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Investigation of the persistence of florfenicol residues in bovine milk and fate during processing
International Dairy Journal 39 (2014) 270e275
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Investigation of the persistence of florfenicol residues in bovine milk and fate during processing Clare Power a, e, Ríona Sayers c, Martin Danaher b, Mary Moloney b, Bernadette O'Brien d, Ambrose Furey e, Kieran Jordan a, * a
Food Safety Department, Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland d Livestock Systems Department, Animal and Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland e Team Elucidate, Department of Chemistry, Cork Institute of Technology, Bishopstown, Cork, Ireland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2013 Received in revised form 25 July 2014 Accepted 25 July 2014 Available online 2 August 2014
The persistence of florfenicol residues in milk and their migration to dairy products was investigated following treatment of six lactating dairy cows with Selectan 300 mg mL1 solution for injection at a dose of 1 mL 15 kg body weight1. Florfenicol residues were measurable (LOQ 1.0 mg kg1) in the milk of three cows for up to 27 days post-treatment but were not detectable at day 34. On day 3 post-treatment, the milk was pooled into two independent aliquots, each containing the milk from three cows. One part of each aliquot was pasteurised. Milk products were manufactured from the pasteurised and unpasteurised aliquots. The florfenicol residue concentration in cheese was similar to the initial milk (989 versus 845 mg kg1, respectively). Residues in skim-milk powder were approximately eight-fold higher than the skim milk used for its manufacture (8927 versus 1089 mg kg1, respectively). © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Florfenicol (FLOR) is a broad spectrum antibiotic licensed for use in veterinary medicine. It has bacteriostatic properties in that it prevents protein synthesis in bacterial cells (Atef, El-Gendi, Amer, & Abd El-Aty, 2010; Lim et al., 2010; Xie et al., 2011). Chloramphenicol, which belongs to the same pharmacological group as FLOR pez, & Gutie rrez, 2010; Schwarz, Kehrenberg, (Ruiz, Zapata, Lo Doublet, & Cloeckaert, 2004), was a widely used human medicine until reports of aplastic anaemia arising from its use led to the introduction of FLOR as a safer antibiotic. Because of the risk of toxicity to humans, chloramphenicol cannot be used in food-producing animals (Settepani, 1984; Stolker & Brinkman, 2005; Su et al., 2011). While the mechanisms of antibacterial activity of FLOR and chloramphenicol are similar (Atef et al., 2010), the presence of a fluorine atom in FLOR makes it resistant to deactivation by bacterial transmissible plasmids (Ruiz et al., 2010). In addition, because of its lipophilicity, FLOR demonstrates good tissue penetration increasing its activity against bovine respiratory disease
* Corresponding author. Tel.: þ353 25 42451. E-mail address: [email protected] (K. Jordan). http://dx.doi.org/10.1016/j.idairyj.2014.07.012 0958-6946/© 2014 Elsevier Ltd. All rights reserved.
(BRD) (Schwarz et al., 2004). Therefore, FLOR is regarded as an important drug in the therapeutic control of BRD infections. The metabolism and persistence of FLOR has been studied by other groups in edible tissues (Atef et al., 2010; Lim et al., 2010). In the EU, the marker residue has been defined as the sum of FLOR and its metabolites measured as florfenicol amine, FLOR-A (Anon, 2010). Although the pharmacokinetics of FLOR have been extensively studied in animals (Atef et al., 2010), there are few reports in the literature concerning the persistence of FLOR residues in milk, or on the migration from milk during manufacture of products. The widespread use of antibiotics, such as FLOR, for therapy and disease prevention in food-producing animals raises concern about the potential of antibiotic residues in food (Franje et al., 2010) that are potentially harmful to consumers (Damte et al., 2012). Maximum residue limits (MRLs), which are the maximum concentration of a veterinary drug residue legally permissible in food, are proposed by the European Medicines Agency (Anon, 2009, 2010) to protect public health. Withdrawal periods can be subsequently defined to ensure residue levels in a foodstuff are below the MRL (Damte et al., 2012; Moreno, Imperiale, Mottier, Alvarez, & Lanusse, 2005; Veterinary Medicines Directorate, 2008). At present, no MRL has been set for FLOR in milk, therefore any concentration level of this drug detected in milk would constitute a noncompliant result.
C. Power et al. / International Dairy Journal 39 (2014) 270e275
To add to the current, limited information on the presence of FLOR residues in milk and dairy products following treatment, the objectives of this study were to investigate the persistence of FLOR residues in milk following the treatment of lactating dairy cows, to measure residue concentrations in dairy products produced from the milk of treated cows, and to assess the stability of such residues in product during storage. 2. Materials and methods 2.1. Chemicals and reagents Methanol (MeOH), acetonitrile (MeCN; LC-MS grade), propan-2ol (IPA; chromasolv grade), sodium hydroxide pellets (puriss p.a.), ammonium formate, magnesium sulphate anhydrous and florfenicol (FLOR) analytical standard for drug analysis were all purchased from SigmaeAldrich (Dublin, Ireland). Florfenicol amine (FLOR-A) came from Witega (Berlin, Germany). Sodium chloride p.a. came from Applichem GmbH (Darmstadt, Germany) and glacial acetic acid, 100% anhydrous was sourced from Merck (Darmstadt, Germany). Ultra-pure water (18.2 MU cm1) was generated inhouse by using a Millipore (Cork, Ireland) water purification system. Syringe filters (0.2 mm, 13 mm, PTFE) were also supplied by Millipore. Polypropylene centrifuge tubes (50 mL and 15 mL) with screw caps were obtained from Sarstedt Ltd. (Wexford, Ireland). A Sartorius Extend top pan balance was employed for weighing milk samples (Sartorius, Dublin, Ireland). A glass dispenser from BrandTech (Essex, CT, USA) was used for aliquoting acetonitrile. A Mistral 3000i centrifuge (Davidson and Hardy, Dublin, Ireland), a multi-vortexer (VWR International, Dublin, Ireland) and a Turbovap
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LV (Caliper Life Sciences, Runcorn, UK) were used during sample extraction. 2.2. Animal trials A total of six lactating dairy cows, three Holstein Friesian, two Montbeliard and one Norwegian Red, was administered Selectan (Laboratorios Hipra S.A., Girona, Spain), a 300 mg mL1 solution of FLOR, by intramuscular injection, as approved by the Irish Medicines Board (IMB). Each individual cow was weighed and Selectan administered at a rate of 20 mg kg1 body weight (1 mL of Selectan 15 kg body weight1). Selectan was administered on two occasions, 48 h apart as per the IMB protocol (Irish Medicines Board, 2014). The appropriate dose for cattle is 20 mg kg1 body weight (1 mL of the product 15 kg1) to be administered twice (48 h apart) by the intramuscular route. For treatment of cattle over 150 kg body weight, the dose is divided so that no more than 10 mL are injected at one site. Prior to administration, a 50 mL milk sample was collected from each cow as a control. After administration, milk samples were taken twice daily (morning and evening) until there was no detectable FLOR marker residues in two consecutive milk samples. Samples were labelled on collection, refrigerated at 4 C and analysed within 4 days of being collected. 2.3. Design of dairy processing experiments The cows were divided into two groups of three cows (balanced by breed as much as possible) and all the milk from each group was collected and pooled on day 3 following the second administration of FLOR. The milk used for the manufacture of dairy products
Fig. 1. Flowchart of the process of product manufacture from milk containing florfenicol residues.
