Accepted Manuscript

Analytical methods

An HPLC-DAD method for the simultaneous determination of nine β-lactam

antibiotics in ewe milk

M. Cámara, A. Gallego-Picó, R.M. Garcinuño, P. Fernández-Hernando, J.S.

Durand-Alegría, P.J. Sánchez

PII: S0308-8146(13)00387-7

DOI: http://dx.doi.org/10.1016/j.foodchem.2013.02.131

Reference: FOCH 13881

To appear in: Food Chemistry

Please cite this article as: Cámara, M., Gallego-Picó, A., Garcinuño, R.M., Fernández-Hernando, P., Durand-

Alegría, J.S., Sánchez, P.J., An HPLC-DAD method for the simultaneous determination of nine β-lactam antibiotics

in ewe milk, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.02.131

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1

An HPLC-DAD method for the simultaneous determination of nine β-lactam 1

antibiotics in ewe milk 2

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M. Cámaraa, A. Gallego-Picó b, R.M. Garcinuñob, P.Fernández-Hernandob, J.S. Durand-Alegríab, P.J. 7

Sáncheza 8

aRegional Centre of Animal Selection and Reproduction (CERSYRA), E-13300 Valdepeñas, Spain 9

bDepartment of Analytical Sciences, Faculty of Sciences, National University of Distance Education 10 (UNED), E-28040 Madrid, Spain 11

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21 22 23 24 25

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28 29 30 *Corresponding author: Dr. Rosa Mª Garcinuño Martínez 31

Address: C/ Senda del Rey nº 9, 28040 Madrid, Spain. 32

Telephone number: + 34 91 398 73 66 33 Fax number: + 34 91 398 83 79 34 E-mail address: rmgarcinuno@ccia.uned.es 35

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Abstract 1

The presence of -lactam residues in foodstuffs constitutes a potential risk to the 2 human health and undesirable effects on consumers, and nowadays these antibiotic 3 residues are also recognized as an emerging environmental problem. In addition, these 4 are of great concern to prestigious Manchego cheese processors (Central Spain 5 denomination of origin) because they reduce the curdling of milk and cause improper 6 cheese ripening, which consequently lead to an important loss of monetary income. 7

This work describes the development of a sensitive and reliable method using liquid 8 chromatography with UV-diode array detection (LC-DAD) for simultaneous 9 determination of the β-lactam antibiotics, ampicillin (AMP), benzylpenicillin (PEG), 10 cephalexin (CFX), cefazolin (CFL), cefoperazone (CFP), cloxacillin (CLO), dicloxacillin 11 (DCL), oxacillin (OXA) and phenoxymethylpenicillin (PEV), in Manchega ewe milk. The 12 column, mobile phase, temperature and flow rate were optimised to provide the best 13 resolution of these analytes. The extraction method of the antibiotic residues involves 14 the deproteinization of the milk sample using acetonitrile and centrifugation followed by 15 a solid-phase extraction (SPE) clean-up. The recoveries for the studied β-lactams 16 ranged from 79 to 96 % with relative standard deviations between 0.5 and 4.9 %. The 17 limits of quantification (LOQs) for all these compounds were in the range of 3.4-8.6 18 μgkg-1, which are lower than the maximum residue limits (MRLs) established by the 19 European Union for the studied β-lactams in milk, making the method suitable for 20 performing routine analyses. 21

The proposed multi-residue LC-UV-diode array detection (LC-DAD) method is a 22 powerful and popular alternative for the determination and confirmation of antibiotic 23 residues in small milk industries and is the first one capable of determining nine β-24 lactam antibiotics in samples of Manchega ewe milk. 25

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Keywords 27

Antibiotic residues, -lactams, ewe milk, Liquid chromatography, UV-diode array 28

detection 29

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1. Introduction 1

Antibiotics are frequently used in veterinary practice to treat and prevent some 2 diseases as mastitis and other microbial infections. The widespread administration of 3 antibiotic in food-producing animals or the non-fulfillment of antibiotic withdrawal 4 periods, which ensure that the drug has been completely excreted from the animal, 5 prior to animal products being available for consumers, may induce the presence of 6 antibiotic residues in the food chain [1, 2]. Continuous sub-lethal levels of antibiotics in 7 food have led to the emergence of harmful bacteria resistant to antibiotics [3,4], cause 8 allergic reactions in sensitized individuals [5], or affect the intestinal flora [6]. Therefore, 9 the presence of residues of antimicrobial agents constitutes a potential risk to the 10 human health and undesirable effects on consumers, and nowadays these residues 11 are also recognized as an emerging environmental problem [7-9]. 12

