- Email: [email protected]
A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces
International Journal of Antimicrobial Agents 23 (2004) 390–393
A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces Julie Sunderland a,∗ , Andrew M. Lovering a , Caroline M. Tobin a , Alasdair P. MacGowan a , John M. Roe b , Anne A. Delsol b a
b
Microbiology Department, Bristol Centre for Antimicrobial Research and Evaluation, Southmead Hostital, North Bristol NHS Trust, Bristol, BS10 5NB, UK Department of Clinical Veterinary Science, Division of Farm Animal Science, University of Bristol, Langford BS40 5DU, UK Received 23 June 2003; accepted 18 July 2003
Abstract A reverse-phase HPLC assay is described for the simultaneous assay of enrofloxacin (ENR) and ciprofloxacin (CPX) in pig faeces. Extraction used dichloromethane, 2-propanol and 0.3 M ortho-phosphoric acid (1:5:4 v/v/v). Separation was achieved using a Spherisorb S5 C8 column, heated to 50 ◦ C and a mobile phase of 0.16% ortho-phosphoric acid (adjusted to pH 3.0 with tetrabutylammonium hydroxide solution) with 20 ml acetonitrile per litre solution. The method used fluorescence detection (Ex 310 nm; Em 445 nm), a flow rate of 1 ml/min and a 20 l injection volume. Retention times were approximately 6 min for ciprofloxacin and 10 min for enrofloxacin. The linearity range for both compounds was 0–20 mg/kg, lowest limit of quantification 0.3 mg/kg and recoveries were >92%. © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Enrofloxacin; Ciprofloxacin; Metabolite; Pig faeces
1. Introduction Enrofloxacin (ENR) is a broad spectrum fluoroquinolone antibiotic available in both oral and parenteral formulations, which is used in veterinary practice for the treatment of respiratory and gastrointestinal infections. In several animal species, including pigs, ENR is de-ethylated to its primary metabolite, ciprofloxacin (CPX) [1] and both ENR and CPX are found in the bile and urine of animals receiving ENR [2]. In recent years, the potential of veterinary antibiotics to select for resistant micro-organisms and to promote transfer of resistance genes to pathogens of human medical importance has become of concern. This has prompted a number of groups to look at both the disposition of antibiotics in the gut, in animals and man, and their effect on the ecology of the bacterial flora found there [3]. Antibiotic disposition studies and research into the impact of antibiotic administration on the emergence of resistance have been hampered by difficulties in determining antibiotic ∗ Corresponding author. Present address: 3/19 Rosslare Promenade, Mindarie, WA6030, Australia. E-mail address: [email protected] (J. Sunderland).
concentrations in faecal material. In general, problems result from both inactivation [4] and binding of drug [5] to faecal material as well as from interference due to endogenous material. Although HPLC has proved a more specific and accurate method than bioassay techniques for the assay of ENR in serum and tissues of animals [6,7] there is only a single paper to support its use in studies to assess the faecal disposition of ENR and its metabolite CPX [8]. In this study, we describe a simple HPLC method for the simultaneous determination of ENR and CPX in pig faeces.