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C. Power et al. / International Dairy Journal 39 (2014) 270e275
contained high FLOR residue concentrations (670 ± 48 mg kg1). Each milk pool, using the milk from 3 cows, was further subdivided and one part was pasteurised at (72 C 15 s) while the second remained unpasteurised. Semi-soft laboratory scale cheese was manufactured using milk from each pasteurised and unpasteurised sub-divided pool. Similarly, a separate portion of the same milk used in cheese manufacture, while another was separated into skim milk and cream fractions and butter and skim-milk powder were manufactured. Buttermilk was collected from this butter manufacturing process. A summary of the milk product manufacture completed in this study is outlined in Fig. 1. 2.4. Manufacture of cheese, skim milk, cream, butter and skim-milk powder Cheese was manufactured from pasteurised and unpasteurised milk using a method previously described (Power et al., 2013). For the separation of whole milk to skim milk and cream, milk was heated to 50 C and separated using a Disc Bowl Centrifuge (Armfield, Ringwood, UK). Cream and skim-milk fractions were collected. Following the separation of milk into skim and cream, the cream was chilled and whisked in a food blender until the cream separated into buttermilk and butter. Buttermilk, butter, skim and cream samples were analysed (as described below) from fresh product at day 0. Skim-milk powder was manufactured using pasteurised and unpasteurised milk using the method previously described (Power et al., 2013). 2.5. Stability of florfenicol residues in dairy products The stability to freezing of FLOR marker residues in samples was studied by analysing fresh product on the day of manufacture, and then freezing it at 20 C and analysing the samples again after 6 and 12 months. Skim-milk powder was stored in 50 mL centrifuge tubes in the dark at ambient temperature (18e22 C). Samples of powder were taken at day 0, 6 and 12 months and analysed for the presence of FLOR residues. The stability of residues during cheese ripening (at 14 C) and butter storage (at 5 C) was studied by analysing samples over several weeks. 2.6. Sample preparation prior to analysis
steel replacement filter (all from Waters, Milford, MA, USA). The column was maintained at a temperature of 50 C and the pump was operated at a flow rate of 0.4 mL min1. A binary gradient system was used to separate analytes comprising mobile phase A, 0.1% acetic acid in H2O (v/v) and mobile phase B, 5 mM ammonium formate in MeOH:MeCN (75:25, v/v). The gradient profile was as follows: 0e1.25 min, 99.5% A; 1.5e2.0 min, 10% A; 2.05e3.0 min, 99.5% A. The injection volume was 2.5 mL. UHPLC weak and strong washes consisted of H2O:MeOH (90:10, v/v, 1000 mL) and MeOH:IPA:H2O (80:10:10, v/v, 750 mL), respectively. FLOR and FLOR-A residues were detected using a Waters Quattro Premier XE triple quadrupole mass spectrometer operating in electrospray ionisation (ESI) mode. Nitrogen was used for nebulisation, desolvation (650 L h1) and cone gas (50 L h1). Argon was used as collision gas (0.013 L h1). The source temperature was set at 90 C and desolvation temperature at 300 C. The capillary voltage was set at 3.0 kV. The SRM windows were time sectored, and dwell time, interscan delay and inter-channel delays were set to get maximum response from the instrument. UHPLC-tandem mass spectrometry (MS/MS) experimental conditions are summarised in Table 1. The UHPLCeMS/MS system was controlled by MassLynx™ software and data were processed using TargetLynx™ Software (both from Waters). 2.8. Calibration and controls FLOR and FLOR-A were prepared in methanol at a concentration of 2 mg mL1. Working standard solutions containing both FLOR and FLOR-A were prepared at concentrations of 100.0, 50.0, 25.0, 10.0, 5.0, 2.5, 1.0, 0.5, 0.25 and 0.1 mg mL1. Extracted milk matrix calibrants were prepared by spiking negative milk samples (10 g) with 100 mL of working standard solutions prior to extraction. This gave 10 point calibration curves in the range 1.0e1,000 mg kg1 (milk, buttermilk, skim milk and whey) and 10.0e10,000 mg kg1 (butter, cheese, cream, curd, and powder). An additional four blank milk samples (recovery controls) were spiked after evaporation, two with 50 mL of standard 2 (0.25 mg mL1) and two with standard 7 (10 mg mL1) to monitor for the loss of analytes during extraction. This gave concentrations of 2.5 and 100 mg kg1 for milk, buttermilk, skim milk and whey, or 25 and 1000 mg kg1 for butter, cheese, cream, curd, and powder, respectively. FLOR marker residues are expressed as FLOR-A equivalents using the equation
X
All samples were prepared for analysis as described by Power et al. (2013). 2.7. Ultrahigh performance liquid chromatography tandem mass spectrometry analysis All sample separations were performed as described by Whelan et al. (2010) using an Acquity ultrahigh performance liquid chromatography (UHPLC) system comprising a stainless steel HSS T3 analytical column (100 mm 2.1 mm, particle size 1.8 mm) equipped with an in-line filter unit containing a 0.2 mm stainless
FLOR marker residues mg kg1
¼ ðFLOR 0:69Þ þ FLOR A
(1)
The factor, 0.69, is calculated by dividing the molecular weight of FLOR-A (247.29 g mol1) by the molecular weight of FLOR (358.21 g mol1). 2.9. Statistical analyses All statistical analyses were carried out using SAS (version 9.1.3, SAS Institute, Cary, NC, USA). A randomised design that
Table 1 Ultrahigh pressure liquid chromatography tandem mass spectrometry conditions used for analysis of florfenicol and florfenicol amine residues.a Analyte
tR (min)
Function (min)
Transition (m/z)
FLOR-A
2.22
1:0e1.8 min
FLOR
2.22
2:1.8e3 min
247.9 247.9 355.7 355.7 357.7 357.7
a
/ / / / / /
129.9 229.9 185.0 335.7 185.0 337.8
Abbreviations are: tR, retention time (min); CE, collision energy; FLOR, florfenicol.
Polarity
Dwell time (s)
Cone (V)
CE (V)
þ
0.02 0.005 0.1
28
22 13 18 10 18 10
30
C. Power et al. / International Dairy Journal 39 (2014) 270e275
incorporated the treatment (pasteurised and unpasteurised) and (effect of residue over time) was used. The response variables related to the cheese and butter manufactured using pasteurised and unpasteurised milk. Pooling of the milk into two groups resulted in small sample sizes, which impacted on the assumptions of normality. Therefore, the medians (50th percentile) and interquartile ranges (IQR; 25e75th percentile), rather than the means and standard deviations, were used to express measures of central tendency, and nonparametric statistical methods were used. Statistically significant differences (p < 0.05) between different treatment levels were determined by using KruskaleWallis nonparametric one-way analysis of variance and the ManneWhitneyeWilcoxon test to compare group differences. 2.10. Validation Validation of the method for accuracy and precision was conducted in bovine milk, ovine milk, caprine milk, cottage cheese manufactured from bovine milk, and organic butter manufactured from bovine milk (all purchased at Superquin, Ashtown, Dublin, Ireland, except for the ovine milk that was obtained directly from the farm of Henry Clifton Brown, Tipperary, Ireland). Concentrations of 2.50 mg kg1, 3.75 mg kg1 and 5.00 mg kg1 for florfenicol (in duplicate) and florfenicol amine were added to the milks and bovine milk products under validation. 3. Results 3.1. Validation The validation results as depicted in Table 2, show that accuracy values were generally within the acceptable limits of 70e110% for ovine milk, caprine milk and cheese for FLOR. However for bovine milk and low butter concentrations, accuracy values were marginally outside the acceptable limits. FLOR-A accuracy values were within range, with the exception of the values obtained for
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cheese, where they were slightly elevated. While precision, measured as the coefficient of variation (CV), showed results between 2% and 15.1% with the exception being the precision values of FLOR-A in cheese, which were slightly elevated at between 17.5% and 22.2%.