Despite there is an increasing concern over the negative effects of inappropriate 13 antibiotic use [10], these are administered at sub-therapeutic levels as feed additives or 14 through drinking water to enhance animal growth and feed efficiency [11, 12], even 15 though these illegal practices are forbidden in European Union since 2006 [13]. In 16 order to control such veterinary drugs in animals producing food, the European Union 17 has established the maximum concentration of a residue resulting from the use of a 18 veterinary medicinal product which may be accepted as being legally permitted or 19 recognized as acceptable in or on a food (maximum residue limits, MRLs) [14]. 20

Antibiotic residues in milk are of great concern to dairy farmers and milk processors, 21 since these residues may interfere and adversely affect the manufacture of some dairy 22 products, such as cheese or yoghurt due to the inhibition of the fermentation processes 23 [15, 16]. From an economic point of view, this can produce an important loss of 24 monetary income to the milk processors. Thus, milk is the most studied matrix and -25 lactams are some of the antibiotics most frequently used in veterinary medicine for the 26 treatment of animals in Europe [17] and Comunidad de Castilla-La Mancha (Cámara et 27 al., unpublished data), due to many β-lactams are resistant to degradation by the 28 enzyme penicillinase and may be reported as unidentified microbial inhibitors [18]. 29

Recent reviews present analytical methods that have been developed during the past 30 decade for some β-lactams in milk (microbiological approaches, biosensors, 31 immunochemical techniques and chromatographic methods) [19] and progress in 32 analysis of residual antibacterial in food and future perspectives, inspired in 33 Commission Decision 2002/657/EC [20], have been described [21]. The literature 34 reports new analytical strategies and confirmatory methods for residue analysis that 35 depend almost completely on LC-MS-MS [22-27]. However, the implementation to 36 control the antimicrobial residues is still limited due to the complex laboratory 37 equipment and the high cost required. In small milk industries, an alternative for the 38 determination and confirmation of antibiotic residues is the use of liquid 39 chromatographic with UV-diode array detection (LC-DAD) methods, and a way to 40 improve cost-effectiveness is to maximize the number of analytes that may be 41 determined by a single procedure using multi-residue methods. 42

Milk is a complex matrix with high protein and fat contents, which often interfere in 43 analytical procedures. The fat content of ewe milk is particularly high (7.9 % vs. 3.7 % 44 in bovine milk), which makes the isolation of β-lactams from it especially complicated 45 and laborious. Extraction of β-lactams from samples typically involves the use of large 46 amounts of organic solvents and an extensive sample clean-up [19, 21, 22, 26-35]. 47

Many analytical methods have appeared in the literature for the determination of 48 antibiotic residues in milk [17, 19], but only a limited number of them focus on multi-49 residue analysis [18, 19, 30, 31]. Although several LC methods for β-lactams using 50 various extraction and deproteinization procedures have been published for bovine milk 51

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[18, 19, 21, 26, 30-33], only a few methods have been reported for ewe milk [24, 36]. 1 However, these methods deal specifically with the determination of macrolide 2 antibiotics. According to our knowledge, there are no reliable methods for the 3 simultaneous determination of β-lactams in ewe milk. 4

Therefore, this study is focussed on the development of a robust, simple and practical 5 method capable of simultaneously extracting and analysing the selected β-lactam 6 antibiotics, ampicillin (AMP), benzylpenicillin (PEG), cephalexin (CFX), cefazolin (CFL), 7 cefoperazone (CFP), cloxacillin (CLO), dicloxacillin (DCL), oxacillin (OXA) and 8 phenoxymethylpenicillin (PEV), in ewe milk from La Mancha region (Central Spain) 9 where milk production is mainly used for processing a prestigious cheese with own 10 denomination of origin, Manchego cheese. 11

The proposed multi-residue LC-UV-diode array detection (LC-DAD) method is a 12 powerful and popular alternative for the determination and confirmation of antibiotic 13 residues in small milk industries and is the first one capable of determining nine β-14 lactam antibiotics in samples of Manchega ewe milk. 15

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2. Experimental 17

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2.1. Chemicals and materials 19

Commercial β-lactam standards were supplied by Fluka (Steinheim, Germany). HPLC 20 gradient grade acetonitrile, sodium hydroxide, hydrochloric acid and mono-basic 21 potassium phosphate were purchased from Panreac (Barcelona, Spain). Ortho-22 phosphoric acid (85 %) was obtained from Merck (Darmstadt, Germany). Deionised 23 water (18.2 Mcm) used for the preparation of all aqueous solutions was obtained 24 using a Milli-Q water system (Millipore Ibérica, Madrid, Spain). 25

Syringe membrane filters 0.45 μm Millex-HN (Millipore, Bedford, MA, USA) and solid 26 phase extraction cartridges Spe-ed SPE C18 (Applied Separation, Allentown, PA, 27 USA) were used for processing samples. 28