2. Materials and methods 2.1. Chemicals and reagents Enrofloxacin, ciprofloxacin (Bayer AG, Wuppertal, Germany), neomycin, lincomycin, tylosin (Sigma, Poole, UK) stock solutions were made up in deionised water and protected from light. Chlortetracycline (Sigma) was dissolved in 0.01 M H3 PO4 (BDH, Poole, UK) and avilamycin (Eli Lilly, Liverpool, UK) in acetonitrile (BDH). The extraction solution was based on that reported by Scholl et al. [9] and
0924-8579/$ – see front matter © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.07.014
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393
consisted of a mixture of dichloromethane, 2-propanol, and 0.3 M ortho-phosphoric acid (1:5:4 v/v/v). 2.2. HPLC Chromatography was performed on a Spherisorb S5 C8 4.6 mm × 100 mm column (Waters, Watford, UK) which was maintained at 50 ◦ C to reduce the retention time of both compounds. The mobile phase was pumped at a flow of 1 ml/min and was composed of 0.16% ortho-phosphoric acid (Sigma), adjusted to pH 3.0 with tetrabutylammonium hydroxide solution with 20 ml/l of acetonitrile added after the pH adjustment. Detection of both compounds was by fluorescence (Ex 310 nm; Em 445 nm) and an injection volume of 20 l was used. 2.3. Sample preparation and extraction method Samples of 100 mg of faeces were placed in glass homogenisers (Jencons Scientific, Leighton Buzzard, UK), 400 l of deionised water added and homogenised manually for 2 min. To each sample, 2 ml of extraction solution was added and the samples mixed. Samples were left at room temperature, in the dark, for 45 min before 1.5 ml of 0.07 M ortho-phosphoric acid was added. Samples were then mixed and allowed to stand for a further 10 min before being centrifuged at 5800 × g for 10 min and 20 l of the supernatant injected into the chromatograph.
391
neomycin, lincomycin, avilamycin and tylosin at a concentration equivalent to 4000 mg/l. Stability of ENR and CPX from faecal and aqueous samples was determined in the extraction solution over a 16.5 h period at +5 ◦ C at three concentrations of 0.3, 1.0 and 10.0 mg/l.
3. Results 3.1. Sensitivity, range and specificity The retention times for ENR and CPX were approximately 6 and 10 min, respectively, with a late running peak (endogenous material) often present at 26 min (Fig. 1). The lowest limit of quantification was 0.3 mg/kg, based on a signal-to-noise ratio of greater than three and a CV of <15%. The lowest limit of detection was 0.1 mg/kg for both compounds with a signal-to-noise ratio of >2 and CV <20%. There were no endogenous peaks from antibiotic-free faeces that could have potentially interfered with the determination
2.4. Assay validation Recovery and intra-assay reproducibility of ENR and CPX from faeces was determined using a set of six samples of antibiotic-free faeces from different pigs, six samples from the same pig and six aqueous samples, each spiked to final concentrations of 0.3, 0.5, 1.0, 5.0 and 20.0 mg/l for recovery studies and 0.3, 1.0 and 10.0 mg/l for reproducibility studies. Inter-assay reproducibility was assessed by extraction and assay on six separate days of spiked aqueous and faeces samples to a final concentration of 1.0 mg/l. The recovery from faeces at each concentration was calculated relative to the aqueous samples and the reproducibility assessed by determination of coefficients of variation for the six replicates. Assay linearity was determined from the visual inspection of the plot of peaks heights against concentration and from the correlation coefficient from the line of best fit. Accuracy, and percentage error, for the determination of both ENR and CPX in faecal samples (final concentrations: 0.7, 8.0 and 12.0 mg/kg) was calculated using a standard curve of prepared aqueous calibrators (0.0, 0.3, 0.5, 1.0, 5.0, 10.0 and 20.0 mg/l). To determine whether there was potential interference from either endogenous material or potentially co-administered antibiotics, faeces from six different pigs given antibiotic-free feed were assayed along with aqueous solutions of chlortetracycline,
Fig. 1. HPLC chromatogram of 10 mg/kg enrofloxacin (ENR) (retention time: 636 s) and ciprofloxacin (CPX) (retention time: 354 s) extracted from spiked pig faeces showing an endogenous late eluting peak (LEP).