3.2. Florfenicol marker residues in milk Control milk samples taken from each animal and analysed prior to administration resulted in no detectable FLOR marker residues. The results in Fig. 2 show an increase in FLOR marker residues at day 2.5 which corresponds to the first milk samples collected following the second treatment. The highest concentration of summed FLOR marker residues (expressed as FLOR-A) measured in samples from individual cows ranged from 865 to 1871 mg kg1. FLOR marker residues were measurable in the milk of three animals (0.8, 1.0 and 1.4 mg kg1, FLOR-A equivalents) at day 27, but were below the limit of quantification of 1.0 mg kg1 at later time-points. The major residue found in the milk of dairy cows following treatment was FLOR parent drug. FLOR-A residues were also measurable in milk but occurred at lower concentrations (highest concentration of 125 to 380 mg kg1 in the milk at 2.5 days posttreatment) and were not quantifiable at >19.5 days. 3.3. Transfer of florfenicol marker residues into cheese The migration of FLOR marker residues during cheesemaking using pasteurised and unpasteurised milk containing residue concentrations is shown in Table 3. FLOR marker residue levels were similar in the starting milk to the curd and whey produced during the cheesemaking process. The transfer of residue to curd, whey and cheese made from pasteurised milk was not significantly different from cheese made with unpasteurised milk (p > 0.05). The concentration of residue detected in the final cheese was similar to that detected in the starting curd.
Table 2 Validation results for florfenicol and florfenicol amine in milk (bovine, ovine and caprine), cheese and butter samples (n ¼ 7). FLOR (mg kg1)
Parameter
Bovine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Ovine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Caprine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Butter Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Cheese Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%)
kg1)
kg1)
kg1)
kg1)
kg1)
Florfenicol amine (mg kg1)
2.5
3.75
5.00
2.5
3.75
5.00
2.92 0.25 8.5 118
4.35 0.31 7.1 116
5.64 0.39 6.9 113
2.34 0.27 11.5 94
3.89 0.59 15.1 104
5.52 0.59 10.7 110
2.63 0.27 10.2 105
3.65 0.32 8.7 97
4.85 0.29 6.0 97
2.75 0.39 14.0 110
3.58 0.54 15.1 96
4.52 0.61 13.4 91
2.69 0.12 4.0 108
3.94 0.14 3.1 105
5.04 0.41 2.0 101
2.53 0.26 10.1 101
3.68 0.23 6.2 98
4.92 0.46 9.3 98
2.80 0.11 4.1 112
3.92 0.23 5.9 105
5.47 0.21 3.8 109
2.61 0.20 7.7 104
3.71 0.27 7.3 99
5.15 0.27 5.3 103
2.49 0.28 9.2 108
3.39 0.59 10.5 99
5.06 0.53 4.4 109
2.85 1.46 17.5 124
4.39 2.47 21.7 132
6.00 6.16 22.2 133
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C. Power et al. / International Dairy Journal 39 (2014) 270e275 Table 4 Concentrations (mg kg1) of florfenicol residues in fresh and frozen pasteurised and unpasteurised milk, butter, buttermilk, skim milk and cream.a Product
Fig. 2. Excretion of florfenicol marker residues (expressed as florfenicol amine, mg kg1) into milk as a function of time (days). The average and standard deviation of six animals is shown.
3.4. Transfer of florfenicol residues into cream, skim milk and subsequent products The migration of FLOR during the separation of milk into skim milk and cream, during the manufacture of butter and during the manufacture of skim-milk powder using pasteurised and unpasteurised milk containing residue concentrations is shown in Table 4. The residue present in the skim milk was approximately double the concentration present in the cream. For skim-milk powder, FLOR marker residues concentrated in the powder by a factor of approximately 8 compared with the starting skim milk. When butter was manufactured from the cream, the residues migrated with the buttermilk, which contained approximately three times greater residue levels than were present in the butter. 3.5. Stability of florfenicol residues in dairy products There was no significant difference (p > 0.05) in the residues measured in products made from pasteurised or unpasteurised milk (Tables 3 and 4). When analysed fresh, there was a significant difference (p < 0.05) in the presence of FLOR residues in cheese and butter during storage for 3 weeks. There was no significant difference (p > 0.05) in the residue concentration of skim-milk powder stored for 6 months at ambient temperature. Table 3 Concentrations (mg kg1) of florfenicol marker residue in fresh and frozen raw and pasteurised milk, curd, whey, cheese during ripening.a Product
Storage time (months) 0
Raw milk Milk Curd Whey Cheese week 0 Cheese week 1 Cheese week 2 Cheese week 3 Pasteurised milk Milk Curd Whey Cheese week 0 Cheese week 1 Cheese week 2 Cheese week 3
845; 835; 957; NT 989; 355; 337; 758; 927; 955; NT 957; 494; 335;
6 745 915 992 980 535 479 804 953 845 900 749 409
12
761; 823; 885; 851; 763; 530; 647;
660 813 746 853 962 867 859
642; 848; 954; 757; 728; 708; 665;
660 766 779 989 827 823 357
745; 888; 754; 916; 703; 659; 625;
646 747 616 734 814 808 746
882; 907; 934; 1002; 837; 787; 724;
702 768 716 703 849 782 753
a Two independent experiments were done and both values are shown. Milk, cheese, curd and whey were stored at 20 C, cheese was also stored at 14 C for 3 weeks. NT, not tested.
Raw milk Milk Skim milk Cream Skim-milk powder Butter week 0 Butter week 1 Butter week 2 Butter week 3 Buttermilk Pasteurised milk Milk Skim milk Cream Skim-milk powder Butter week 0 Butter week 1 Butter week 2 Butter week 3 Buttermilk
Storage time (months) 0
6
12
656; 542 1089; 1046 562; 571 8927; 7142 375; 279 352; 315 226; 180 210; 149 1041; 866
729; 697 779; 712 413; 412 9279; 6376 293; 304 281; 227 240; 203 230; 140 769; 740
827; 763 990; 864 380; 390 7001; 6490 268; 249 258; 226 235; 191 232; 165 917; 885
738; 662 1021; 1034 588; 452 8477; 6956 290; 320 340; 332 195; 183 201; 172 870; 902
740; 647 862; 650 477; 415 8689; 7416 306; 321 269; 282 241; 237 234; 286 702; 704
730; 697 855; 928 497; 388 6808; 6537 284; 269 244; 232 261; 209 234; 232 913; 789
a Two independent experiments were done and both values are shown. Milk, skim milk, cream, butter and buttermilk were stored at 20 C, skim powder was stored at ambient temperature (18e22 C) and butter was stored at 5 C.