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2.2. Samples 30

Antimicrobial-free milk samples were collected once or twice a week from bulk tanks in 31 the research farm of Regional Centre of Animal Selection and Reproduction 32 (CERSYRA) in Valdepeñas (Ciudad Real, Spain), and were stored at -30 ºC until use. 33 Before the analysis, the samples were thawed by refrigeration for one day and 34 homogenised in a water bath at 25 ºC for 3 minutes. 35

For recovery experiments, samples were spiked with the desired amount of each 36 antibiotic. Then the spiked samples were homogenised by manual shaking and 37 maintained at room temperature for approximately 20 minutes to allow the equilibration 38 of the β-lactams with the milk matrix before their extraction. 39

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2.3. Preparation of standard solutions 41

Stock standard solutions of individual β-lactams (1 gL-1) were prepared by dissolving 42 50 mg of each compound in 50 mL of water/acetonitrile (50:50, v/v). These solutions 43 were stored in dark glass bottles at 4 ºC and were stable for at least one month. A 44 standard stock mixture of β-lactams (20 mgL-1) was prepared in water from the 45 individual stocks solutions and stored at 4-6 ºC. Working standard solutions at different 46 concentrations were prepared daily in water by appropriate dilution. 47

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2.4. Processing of milk samples 1

A volume of 5 mL of homogenised and fortified raw milk sample was placed in a 15 mL 2 test tube and 5 mL of acetonitrile was added to promote protein precipitation. The 3 mixture was stirred for 1 minute and then left to stand for 10 minutes at room 4 temperature. After centrifugation at 3500 rpm for 20 minutes at 4 ºC, the supernatant 5 was collected onto a glass tube, filtered through a 0.45 μm membrane filter and loaded 6 onto a SPE cartridge, where the extract was allowed to pass through. The cartridge 7 was initially preconditioned sequentially with 5 mL of acetonitrile followed by 5 mL of 8 water two times under vacuum conditions for 15 s. 9

The retained analytes were then eluted with two volumes of 3 mL of phosphate buffer 10 at pH 3.4 and acetonitrile (30:70, v/v) and collected in another test tube. The SPE 11 eluates were evaporated to dryness under a gentle stream of nitrogen at room 12 temperature. Finally, the obtained residues were redissolved in a volume of 500 μL of 13 deionised water, and a 100 μL aliquot was injected onto the LC-UV-DAD-system. All 14 the analyses were carried out in triplicate. A scheme for sample preparation is shown in 15 Figure 1. 16

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2.5. Instrumentation 18

All measurements were performed using an HP 1100 Series LC system (Hewlett 19 Packard, Palo Alto, CA, USA) equipped with a quaternary pump, a vacuum degasser, a 20 column compartment, an autosampler with a 100 μL loop and a diode-array detector. 21 Hewlett-Packard ChemStation software was used by the instrument control and data 22 processing utilities. The stainless analytical column was packed with Supelcosil LC 18 23 DB 5 μm (15 cm x 4.6 mm) from Scharlau (Barcelona, Spain). For the preparation of 24 the samples, a Vortex VibraMix from Ovan (Barcelona, Spain) was used. The pH of the 25 running buffer was adjusting using a Crison pH meter model basic 20 (Barcelona, 26 Spain). A Medifriger model P Selecta (Barcelona, Spain) centrifuge was used to 27 separate the supernatant from the solid phase after protein precipitation. 28

Millex-HN syringe membrane filters (0.45 μm) (Millipore, Bedford, MA, USA) and Spe-29 ed SPE C18 solid phase extraction cartridges (Applied Separation, Allentown, PA, 30 USA) were used for processing samples. 31

Solid-phase extractions of the antibiotics were carried out using a vacuum manifold 32 model Vac Elut 20 from Varian (Palo Alto, CA, USA) coupled with a vacuum pump. 33

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2.6. Chromatographic conditions 35

The LC gradient elution was performed using a mobile phase of 25 mM phosphate 36 buffer solution at pH 3.4 (eluent A) and acetonitrile (eluent B) at a flow rate of 1 mLmin-37 1. Chromatographic separation of the analytes was achieved with the following 38 gradient: 0-2 min, 15 % B; 2-10 min, 15-75% B; 10-13 min, 75% B. The autosampler 39 and the column were maintained at 22 ºC. Quantitative measurements of the peak 40 areas were performed by selecting the appropriate detection wavelength for the 41 compounds to achieve maximum sensitivity. Therefore, β-lactams were quantified at 42 210 nm. 43

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3. Results and discussion 1

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3.1. Optimisation of chromatographic separation 3

The optimisation of HPLC conditions for the separation and simultaneous quantification 4 of the nine β-lactam antibiotics was carried out by the evaluation of the variables that 5 mainly affect chromatographic processes. 6