392
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393
of ENR and CPX or from the assay of aqueous solutions of the following veterinary antibiotics: chlortetracycline, neomycin, lincomycin, avilamycin and tylosin. 3.2. Recovery and reproducibility The mean percentage recovery of ENR and CPX from six different pig faecal samples at 0.3, 0.5, 1.0, 5.0 and 20.0 mg/kg were; 97.6, 96.8, 98.9, 96.5 and 94.2% (ENR) and 92.7, 106.8, 101.2, 100.7 and 103.9% (CPX), respectively. No differences were seen in reproducibility between the six different and six identical faecal samples, with intra-assay reproducibility for aqueous samples spiked to final concentrations of 1.0 and 10.0 mg/l of <3.5% CV and spiked faecal samples <5% CV for both compounds. Intra-assay aqueous and faecal samples spiked to a final concentration of 0.3 mg/l had %CVs <11.5%. Inter-assay reproducibility over six separate experiments on different days for samples spiked with 1.0 mg/l of both compounds had %CVs <9% in faeces and <4.5% in aqueous. 3.3. Linearity and accuracy Assay linearity for faecal and aqueous samples was good over the range 0–20 mg/kg with correlation coefficients r 2 = 0.9989 (ENR aqueous), 0.9995 (CPX aqueous), 0.9995 (ENR faeces), 0.9998 (CPX faeces). Assay accuracy was also good with percentage errors for the assay of ENR and CPX in faeces of +3.8% (ENR), −6.9% (CPX) (0.7 mg/kg), −7.2% (ENR), −5.5% (CPX) (8.0 mg/kg) and −5.6% (ENR), −6.5% (CIP) (12.0 mg/kg). 3.4. Stability Over a period of 16.5 h at 5 ◦ C there was no significant degradation of either ENR or CPX following extraction from faecal material; the slope of the linear regression lines ranged from −0.00005 to 0.00005 which equates to <6.0% loss for both compounds at 1.0 and 10.0 mg/kg and <9% loss at 0.3 mg/kg.
the disposition and clearance of antimicrobials in the animal species receiving the agent. Although there is a large body of literature describing the pharmacokinetics of antimicrobials in animals, and also reporting the residue levels found in tissues at slaughter, relatively little has been published on either antimicrobial concentrations in the gut or the quantities of antimicrobial excreted in faeces. In those studies where faecal excretion of antimicrobials has been evaluated, issues such as recovery and binding to faecal material may give variable and potentially misleading results when a non-specific method such as bioassay is used. The quinolones appear to be particularly affected by these problems, with up to 95% of agents such as norfloxacin being reported as bound to faeces [10]. In this study, we have been able to develop and validate a HPLC method, which is both accurate and sensitive, and which allows complete recovery of enrofloxacin and its metabolite, ciprofloxacin, from pig faeces. The method used the extraction procedure described by Scholl et al. [9] for the recovery of ciprofloxacin from human faeces and gave extraction solutions in which both analytes were stable for at least 16 h at 5 ◦ C, permitting method automation for large-scale studies. The assay was specific for the assay of enrofloxacin and ciprofloxacin in the presence of a range of antimicrobials that are commonly administered in animal husbandry and allowed detection down to a limit of 0.3 mg/kg. Although this is higher than the typical MICs for sensitive strains, of <0.06 mg/l, it is below the concentrations reported for enrofloxacin in pig faeces and gut contents, where mean levels of up to 11.3 mg/kg have been reported [8]. We conclude, that HPLC analysis of enrofloxacin and ciprofloxacin is a sensitive method that is suitable for use in studies to examine the disposition of these agents in pig faeces and may be applicable to faecal recovery investigations in other species.
Acknowledgements We would like to acknowledge the contribution of our colleague Dr. Les White who initiated these studies. This study was funded by a grant from DEFRA.