However, after 12 months storage there was a decrease in the residue concentration in the skim-milk powder. When stored at 20 C, for 6 or 12 months, residue concentrations in milk, curd, buttermilk, cream and whey samples were similar to the fresh product after 6 or 12 months frozen. In butter, and particularly in cheese, there were differences in the results between fresh and stored or frozen product. In cheese stored at 14 C, the residue concentration decreased on storage. However, after 6 or 12 months at 20 C, the concentrations appeared to increase during frozen storage. 4. Discussion This study demonstrated that following administration FLOR marker residues were excreted in milk and persisted in milk for at least 27 days post-treatment. Residues were not measurable following a withdrawal period of 34 days. Limited studies have been conducted on the migration of FLOR residues from milk to dairy products, or on the stability of residues in product. A study was conducted on FLOR residues levels in milk of lactating cows following treatment by intramuscular or intramammary routes, to establish withdrawal times, therapeutic effect and potential influence on milk yield (Ruiz et al., 2010). The milk was sampled in that study for 9 days post administration with a limit of detection (LOD) on the analytical method used of 100 mg L1. In this current study, the milk was sampled for 41 days. However, an additional study on FLOR in pigs, (Damte et al., 2012), considered the withdrawal of FLOR residues where 30 pigs were administered a combination antibiotic of FLOR and tylosin intramuscularly and were sacrificed at different time-points up to 28 days post administration. The liver, kidney, muscle and fat were analysed for the presence of florfenicol residues using an analytical method with a limit of quantification (LOQ) for FLOR of 0.1 mg L1. The results showed the presence of FLOR residues for up to 28 days post administration, which would be consistent with the findings of this current study. Heat stability of FLOR in water, salt water, soybean sauce and chicken meat has been studied at 100 C for 30 min, 1 h and 2 h
C. Power et al. / International Dairy Journal 39 (2014) 270e275
(Franje et al., 2010). The results demonstrated that FLOR degraded more when heated in chicken muscle in comparison to heating in water/salt water or soybean sauce. In this current study, heat treatment during pasteurisation (72 C for 15 s) of milk containing FLOR, or during powder manufacture had no effect on stability in the different products. During powder manufacture, the inlet temperature of 185 C with an outlet air-temperature which was maintained at 90 ± 2 C. However, during the manufacture of dry powder, the high temperatures reached were for fractions of a second, the times for which the temperatures were maintained were therefore considerably shorter than in the study of Franje et al. (2010). Even though there was no significant heating during cheesemaking, the pH was acidic as part of the cheesemaking process, and this had little effect on the stability of FLOR residues. The significance of these results is that FLOR residues, if present in milk, remain stable during the manufacturing processes of dairy products, whether pasteurised or not. However, storage of butter or particularly cheese for up to 3 weeks resulted in a decrease in the residue concentrations observed; such a decrease was not observed for storage of skim-milk powder for 6 months. In contrast, residue concentrations in cheese appeared to increase again if the samples were frozen. These changes may be caused by binding of the FLOR residues in the sample matrix, which may have affected the extractability of the analytes. Although the profile of the analytical results suggests that there may also be degradation or conversion of the analytes to unknown products. Further research would be required to understand the effect of freezing on the residues and to determine if the increased values are real or are an artefact of methodology. Examination of the behaviour of FLOR residues in milk following administration in the drying off period could be studied to determine if residues are detected and persist in the milk post-calving. 5. Conclusions The results from this fully licensed study demonstrate that FLOR marker residues persist in milk for 27 days post-treatment. As there is no MRL for FLOR in milk, once administered to a lactating animal within a lactating period, the complete withdrawal of residue in milk would have to be confirmed post administration before milk could be deemed acceptable for human consumption. In addition, the results from the experiments on dairy products manufactured from incurred milk demonstrate that residues of FLOR concentrated in the skim as opposed to cream and buttermilk as opposed to butter. There was also an increase in residues in powder produced from skim milk. Stability studies highlighted some issues with instability that were observed in cheese and butter, and also with freezing at 20 C. This aspect requires further research. Acknowledgements This work was supported by the Dairy Levy Trust. The authors wish to acknowledge the staff at Ashtown and the farm staff at
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Moorepark, in particular Noel Byrne and John Paul Murphy, for their valuable assistance. Clare Power was in receipt of a Teagasc Walsh Fellowship. References Anon. (2009). Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/ 90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council. Official Journal of European Communities, L152, 11e22. Anon. (2010). Commission implementing regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin (pp. 1e70). Brussels, Belgium: European Commission. Atef, M., El-Gendi, A. Y. I., Amer, A. M., & Abd El-Aty, A. M. (2010). Effect of three anthelmentics on disposition kinetics of florfenicol in goats. Food and Chemical Toxicology, 48, 3340e3344. Damte, D., Jeong, H. J., Lee, S. J., Cho, B. H., Kim, J. C., & Park, S. C. (2012). Evaluation of linear regression statistical approaches for withdrawal time estimation of veterinary drugs. Food and Chemical Toxicology, 50, 773e778. Franje, C. A., Chang, S. K., Shyu, C. L., Davis, J. L., Lee, Y. W., Lee, R. J., et al. (2010). Differential heat stability of amphenicols characterized by structural degradation, mass spectrometry and antimicrobial activity. Journal of Pharmaceutical and Biomedical Analysis, 53, 869e877. Irish Medicines Board. (2014). Available at: http://www.imb.ie/images/uploaded/ swedocuments/LicenseSPC_10846-008-001_06032014131949.pdf Accessed 23.05.14. Lim, J. H., Kim, M. S., Hwang, Y. H., Song, I. N., Park, B. K., & Yun, H. I. (2010). Plasma and tissue depletion of florfenicol in olive flounder (Paralichthys olivaceus) after oral administration. Aquaculture, 30, 71e74. Moreno, L., Imperiale, F., Mottier, L., Alvarez, L., & Lanusse, C. (2005). Comparison of milk residue profiles after oral and subcutaneous administration of benzimidazole anthelmintics to dairy cows. Analytica Chimica Acta, 536, 91e99. Power, C., Danaher, M., Sayers, R., O'Brien, B., Clancy, C., Furey, A., et al. (2013). Investigation of the migration of triclabendazole residues to milk products manufactured from bovine milk, and stability therein, following lactating cow treatment. Journal of Dairy Science, 96, 6223e6232. pez, C. C., & Gutie rrez, H. (2010). Florfenicol concenRuiz, J. B., Zapata, M. N., Lo trations in milk of lactating cows postreated by intramuscular or intrardoba, 15, 2041e2050. mammary routes. Revista Mvz Co Schwarz, S., Kehrenberg, C., Doublet, B., & Cloeckaert, A. (2004). Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiology Reviews, 28, 519e542. Settepani, J. A. (1984). The hazard of using chloramphenicol in food animals. Journal of the American Veterinary Medicine Association, 184, 930e931. Stolker, A. A. M., & Brinkman, U. A. (2005). Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animalsda review. Journal of Chromatography A, 1067, 15e53. Su, P., Liu, N., Zhu, M., Ning, B., Liu, M., Yang, Z., et al. (2011). Simultaneous detection of five antibiotics in milk by high-throughput suspension array technology. Talanta, 85, 1160e1165. Veterinary Medicines Directorate. (2008). National food residue database e Residue testing plan for Ireland (targeted samples). Available at: http://nfrd.teagasc.ie/ study_main.asp?Year¼2008%7C2009&SType¼5&FCat¼&FSCat¼& NoFiltF¼&NoFiltNC¼&NoPreFilter¼1&Food¼&ResCat¼&ResSCat¼&Res¼& filt¼1&StudyID¼222&caller¼2 Accessed 10.09.13. Whelan, M., Chirollo, C., Furey, A., Cortesi, M. L., Anastasio, A., & Danaher, M. (2010). Investigation of the persistence of levamisole and oxyclozanide in milk and fate in cheese. Journal of Agricultural and Food Chemistry, 58, 12204e12209. Xie, K., Jia, L., Yao, Y., Xu, D., Chen, S., Xie, X., et al. (2011). Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversedphase high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B, 879, 2351e2354.
