play an important role in the treatment of respiratory infections.
A combination of three plates, seeded with resp. Micrococcus luteus, Bacillus cereus and Escherichia coli, can be used for detection of beta-lactam antibiotics, cephalosporins, tetracyclines and quinolones in pork, veal, beef, poultry and fish muscle tissue. The sensitivity of each plate is optimal for one or two antibiotic families, resulting in detection limits lower than the corresponding maximal residue limits and in distinct inhibition patterns typical for each antibiotic family. Beta-lactam antibiotics such as penillin G, ampicillin and amoxicillin, and the cephalosporin cephalexin give only inhibition zones on the plate with M. luteus. Two other cephalosporins, ceftiofur and cefquinome, are detected on this plate and on the plate seeded with E. coli. Tetracyclines are only detected up to the MRL level with B. cereus, and quinolones with E. coli. The method has important advantages compared to the four plate test, described in the EUR 15127-EN report Residues in food producing animals and their products: Reference Materials and Methods: the limits of detection are lower with the new plate combination and the inhibition pattern allows a presumptive identification of the antibiotic family involved. This enables the laboratory to select an appropriate chromatographic technique for identification and quantification of the growth inhibiting substance. Moreover the method can be used for examination of muscle tissue from mammals, birds as well as from fish.
The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.
The fluoroquinolones are a series of synthetic antibacterial agents that are undergoing extensive investigation for both human and veterinary use in the treatment of a variety of bacterial infections. These agents work through the inhibition of DNA gyrase, interfering with the supercoiling of bacterial chromosomal material. As a result, these agents are rapidly bactericidal primarily against gram-negative bacteria, mycoplasma, and some gram-positive bacteria, with most having little to no activity against group D streptococci and obligate anaerobic bacteria. Resistance develops slowly and is almost always chromosomal and not plasmid-mediated. However, development of resistance to the fluoroquinolones and transfer of that resistance among animal and human pathogens have become a hotly debated issue among microbiologists. The fluoroquinolones are a current antimicrobial class whose use in veterinary medicine is being scrutinized. From a pharmacokinetic perspective, these agents are variably but well absorbed from the gastrointestinal tract and almost completely absorbed from parenteral injection sites, and they are well distributed to various tissues in the body. The fluoroquinolones are metabolized and renally excreted, with many of them having approximately equal excretion by the hepatic and the renal excretory systems. The primary toxicity observed at therapeutic doses involves the gastrointestinal system and phototoxicity, although at higher doses central nervous system toxicity and ocular cataracts are observed. Administration to immature animals may result in erosive arthropathies at weight-bearing joints, and administration of high doses to pregnant animals results in maternotoxicity and occasionally embryonic death. The fluoroquinolones are approved for indications such as urinary tract infections and soft tissue infections in dogs and cats and colibacillosis in poultry. Approval for bovine respiratory disease in the United States is being sought. Other indications for which the fluoroquinolones have been used in animal health include deep-seated infections, prostatitis, and other bacterial infections resistant to standard antimicrobial therapy.
The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended for muscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated with aqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solution without tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones of the beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels the differences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappeared completely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected in spiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These results indicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intact meat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initially intended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screening purposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitive for beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolones enrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.
The history of the development of the quinolones is described from the first quinolone, nalidixic acid, via the first 6-fluorinated quinolone norfloxacin, to the latest extended-spectrum fluoroquinolones. The structural modifications made to the basic quinolone and naphthyridone nucleus and to the side chains have allowed improvements to be made such that the next group of fluoroquinolones after norfloxacin, exemplified by ciprofloxacin, had high activity against gram-negative species and a number of atypical pathogens, good-to-moderate activity against gram-positive species and were well absorbed and distributed. These compounds have been successfully used in the clinic for a decade and the size of the market has risen in recent years to only a little less than that for penicillins and macrolides. Notwithstanding the broad spectrum of these compounds, defects became evident. The growth in understanding of structure activity relationships with fluoroquinolones has enabled the development of even better compounds. The targets in fluoroquinolone research during the last few years include: improvements in pharmacokinetic properties, greater activity against gram-positive cocci and anaerobes, activity against fluoroquinolone-resistant strains, and improvements in activity against non-fermentative gram-negative species. The compounds developed in the recent years have fulfilled some but not all of these goals; improved bioavailability is one target achieved with most of the more recent compounds allowing for once-daily dosing. Gatifloxacin, moxifoxacin and trovafloxacin have all greatly improved the activity against gram-positive cocci, particularly pneumococci, and against anaerobes. They are not quite as active as ciprofloxacin against Enterobacteriaceae, and show no substantial improvements in activity against non-fermentative species. Clinafloxacin, gemifloxacin and sitafloxacin have even better activity against gram-positive cocci and are as active as ciprofloxacin against most gram-negatives, though gemifloxacin is less active than the other new compounds against gram-negative anaerobes. These three compounds do retain some activity against a number of ciprofloxacin-resistant species (gram-positive and gram-negative), but whether this activity will be adequate for clinical use is at present unclear. Both clinafloxacin and sitafloxacin contain a chloro substituent at position 8 of the quinolone nucleus. A halogen at this position in a number of compounds, though giving good activity, has also been associated with phototoxicity. Several fluoroquinolones have had to be withdrawn or strictly limited in their use post-marketing and in some cases no obvious relationship can be seen between the adverse effects and structural features, making this an area for urgent research.
A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C(18) column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile-0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile-0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g(-1), except for sarafloxacin which had a limit of 10 ng g(-1). Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r(2) higher than 0.999 for all quinolones.
To assess if microbiological inhibition tests for detection of antibiotic residues are suitable for routine screening for quinolone residues, the limit of detection (LOD) of 10 different quinolones and fluoroquinolones was determined. Two media were tested, one at pH 6 and the other at pH 8, each seeded with one of the following test strains: Bacillus subtilis, Escherichia coli or Bacillus cereus. LODs of the 10 substances were highest on plates seeded with B. cereus, intended for selective detection of tetracycline residues. The pattern of zones on the other four plates differed for the targeted quinolones: flumequine and oxolinic acid were detected at lower concentrations at pH 6, while the LODs of ciprofloxacin, enrofloxacin, danofloxacin, marbofloxacin, sarafloxacin and norfloxacin were lower at pH 8. Nine of the 10 quinolones were detected more easily with E. coli, but the LOD of difloxacin was lower with B. subtilis. Finally, the three most sensitive media were selected and fluid from chicken meat, spiked with eight quinolones near maximum residue limits (MRL), analysed on each plate. The plate seeded with E. coli at pH 8 detected five of eight quinolones at levels of interest, but an additional E. coli plate at pH 6 was necessary for detection of flumequine in species other than poultry and fish. None of the plates detected oxolinic acid and difloxacin at MRLs in muscle tissue.