4. Discussion For a number of years now, concerns have been expressed about the use, in animal husbandry, of antimicrobials with close analogues that are used in human medicine. Although in a number of cases, direct transfer of resistant organisms, and resistance genes from animal to human sources has been demonstrated [3], the impact on antimicrobial resistance in human pathogens from antimicrobial use in animals remains largely unquantified. This selective pressure can be exerted either directly from the action of antimicrobials on the gut flora of the animals receiving the antimicrobial, or indirectly from antimicrobials excreted into the environment. However, any attempts to study these need to address
References [1] Nielsen P, Gyrd-Hansen N. Bioavailability of enrofloxacin after oral administration to fed and fasted pigs. Pharmacol Toxicol 1997;80: 246–50. [2] Brown SA. Fluoroquinolones in animal health. J Vet Pharmacol Ther 1996;19:1–14. [3] Van den Bogaard AE, Stobberingh EE. Antibiotic usage in animals: impact on bacterial resistance and public health. Drugs 1999;58:589– 607. [4] Welling GW, Holtrop A, Slootmaker-van der Meulen C, MeijerSevers GJ, van Santen E, Tonk RH, et al. Inactivation of ceftriaxone by faecal enzyme preparations during ceftriaxone treatment. J Antimicrob Chemother 1992;30:234–6.
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393 [5] Hazenberg MP, Pennock-Schroder AM, Van den Boom M, Van de Merwe JP. Binding to and antibacterial effect of ampicillin, neomycin and polymyxin B on human faeces. J Hyg 1984;93:27–34. [6] Kung K, Riond J-L, Wolffram S, Wanner M. Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs. Res Vet Sci 1993;54:247–8. [7] Garcia MA, Solans C, Aramayona JJ, Reuda S, Bregante MA, de Jong A. Simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in plasma by HPLC with fluorescence detection. Biomed Chromatogr 1999;13:350–3.
393
[8] Wiuff C, Lykkesfeldt J, Aarestrup FM, Svendsen O. Distribution of enrofloxacin in intestinal tissue and contents of healthy pigs after oral and intramuscular administrations. J Vet Pharmacol Ther 2002;25:335–42. [9] Scholl H, Schmidt K, Weber B. Sensitive and selective determination of picogram amounts of ciprofloxacin and its metabolites in biological samples using high-performance liquid chromatography and photothermal post-column derivatization. J Chromatogr 1987;416: 321–30. [10] Edlund C, Lindqvist L, Nord CE. Norfloxacin binds to human fecal material. Antimicrob Agents Chemother 1988;32:1869–74.
A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces Julie Sunderland a,∗ , Andrew M. Lovering a , Caroline M. Tobin a , Alasdair P. MacGowan a , John M. Roe b , Anne A. Delsol b a
b
Microbiology Department, Bristol Centre for Antimicrobial Research and Evaluation, Southmead Hostital, North Bristol NHS Trust, Bristol, BS10 5NB, UK Department of Clinical Veterinary Science, Division of Farm Animal Science, University of Bristol, Langford BS40 5DU, UK Received 23 June 2003; accepted 18 July 2003
Abstract A reverse-phase HPLC assay is described for the simultaneous assay of enrofloxacin (ENR) and ciprofloxacin (CPX) in pig faeces. Extraction used dichloromethane, 2-propanol and 0.3 M ortho-phosphoric acid (1:5:4 v/v/v). Separation was achieved using a Spherisorb S5 C8 column, heated to 50 ◦ C and a mobile phase of 0.16% ortho-phosphoric acid (adjusted to pH 3.0 with tetrabutylammonium hydroxide solution) with 20 ml acetonitrile per litre solution. The method used fluorescence detection (Ex 310 nm; Em 445 nm), a flow rate of 1 ml/min and a 20 l injection volume. Retention times were approximately 6 min for ciprofloxacin and 10 min for enrofloxacin. The linearity range for both compounds was 0–20 mg/kg, lowest limit of quantification 0.3 mg/kg and recoveries were >92%. © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Enrofloxacin; Ciprofloxacin; Metabolite; Pig faeces
1. Introduction Enrofloxacin (ENR) is a broad spectrum fluoroquinolone antibiotic available in both oral and parenteral formulations, which is used in veterinary practice for the treatment of respiratory and gastrointestinal infections. In several animal species, including pigs, ENR is de-ethylated to its primary metabolite, ciprofloxacin (CPX) [1] and both ENR and CPX are found in the bile and urine of animals receiving ENR [2]. In recent years, the potential of veterinary antibiotics to select for resistant micro-organisms and to promote transfer of resistance genes to pathogens of human medical importance has become of concern. This has prompted a number of groups to look at both the disposition of antibiotics in the gut, in animals and man, and their effect on the ecology of the bacterial flora found there [3]. Antibiotic disposition studies and research into the impact of antibiotic administration on the emergence of resistance have been hampered by difficulties in determining antibiotic ∗ Corresponding author. Present address: 3/19 Rosslare Promenade, Mindarie, WA6030, Australia. E-mail address: [email protected] (J. Sunderland).