Contents lists available at ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Investigation of the persistence of florfenicol residues in bovine milk and fate during processing Clare Power a, e, Ríona Sayers c, Martin Danaher b, Mary Moloney b, Bernadette O'Brien d, Ambrose Furey e, Kieran Jordan a, * a
Food Safety Department, Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland d Livestock Systems Department, Animal and Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland e Team Elucidate, Department of Chemistry, Cork Institute of Technology, Bishopstown, Cork, Ireland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2013 Received in revised form 25 July 2014 Accepted 25 July 2014 Available online 2 August 2014
The persistence of florfenicol residues in milk and their migration to dairy products was investigated following treatment of six lactating dairy cows with Selectan 300 mg mL1 solution for injection at a dose of 1 mL 15 kg body weight1. Florfenicol residues were measurable (LOQ 1.0 mg kg1) in the milk of three cows for up to 27 days post-treatment but were not detectable at day 34. On day 3 post-treatment, the milk was pooled into two independent aliquots, each containing the milk from three cows. One part of each aliquot was pasteurised. Milk products were manufactured from the pasteurised and unpasteurised aliquots. The florfenicol residue concentration in cheese was similar to the initial milk (989 versus 845 mg kg1, respectively). Residues in skim-milk powder were approximately eight-fold higher than the skim milk used for its manufacture (8927 versus 1089 mg kg1, respectively). © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Florfenicol (FLOR) is a broad spectrum antibiotic licensed for use in veterinary medicine. It has bacteriostatic properties in that it prevents protein synthesis in bacterial cells (Atef, El-Gendi, Amer, & Abd El-Aty, 2010; Lim et al., 2010; Xie et al., 2011). Chloramphenicol, which belongs to the same pharmacological group as FLOR pez, & Gutie rrez, 2010; Schwarz, Kehrenberg, (Ruiz, Zapata, Lo Doublet, & Cloeckaert, 2004), was a widely used human medicine until reports of aplastic anaemia arising from its use led to the introduction of FLOR as a safer antibiotic. Because of the risk of toxicity to humans, chloramphenicol cannot be used in food-producing animals (Settepani, 1984; Stolker & Brinkman, 2005; Su et al., 2011). While the mechanisms of antibacterial activity of FLOR and chloramphenicol are similar (Atef et al., 2010), the presence of a fluorine atom in FLOR makes it resistant to deactivation by bacterial transmissible plasmids (Ruiz et al., 2010). In addition, because of its lipophilicity, FLOR demonstrates good tissue penetration increasing its activity against bovine respiratory disease
* Corresponding author. Tel.: þ353 25 42451. E-mail address: [email protected] (K. Jordan). http://dx.doi.org/10.1016/j.idairyj.2014.07.012 0958-6946/© 2014 Elsevier Ltd. All rights reserved.
(BRD) (Schwarz et al., 2004). Therefore, FLOR is regarded as an important drug in the therapeutic control of BRD infections. The metabolism and persistence of FLOR has been studied by other groups in edible tissues (Atef et al., 2010; Lim et al., 2010). In the EU, the marker residue has been defined as the sum of FLOR and its metabolites measured as florfenicol amine, FLOR-A (Anon, 2010). Although the pharmacokinetics of FLOR have been extensively studied in animals (Atef et al., 2010), there are few reports in the literature concerning the persistence of FLOR residues in milk, or on the migration from milk during manufacture of products. The widespread use of antibiotics, such as FLOR, for therapy and disease prevention in food-producing animals raises concern about the potential of antibiotic residues in food (Franje et al., 2010) that are potentially harmful to consumers (Damte et al., 2012). Maximum residue limits (MRLs), which are the maximum concentration of a veterinary drug residue legally permissible in food, are proposed by the European Medicines Agency (Anon, 2009, 2010) to protect public health. Withdrawal periods can be subsequently defined to ensure residue levels in a foodstuff are below the MRL (Damte et al., 2012; Moreno, Imperiale, Mottier, Alvarez, & Lanusse, 2005; Veterinary Medicines Directorate, 2008). At present, no MRL has been set for FLOR in milk, therefore any concentration level of this drug detected in milk would constitute a noncompliant result.
C. Power et al. / International Dairy Journal 39 (2014) 270e275
To add to the current, limited information on the presence of FLOR residues in milk and dairy products following treatment, the objectives of this study were to investigate the persistence of FLOR residues in milk following the treatment of lactating dairy cows, to measure residue concentrations in dairy products produced from the milk of treated cows, and to assess the stability of such residues in product during storage. 2. Materials and methods 2.1. Chemicals and reagents Methanol (MeOH), acetonitrile (MeCN; LC-MS grade), propan-2ol (IPA; chromasolv grade), sodium hydroxide pellets (puriss p.a.), ammonium formate, magnesium sulphate anhydrous and florfenicol (FLOR) analytical standard for drug analysis were all purchased from SigmaeAldrich (Dublin, Ireland). Florfenicol amine (FLOR-A) came from Witega (Berlin, Germany). Sodium chloride p.a. came from Applichem GmbH (Darmstadt, Germany) and glacial acetic acid, 100% anhydrous was sourced from Merck (Darmstadt, Germany). Ultra-pure water (18.2 MU cm1) was generated inhouse by using a Millipore (Cork, Ireland) water purification system. Syringe filters (0.2 mm, 13 mm, PTFE) were also supplied by Millipore. Polypropylene centrifuge tubes (50 mL and 15 mL) with screw caps were obtained from Sarstedt Ltd. (Wexford, Ireland). A Sartorius Extend top pan balance was employed for weighing milk samples (Sartorius, Dublin, Ireland). A glass dispenser from BrandTech (Essex, CT, USA) was used for aliquoting acetonitrile. A Mistral 3000i centrifuge (Davidson and Hardy, Dublin, Ireland), a multi-vortexer (VWR International, Dublin, Ireland) and a Turbovap
271
LV (Caliper Life Sciences, Runcorn, UK) were used during sample extraction. 2.2. Animal trials A total of six lactating dairy cows, three Holstein Friesian, two Montbeliard and one Norwegian Red, was administered Selectan (Laboratorios Hipra S.A., Girona, Spain), a 300 mg mL1 solution of FLOR, by intramuscular injection, as approved by the Irish Medicines Board (IMB). Each individual cow was weighed and Selectan administered at a rate of 20 mg kg1 body weight (1 mL of Selectan 15 kg body weight1). Selectan was administered on two occasions, 48 h apart as per the IMB protocol (Irish Medicines Board, 2014). The appropriate dose for cattle is 20 mg kg1 body weight (1 mL of the product 15 kg1) to be administered twice (48 h apart) by the intramuscular route. For treatment of cattle over 150 kg body weight, the dose is divided so that no more than 10 mL are injected at one site. Prior to administration, a 50 mL milk sample was collected from each cow as a control. After administration, milk samples were taken twice daily (morning and evening) until there was no detectable FLOR marker residues in two consecutive milk samples. Samples were labelled on collection, refrigerated at 4 C and analysed within 4 days of being collected. 2.3. Design of dairy processing experiments The cows were divided into two groups of three cows (balanced by breed as much as possible) and all the milk from each group was collected and pooled on day 3 following the second administration of FLOR. The milk used for the manufacture of dairy products
Fig. 1. Flowchart of the process of product manufacture from milk containing florfenicol residues.
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C. Power et al. / International Dairy Journal 39 (2014) 270e275
contained high FLOR residue concentrations (670 ± 48 mg kg1). Each milk pool, using the milk from 3 cows, was further subdivided and one part was pasteurised at (72 C 15 s) while the second remained unpasteurised. Semi-soft laboratory scale cheese was manufactured using milk from each pasteurised and unpasteurised sub-divided pool. Similarly, a separate portion of the same milk used in cheese manufacture, while another was separated into skim milk and cream fractions and butter and skim-milk powder were manufactured. Buttermilk was collected from this butter manufacturing process. A summary of the milk product manufacture completed in this study is outlined in Fig. 1. 2.4. Manufacture of cheese, skim milk, cream, butter and skim-milk powder Cheese was manufactured from pasteurised and unpasteurised milk using a method previously described (Power et al., 2013). For the separation of whole milk to skim milk and cream, milk was heated to 50 C and separated using a Disc Bowl Centrifuge (Armfield, Ringwood, UK). Cream and skim-milk fractions were collected. Following the separation of milk into skim and cream, the cream was chilled and whisked in a food blender until the cream separated into buttermilk and butter. Buttermilk, butter, skim and cream samples were analysed (as described below) from fresh product at day 0. Skim-milk powder was manufactured using pasteurised and unpasteurised milk using the method previously described (Power et al., 2013). 2.5. Stability of florfenicol residues in dairy products The stability to freezing of FLOR marker residues in samples was studied by analysing fresh product on the day of manufacture, and then freezing it at 20 C and analysing the samples again after 6 and 12 months. Skim-milk powder was stored in 50 mL centrifuge tubes in the dark at ambient temperature (18e22 C). Samples of powder were taken at day 0, 6 and 12 months and analysed for the presence of FLOR residues. The stability of residues during cheese ripening (at 14 C) and butter storage (at 5 C) was studied by analysing samples over several weeks. 2.6. Sample preparation prior to analysis
steel replacement filter (all from Waters, Milford, MA, USA). The column was maintained at a temperature of 50 C and the pump was operated at a flow rate of 0.4 mL min1. A binary gradient system was used to separate analytes comprising mobile phase A, 0.1% acetic acid in H2O (v/v) and mobile phase B, 5 mM ammonium formate in MeOH:MeCN (75:25, v/v). The gradient profile was as follows: 0e1.25 min, 99.5% A; 1.5e2.0 min, 10% A; 2.05e3.0 min, 99.5% A. The injection volume was 2.5 mL. UHPLC weak and strong washes consisted of H2O:MeOH (90:10, v/v, 1000 mL) and MeOH:IPA:H2O (80:10:10, v/v, 750 mL), respectively. FLOR and FLOR-A residues were detected using a Waters Quattro Premier XE triple quadrupole mass spectrometer operating in electrospray ionisation (ESI) mode. Nitrogen was used for nebulisation, desolvation (650 L h1) and cone gas (50 L h1). Argon was used as collision gas (0.013 L h1). The source temperature was set at 90 C and desolvation temperature at 300 C. The capillary voltage was set at 3.0 kV. The SRM windows were time sectored, and dwell time, interscan delay and inter-channel delays were set to get maximum response from the instrument. UHPLC-tandem mass spectrometry (MS/MS) experimental conditions are summarised in Table 1. The UHPLCeMS/MS system was controlled by MassLynx™ software and data were processed using TargetLynx™ Software (both from Waters). 2.8. Calibration and controls FLOR and FLOR-A were prepared in methanol at a concentration of 2 mg mL1. Working standard solutions containing both FLOR and FLOR-A were prepared at concentrations of 100.0, 50.0, 25.0, 10.0, 5.0, 2.5, 1.0, 0.5, 0.25 and 0.1 mg mL1. Extracted milk matrix calibrants were prepared by spiking negative milk samples (10 g) with 100 mL of working standard solutions prior to extraction. This gave 10 point calibration curves in the range 1.0e1,000 mg kg1 (milk, buttermilk, skim milk and whey) and 10.0e10,000 mg kg1 (butter, cheese, cream, curd, and powder). An additional four blank milk samples (recovery controls) were spiked after evaporation, two with 50 mL of standard 2 (0.25 mg mL1) and two with standard 7 (10 mg mL1) to monitor for the loss of analytes during extraction. This gave concentrations of 2.5 and 100 mg kg1 for milk, buttermilk, skim milk and whey, or 25 and 1000 mg kg1 for butter, cheese, cream, curd, and powder, respectively. FLOR marker residues are expressed as FLOR-A equivalents using the equation
X
All samples were prepared for analysis as described by Power et al. (2013). 2.7. Ultrahigh performance liquid chromatography tandem mass spectrometry analysis All sample separations were performed as described by Whelan et al. (2010) using an Acquity ultrahigh performance liquid chromatography (UHPLC) system comprising a stainless steel HSS T3 analytical column (100 mm 2.1 mm, particle size 1.8 mm) equipped with an in-line filter unit containing a 0.2 mm stainless
FLOR marker residues mg kg1
¼ ðFLOR 0:69Þ þ FLOR A
(1)
The factor, 0.69, is calculated by dividing the molecular weight of FLOR-A (247.29 g mol1) by the molecular weight of FLOR (358.21 g mol1). 2.9. Statistical analyses All statistical analyses were carried out using SAS (version 9.1.3, SAS Institute, Cary, NC, USA). A randomised design that
Table 1 Ultrahigh pressure liquid chromatography tandem mass spectrometry conditions used for analysis of florfenicol and florfenicol amine residues.a Analyte
tR (min)
Function (min)
Transition (m/z)
FLOR-A
2.22
1:0e1.8 min
FLOR
2.22
2:1.8e3 min
247.9 247.9 355.7 355.7 357.7 357.7
a
/ / / / / /
129.9 229.9 185.0 335.7 185.0 337.8
Abbreviations are: tR, retention time (min); CE, collision energy; FLOR, florfenicol.
Polarity
Dwell time (s)
Cone (V)
CE (V)
þ
0.02 0.005 0.1
28
22 13 18 10 18 10
30
C. Power et al. / International Dairy Journal 39 (2014) 270e275
incorporated the treatment (pasteurised and unpasteurised) and (effect of residue over time) was used. The response variables related to the cheese and butter manufactured using pasteurised and unpasteurised milk. Pooling of the milk into two groups resulted in small sample sizes, which impacted on the assumptions of normality. Therefore, the medians (50th percentile) and interquartile ranges (IQR; 25e75th percentile), rather than the means and standard deviations, were used to express measures of central tendency, and nonparametric statistical methods were used. Statistically significant differences (p < 0.05) between different treatment levels were determined by using KruskaleWallis nonparametric one-way analysis of variance and the ManneWhitneyeWilcoxon test to compare group differences. 2.10. Validation Validation of the method for accuracy and precision was conducted in bovine milk, ovine milk, caprine milk, cottage cheese manufactured from bovine milk, and organic butter manufactured from bovine milk (all purchased at Superquin, Ashtown, Dublin, Ireland, except for the ovine milk that was obtained directly from the farm of Henry Clifton Brown, Tipperary, Ireland). Concentrations of 2.50 mg kg1, 3.75 mg kg1 and 5.00 mg kg1 for florfenicol (in duplicate) and florfenicol amine were added to the milks and bovine milk products under validation. 3. Results 3.1. Validation The validation results as depicted in Table 2, show that accuracy values were generally within the acceptable limits of 70e110% for ovine milk, caprine milk and cheese for FLOR. However for bovine milk and low butter concentrations, accuracy values were marginally outside the acceptable limits. FLOR-A accuracy values were within range, with the exception of the values obtained for
273
cheese, where they were slightly elevated. While precision, measured as the coefficient of variation (CV), showed results between 2% and 15.1% with the exception being the precision values of FLOR-A in cheese, which were slightly elevated at between 17.5% and 22.2%.