concentrations in faecal material. In general, problems result from both inactivation [4] and binding of drug [5] to faecal material as well as from interference due to endogenous material. Although HPLC has proved a more specific and accurate method than bioassay techniques for the assay of ENR in serum and tissues of animals [6,7] there is only a single paper to support its use in studies to assess the faecal disposition of ENR and its metabolite CPX [8]. In this study, we describe a simple HPLC method for the simultaneous determination of ENR and CPX in pig faeces.
2. Materials and methods 2.1. Chemicals and reagents Enrofloxacin, ciprofloxacin (Bayer AG, Wuppertal, Germany), neomycin, lincomycin, tylosin (Sigma, Poole, UK) stock solutions were made up in deionised water and protected from light. Chlortetracycline (Sigma) was dissolved in 0.01 M H3 PO4 (BDH, Poole, UK) and avilamycin (Eli Lilly, Liverpool, UK) in acetonitrile (BDH). The extraction solution was based on that reported by Scholl et al. [9] and
0924-8579/$ – see front matter © 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2003.07.014
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393
consisted of a mixture of dichloromethane, 2-propanol, and 0.3 M ortho-phosphoric acid (1:5:4 v/v/v). 2.2. HPLC Chromatography was performed on a Spherisorb S5 C8 4.6 mm × 100 mm column (Waters, Watford, UK) which was maintained at 50 ◦ C to reduce the retention time of both compounds. The mobile phase was pumped at a flow of 1 ml/min and was composed of 0.16% ortho-phosphoric acid (Sigma), adjusted to pH 3.0 with tetrabutylammonium hydroxide solution with 20 ml/l of acetonitrile added after the pH adjustment. Detection of both compounds was by fluorescence (Ex 310 nm; Em 445 nm) and an injection volume of 20 l was used. 2.3. Sample preparation and extraction method Samples of 100 mg of faeces were placed in glass homogenisers (Jencons Scientific, Leighton Buzzard, UK), 400 l of deionised water added and homogenised manually for 2 min. To each sample, 2 ml of extraction solution was added and the samples mixed. Samples were left at room temperature, in the dark, for 45 min before 1.5 ml of 0.07 M ortho-phosphoric acid was added. Samples were then mixed and allowed to stand for a further 10 min before being centrifuged at 5800 × g for 10 min and 20 l of the supernatant injected into the chromatograph.
391
neomycin, lincomycin, avilamycin and tylosin at a concentration equivalent to 4000 mg/l. Stability of ENR and CPX from faecal and aqueous samples was determined in the extraction solution over a 16.5 h period at +5 ◦ C at three concentrations of 0.3, 1.0 and 10.0 mg/l.
3. Results 3.1. Sensitivity, range and specificity The retention times for ENR and CPX were approximately 6 and 10 min, respectively, with a late running peak (endogenous material) often present at 26 min (Fig. 1). The lowest limit of quantification was 0.3 mg/kg, based on a signal-to-noise ratio of greater than three and a CV of <15%. The lowest limit of detection was 0.1 mg/kg for both compounds with a signal-to-noise ratio of >2 and CV <20%. There were no endogenous peaks from antibiotic-free faeces that could have potentially interfered with the determination
2.4. Assay validation Recovery and intra-assay reproducibility of ENR and CPX from faeces was determined using a set of six samples of antibiotic-free faeces from different pigs, six samples from the same pig and six aqueous samples, each spiked to final concentrations of 0.3, 0.5, 1.0, 5.0 and 20.0 mg/l for recovery studies and 0.3, 1.0 and 10.0 mg/l for reproducibility studies. Inter-assay reproducibility was assessed by extraction and assay on six separate days of spiked aqueous and faeces samples to a final concentration of 1.0 mg/l. The recovery from faeces at each concentration was calculated relative to the aqueous samples and the reproducibility assessed by determination of coefficients of variation for the six replicates. Assay linearity was determined from the visual inspection of the plot of peaks heights against concentration and from the correlation coefficient from the line of best fit. Accuracy, and percentage error, for the determination of both ENR and CPX in faecal samples (final concentrations: 0.7, 8.0 and 12.0 mg/kg) was calculated using a standard curve of prepared aqueous calibrators (0.0, 0.3, 0.5, 1.0, 5.0, 10.0 and 20.0 mg/l). To determine whether there was potential interference from either endogenous material or potentially co-administered antibiotics, faeces from six different pigs given antibiotic-free feed were assayed along with aqueous solutions of chlortetracycline,
Fig. 1. HPLC chromatogram of 10 mg/kg enrofloxacin (ENR) (retention time: 636 s) and ciprofloxacin (CPX) (retention time: 354 s) extracted from spiked pig faeces showing an endogenous late eluting peak (LEP).
392
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393
of ENR and CPX or from the assay of aqueous solutions of the following veterinary antibiotics: chlortetracycline, neomycin, lincomycin, avilamycin and tylosin. 3.2. Recovery and reproducibility The mean percentage recovery of ENR and CPX from six different pig faecal samples at 0.3, 0.5, 1.0, 5.0 and 20.0 mg/kg were; 97.6, 96.8, 98.9, 96.5 and 94.2% (ENR) and 92.7, 106.8, 101.2, 100.7 and 103.9% (CPX), respectively. No differences were seen in reproducibility between the six different and six identical faecal samples, with intra-assay reproducibility for aqueous samples spiked to final concentrations of 1.0 and 10.0 mg/l of <3.5% CV and spiked faecal samples <5% CV for both compounds. Intra-assay aqueous and faecal samples spiked to a final concentration of 0.3 mg/l had %CVs <11.5%. Inter-assay reproducibility over six separate experiments on different days for samples spiked with 1.0 mg/l of both compounds had %CVs <9% in faeces and <4.5% in aqueous. 3.3. Linearity and accuracy Assay linearity for faecal and aqueous samples was good over the range 0–20 mg/kg with correlation coefficients r 2 = 0.9989 (ENR aqueous), 0.9995 (CPX aqueous), 0.9995 (ENR faeces), 0.9998 (CPX faeces). Assay accuracy was also good with percentage errors for the assay of ENR and CPX in faeces of +3.8% (ENR), −6.9% (CPX) (0.7 mg/kg), −7.2% (ENR), −5.5% (CPX) (8.0 mg/kg) and −5.6% (ENR), −6.5% (CIP) (12.0 mg/kg). 3.4. Stability Over a period of 16.5 h at 5 ◦ C there was no significant degradation of either ENR or CPX following extraction from faecal material; the slope of the linear regression lines ranged from −0.00005 to 0.00005 which equates to <6.0% loss for both compounds at 1.0 and 10.0 mg/kg and <9% loss at 0.3 mg/kg.