3.2. Florfenicol marker residues in milk Control milk samples taken from each animal and analysed prior to administration resulted in no detectable FLOR marker residues. The results in Fig. 2 show an increase in FLOR marker residues at day 2.5 which corresponds to the first milk samples collected following the second treatment. The highest concentration of summed FLOR marker residues (expressed as FLOR-A) measured in samples from individual cows ranged from 865 to 1871 mg kg1. FLOR marker residues were measurable in the milk of three animals (0.8, 1.0 and 1.4 mg kg1, FLOR-A equivalents) at day 27, but were below the limit of quantification of 1.0 mg kg1 at later time-points. The major residue found in the milk of dairy cows following treatment was FLOR parent drug. FLOR-A residues were also measurable in milk but occurred at lower concentrations (highest concentration of 125 to 380 mg kg1 in the milk at 2.5 days posttreatment) and were not quantifiable at >19.5 days. 3.3. Transfer of florfenicol marker residues into cheese The migration of FLOR marker residues during cheesemaking using pasteurised and unpasteurised milk containing residue concentrations is shown in Table 3. FLOR marker residue levels were similar in the starting milk to the curd and whey produced during the cheesemaking process. The transfer of residue to curd, whey and cheese made from pasteurised milk was not significantly different from cheese made with unpasteurised milk (p > 0.05). The concentration of residue detected in the final cheese was similar to that detected in the starting curd.
Table 2 Validation results for florfenicol and florfenicol amine in milk (bovine, ovine and caprine), cheese and butter samples (n ¼ 7). FLOR (mg kg1)
Parameter
Bovine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Ovine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Caprine milk Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Butter Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%) Cheese Mean (mg kg1) Standard deviation (mg Coefficient of variation Accuracy (%)
kg1)
kg1)
kg1)
kg1)
kg1)
Florfenicol amine (mg kg1)
2.5
3.75
5.00
2.5
3.75
5.00
2.92 0.25 8.5 118
4.35 0.31 7.1 116
5.64 0.39 6.9 113
2.34 0.27 11.5 94
3.89 0.59 15.1 104
5.52 0.59 10.7 110
2.63 0.27 10.2 105
3.65 0.32 8.7 97
4.85 0.29 6.0 97
2.75 0.39 14.0 110
3.58 0.54 15.1 96
4.52 0.61 13.4 91
2.69 0.12 4.0 108
3.94 0.14 3.1 105
5.04 0.41 2.0 101
2.53 0.26 10.1 101
3.68 0.23 6.2 98
4.92 0.46 9.3 98
2.80 0.11 4.1 112
3.92 0.23 5.9 105
5.47 0.21 3.8 109
2.61 0.20 7.7 104
3.71 0.27 7.3 99
5.15 0.27 5.3 103
2.49 0.28 9.2 108
3.39 0.59 10.5 99
5.06 0.53 4.4 109
2.85 1.46 17.5 124
4.39 2.47 21.7 132
6.00 6.16 22.2 133
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C. Power et al. / International Dairy Journal 39 (2014) 270e275 Table 4 Concentrations (mg kg1) of florfenicol residues in fresh and frozen pasteurised and unpasteurised milk, butter, buttermilk, skim milk and cream.a Product
Fig. 2. Excretion of florfenicol marker residues (expressed as florfenicol amine, mg kg1) into milk as a function of time (days). The average and standard deviation of six animals is shown.
3.4. Transfer of florfenicol residues into cream, skim milk and subsequent products The migration of FLOR during the separation of milk into skim milk and cream, during the manufacture of butter and during the manufacture of skim-milk powder using pasteurised and unpasteurised milk containing residue concentrations is shown in Table 4. The residue present in the skim milk was approximately double the concentration present in the cream. For skim-milk powder, FLOR marker residues concentrated in the powder by a factor of approximately 8 compared with the starting skim milk. When butter was manufactured from the cream, the residues migrated with the buttermilk, which contained approximately three times greater residue levels than were present in the butter. 3.5. Stability of florfenicol residues in dairy products There was no significant difference (p > 0.05) in the residues measured in products made from pasteurised or unpasteurised milk (Tables 3 and 4). When analysed fresh, there was a significant difference (p < 0.05) in the presence of FLOR residues in cheese and butter during storage for 3 weeks. There was no significant difference (p > 0.05) in the residue concentration of skim-milk powder stored for 6 months at ambient temperature. Table 3 Concentrations (mg kg1) of florfenicol marker residue in fresh and frozen raw and pasteurised milk, curd, whey, cheese during ripening.a Product
Storage time (months) 0
Raw milk Milk Curd Whey Cheese week 0 Cheese week 1 Cheese week 2 Cheese week 3 Pasteurised milk Milk Curd Whey Cheese week 0 Cheese week 1 Cheese week 2 Cheese week 3
845; 835; 957; NT 989; 355; 337; 758; 927; 955; NT 957; 494; 335;
6 745 915 992 980 535 479 804 953 845 900 749 409
12
761; 823; 885; 851; 763; 530; 647;
660 813 746 853 962 867 859
642; 848; 954; 757; 728; 708; 665;
660 766 779 989 827 823 357
745; 888; 754; 916; 703; 659; 625;
646 747 616 734 814 808 746
882; 907; 934; 1002; 837; 787; 724;
702 768 716 703 849 782 753
a Two independent experiments were done and both values are shown. Milk, cheese, curd and whey were stored at 20 C, cheese was also stored at 14 C for 3 weeks. NT, not tested.
Raw milk Milk Skim milk Cream Skim-milk powder Butter week 0 Butter week 1 Butter week 2 Butter week 3 Buttermilk Pasteurised milk Milk Skim milk Cream Skim-milk powder Butter week 0 Butter week 1 Butter week 2 Butter week 3 Buttermilk
Storage time (months) 0
6
12
656; 542 1089; 1046 562; 571 8927; 7142 375; 279 352; 315 226; 180 210; 149 1041; 866
729; 697 779; 712 413; 412 9279; 6376 293; 304 281; 227 240; 203 230; 140 769; 740
827; 763 990; 864 380; 390 7001; 6490 268; 249 258; 226 235; 191 232; 165 917; 885
738; 662 1021; 1034 588; 452 8477; 6956 290; 320 340; 332 195; 183 201; 172 870; 902
740; 647 862; 650 477; 415 8689; 7416 306; 321 269; 282 241; 237 234; 286 702; 704
730; 697 855; 928 497; 388 6808; 6537 284; 269 244; 232 261; 209 234; 232 913; 789
a Two independent experiments were done and both values are shown. Milk, skim milk, cream, butter and buttermilk were stored at 20 C, skim powder was stored at ambient temperature (18e22 C) and butter was stored at 5 C.