the disposition and clearance of antimicrobials in the animal species receiving the agent. Although there is a large body of literature describing the pharmacokinetics of antimicrobials in animals, and also reporting the residue levels found in tissues at slaughter, relatively little has been published on either antimicrobial concentrations in the gut or the quantities of antimicrobial excreted in faeces. In those studies where faecal excretion of antimicrobials has been evaluated, issues such as recovery and binding to faecal material may give variable and potentially misleading results when a non-specific method such as bioassay is used. The quinolones appear to be particularly affected by these problems, with up to 95% of agents such as norfloxacin being reported as bound to faeces [10]. In this study, we have been able to develop and validate a HPLC method, which is both accurate and sensitive, and which allows complete recovery of enrofloxacin and its metabolite, ciprofloxacin, from pig faeces. The method used the extraction procedure described by Scholl et al. [9] for the recovery of ciprofloxacin from human faeces and gave extraction solutions in which both analytes were stable for at least 16 h at 5 ◦ C, permitting method automation for large-scale studies. The assay was specific for the assay of enrofloxacin and ciprofloxacin in the presence of a range of antimicrobials that are commonly administered in animal husbandry and allowed detection down to a limit of 0.3 mg/kg. Although this is higher than the typical MICs for sensitive strains, of <0.06 mg/l, it is below the concentrations reported for enrofloxacin in pig faeces and gut contents, where mean levels of up to 11.3 mg/kg have been reported [8]. We conclude, that HPLC analysis of enrofloxacin and ciprofloxacin is a sensitive method that is suitable for use in studies to examine the disposition of these agents in pig faeces and may be applicable to faecal recovery investigations in other species.
Acknowledgements We would like to acknowledge the contribution of our colleague Dr. Les White who initiated these studies. This study was funded by a grant from DEFRA.
4. Discussion For a number of years now, concerns have been expressed about the use, in animal husbandry, of antimicrobials with close analogues that are used in human medicine. Although in a number of cases, direct transfer of resistant organisms, and resistance genes from animal to human sources has been demonstrated [3], the impact on antimicrobial resistance in human pathogens from antimicrobial use in animals remains largely unquantified. This selective pressure can be exerted either directly from the action of antimicrobials on the gut flora of the animals receiving the antimicrobial, or indirectly from antimicrobials excreted into the environment. However, any attempts to study these need to address
References [1] Nielsen P, Gyrd-Hansen N. Bioavailability of enrofloxacin after oral administration to fed and fasted pigs. Pharmacol Toxicol 1997;80: 246–50. [2] Brown SA. Fluoroquinolones in animal health. J Vet Pharmacol Ther 1996;19:1–14. [3] Van den Bogaard AE, Stobberingh EE. Antibiotic usage in animals: impact on bacterial resistance and public health. Drugs 1999;58:589– 607. [4] Welling GW, Holtrop A, Slootmaker-van der Meulen C, MeijerSevers GJ, van Santen E, Tonk RH, et al. Inactivation of ceftriaxone by faecal enzyme preparations during ceftriaxone treatment. J Antimicrob Chemother 1992;30:234–6.
J. Sunderland et al. / International Journal of Antimicrobial Agents 23 (2004) 390–393 [5] Hazenberg MP, Pennock-Schroder AM, Van den Boom M, Van de Merwe JP. Binding to and antibacterial effect of ampicillin, neomycin and polymyxin B on human faeces. J Hyg 1984;93:27–34. [6] Kung K, Riond J-L, Wolffram S, Wanner M. Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs. Res Vet Sci 1993;54:247–8. [7] Garcia MA, Solans C, Aramayona JJ, Reuda S, Bregante MA, de Jong A. Simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in plasma by HPLC with fluorescence detection. Biomed Chromatogr 1999;13:350–3.
393
[8] Wiuff C, Lykkesfeldt J, Aarestrup FM, Svendsen O. Distribution of enrofloxacin in intestinal tissue and contents of healthy pigs after oral and intramuscular administrations. J Vet Pharmacol Ther 2002;25:335–42. [9] Scholl H, Schmidt K, Weber B. Sensitive and selective determination of picogram amounts of ciprofloxacin and its metabolites in biological samples using high-performance liquid chromatography and photothermal post-column derivatization. J Chromatogr 1987;416: 321–30. [10] Edlund C, Lindqvist L, Nord CE. Norfloxacin binds to human fecal material. Antimicrob Agents Chemother 1988;32:1869–74.