However, after 12 months storage there was a decrease in the residue concentration in the skim-milk powder. When stored at 20 C, for 6 or 12 months, residue concentrations in milk, curd, buttermilk, cream and whey samples were similar to the fresh product after 6 or 12 months frozen. In butter, and particularly in cheese, there were differences in the results between fresh and stored or frozen product. In cheese stored at 14 C, the residue concentration decreased on storage. However, after 6 or 12 months at 20 C, the concentrations appeared to increase during frozen storage. 4. Discussion This study demonstrated that following administration FLOR marker residues were excreted in milk and persisted in milk for at least 27 days post-treatment. Residues were not measurable following a withdrawal period of 34 days. Limited studies have been conducted on the migration of FLOR residues from milk to dairy products, or on the stability of residues in product. A study was conducted on FLOR residues levels in milk of lactating cows following treatment by intramuscular or intramammary routes, to establish withdrawal times, therapeutic effect and potential influence on milk yield (Ruiz et al., 2010). The milk was sampled in that study for 9 days post administration with a limit of detection (LOD) on the analytical method used of 100 mg L1. In this current study, the milk was sampled for 41 days. However, an additional study on FLOR in pigs, (Damte et al., 2012), considered the withdrawal of FLOR residues where 30 pigs were administered a combination antibiotic of FLOR and tylosin intramuscularly and were sacrificed at different time-points up to 28 days post administration. The liver, kidney, muscle and fat were analysed for the presence of florfenicol residues using an analytical method with a limit of quantification (LOQ) for FLOR of 0.1 mg L1. The results showed the presence of FLOR residues for up to 28 days post administration, which would be consistent with the findings of this current study. Heat stability of FLOR in water, salt water, soybean sauce and chicken meat has been studied at 100 C for 30 min, 1 h and 2 h
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(Franje et al., 2010). The results demonstrated that FLOR degraded more when heated in chicken muscle in comparison to heating in water/salt water or soybean sauce. In this current study, heat treatment during pasteurisation (72 C for 15 s) of milk containing FLOR, or during powder manufacture had no effect on stability in the different products. During powder manufacture, the inlet temperature of 185 C with an outlet air-temperature which was maintained at 90 ± 2 C. However, during the manufacture of dry powder, the high temperatures reached were for fractions of a second, the times for which the temperatures were maintained were therefore considerably shorter than in the study of Franje et al. (2010). Even though there was no significant heating during cheesemaking, the pH was acidic as part of the cheesemaking process, and this had little effect on the stability of FLOR residues. The significance of these results is that FLOR residues, if present in milk, remain stable during the manufacturing processes of dairy products, whether pasteurised or not. However, storage of butter or particularly cheese for up to 3 weeks resulted in a decrease in the residue concentrations observed; such a decrease was not observed for storage of skim-milk powder for 6 months. In contrast, residue concentrations in cheese appeared to increase again if the samples were frozen. These changes may be caused by binding of the FLOR residues in the sample matrix, which may have affected the extractability of the analytes. Although the profile of the analytical results suggests that there may also be degradation or conversion of the analytes to unknown products. Further research would be required to understand the effect of freezing on the residues and to determine if the increased values are real or are an artefact of methodology. Examination of the behaviour of FLOR residues in milk following administration in the drying off period could be studied to determine if residues are detected and persist in the milk post-calving. 5. Conclusions The results from this fully licensed study demonstrate that FLOR marker residues persist in milk for 27 days post-treatment. As there is no MRL for FLOR in milk, once administered to a lactating animal within a lactating period, the complete withdrawal of residue in milk would have to be confirmed post administration before milk could be deemed acceptable for human consumption. In addition, the results from the experiments on dairy products manufactured from incurred milk demonstrate that residues of FLOR concentrated in the skim as opposed to cream and buttermilk as opposed to butter. There was also an increase in residues in powder produced from skim milk. Stability studies highlighted some issues with instability that were observed in cheese and butter, and also with freezing at 20 C. This aspect requires further research. Acknowledgements This work was supported by the Dairy Levy Trust. The authors wish to acknowledge the staff at Ashtown and the farm staff at
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Moorepark, in particular Noel Byrne and John Paul Murphy, for their valuable assistance. Clare Power was in receipt of a Teagasc Walsh Fellowship. References Anon. (2009). Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/ 90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council. Official Journal of European Communities, L152, 11e22. Anon. (2010). Commission implementing regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin (pp. 1e70). Brussels, Belgium: European Commission. Atef, M., El-Gendi, A. Y. I., Amer, A. M., & Abd El-Aty, A. M. (2010). Effect of three anthelmentics on disposition kinetics of florfenicol in goats. Food and Chemical Toxicology, 48, 3340e3344. Damte, D., Jeong, H. J., Lee, S. J., Cho, B. H., Kim, J. C., & Park, S. C. (2012). Evaluation of linear regression statistical approaches for withdrawal time estimation of veterinary drugs. Food and Chemical Toxicology, 50, 773e778. Franje, C. A., Chang, S. K., Shyu, C. L., Davis, J. L., Lee, Y. W., Lee, R. J., et al. (2010). Differential heat stability of amphenicols characterized by structural degradation, mass spectrometry and antimicrobial activity. Journal of Pharmaceutical and Biomedical Analysis, 53, 869e877. Irish Medicines Board. (2014). Available at: http://www.imb.ie/images/uploaded/ swedocuments/LicenseSPC_10846-008-001_06032014131949.pdf Accessed 23.05.14. Lim, J. H., Kim, M. S., Hwang, Y. H., Song, I. N., Park, B. K., & Yun, H. I. (2010). Plasma and tissue depletion of florfenicol in olive flounder (Paralichthys olivaceus) after oral administration. Aquaculture, 30, 71e74. Moreno, L., Imperiale, F., Mottier, L., Alvarez, L., & Lanusse, C. (2005). Comparison of milk residue profiles after oral and subcutaneous administration of benzimidazole anthelmintics to dairy cows. Analytica Chimica Acta, 536, 91e99. Power, C., Danaher, M., Sayers, R., O'Brien, B., Clancy, C., Furey, A., et al. (2013). Investigation of the migration of triclabendazole residues to milk products manufactured from bovine milk, and stability therein, following lactating cow treatment. Journal of Dairy Science, 96, 6223e6232. pez, C. C., & Gutie rrez, H. (2010). Florfenicol concenRuiz, J. B., Zapata, M. N., Lo trations in milk of lactating cows postreated by intramuscular or intrardoba, 15, 2041e2050. mammary routes. Revista Mvz Co Schwarz, S., Kehrenberg, C., Doublet, B., & Cloeckaert, A. (2004). Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiology Reviews, 28, 519e542. Settepani, J. A. (1984). The hazard of using chloramphenicol in food animals. Journal of the American Veterinary Medicine Association, 184, 930e931. Stolker, A. A. M., & Brinkman, U. A. (2005). Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animalsda review. Journal of Chromatography A, 1067, 15e53. Su, P., Liu, N., Zhu, M., Ning, B., Liu, M., Yang, Z., et al. (2011). Simultaneous detection of five antibiotics in milk by high-throughput suspension array technology. Talanta, 85, 1160e1165. Veterinary Medicines Directorate. (2008). National food residue database e Residue testing plan for Ireland (targeted samples). Available at: http://nfrd.teagasc.ie/ study_main.asp?Year¼2008%7C2009&SType¼5&FCat¼&FSCat¼& NoFiltF¼&NoFiltNC¼&NoPreFilter¼1&Food¼&ResCat¼&ResSCat¼&Res¼& filt¼1&StudyID¼222&caller¼2 Accessed 10.09.13. Whelan, M., Chirollo, C., Furey, A., Cortesi, M. L., Anastasio, A., & Danaher, M. (2010). Investigation of the persistence of levamisole and oxyclozanide in milk and fate in cheese. Journal of Agricultural and Food Chemistry, 58, 12204e12209. Xie, K., Jia, L., Yao, Y., Xu, D., Chen, S., Xie, X., et al. (2011). Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversedphase high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B, 879, 2351e2354.