Based on the reported literature, β-lactam antibiotics have usually been separated on a 7 reverse-phase column using acidic mobile phases [19]. Preliminary experiments were 8 performed using a Supelcosil TM LC-18 column (25 cm x 4.6 mm, 5 μm). Due to the 9 large number of analytes and their different affinities for the column, several elution 10 gradient programs using formic acid (0.1%) as eluent A and different ratios of 11 water/acetonitrile with formic acid (0.1%) as eluent B were tested. The chromatograms 12 obtained did not satisfactorily resolve all the signals. Resolution of the compounds was 13 clearly affected by the acidity of the mobile phase. To prevent this, formic acid was 14 replaced with phosphate buffer, and pH was adjusted to 3.4. Then, different gradient 15 programmes using phosphate buffer solution (pH 3.4)/acetonitrile or phosphate buffer 16 solution (pH 3.4)/water/acetonitrile as mobile phases were tested. The best results 17 were obtained by using 25 mM phosphate buffer solution (pH 3.4) as eluent A and 18 acetonitrile as eluent B, running the gradient programme specified in section 2.6. 19 Although a good separation of the nine analytes was achieved under these conditions 20 in 22 minutes, width and symmetry peak were not satisfactory. To improve peak 21 resolution and decrease run time, a shorter column of similar technical characteristics 22 (15 cm x 4.6 mm, 5 μm) was tested using the above-mentioned gradient programme. 23 As a result, resolution improved considerably, and narrow and symmetrical shape 24 peaks were achieved. 25

The efficiency of analyte elution at different temperatures (range 18-60ºC) was 26 evaluated and results indicated that temperature did not affect significantly the results. 27 Therefore, the autosampler and the column were maintained at 22ºC to achieve 28 temperature reproducibility. 29

Finally, flow rate was varied between 0.5 and 1.5 mLmin-1. At low rates, the peak size 30 and tailing off of the final peak increased significantly, as well as the retention times of 31 the compounds. A flow rate of 1 mLmin-1 was selected and separation achieved within 32 10 min. Under these conditions, the elution order was AMP, CFX, CFL, CFP, PEG, 33 PEV OXA, CLO, DCL (Figure 2). 34

Identification of the nine -lactams was carried out by comparing their retention times 35 and their UV spectra with those of the standards. 36

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3. 2. Optimisation of β-lactam extraction 38

Chromatographic analysis of drugs from a complex matrix such as ewe milk is possible 39 only after a pretreatment of the sample, which allows the elimination of interference 40 and even analyte preconcentration facilitating its subsequent analytical determination 41 since, indirectly, the sensitivity and selectivity of the method is increased [19]. Ewe milk 42 contains high concentration of protein and other substances (e.g., fats and 43 carbohydrates). Thus, procedure for isolation of β-lactams from these samples 44 frequently involves protein precipitation using organic solvents like acetonitrile, 45 methanol or iso-octanol or combinations of sulphuric acid and sodium tungstate 46 solutions prior to SPE clean-up [18, 19, 21, 22, 26-35]. 47

The presence of an unstable ring in some β-lactam antibiotics makes them highly 48 sensitive to the presence of alcohols and acidic solutions or prone to degradation by 49

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heat [22] or by cold [32]. Therefore, it is necessary to control pH and temperature 1 during all stages of sample pre-treatment to prevent the degradation of antibiotics. 2

Initially, protein precipitation was performed using acetonitrile. Different volumes of this 3 organic solvent were tested using 5 mL of sample by 1 min vortex-mixing and 4 subsequent centrifugation. A volume of 5 mL of acetonitrile followed by a centrifugation 5 step for 20 min at low temperature was enough to obtain extracts that were completely 6 transparent, suggesting that the precipitation of proteins was completed. 7

Due to the wide range of polarities of β-lactam antibiotics [33], Spe-ed SPE C-18 8 cartridges were selected for further optimization to extract the highly polar amphoteric β 9 -lactams and the less polar monobasic β –lactams, which agrees with a previous 10 studies [26, 27], 11

Free protein spiked sample (approximately 8 mL) was directly applied to the 12 preconditioned C18 cartridge. Because β-lactams are readily degraded under strongly 13 acidic or basic conditions as a result of hydrolysis of the electrophillic β-lactam ring, a 14 suitable elution solvent had to be carefully selected to achieve the highest recovery for 15 the antibiotics containing in the milk samples. Preliminary SPE elution experiments 16 were performed using as extraction solvents mixtures of water/acetonitrile, phosphate 17 buffer (pH 3.4)/acetonitrile, phosphate buffer (pH 3.4)/methanol, or phosphate buffer 18 (pH 3.4)/methanol/acetonitrile at different ratios. Elution of the target compounds was 19 carried out using two volumes of 3 mL of extraction solvent. Simultaneously, and after 20 evaporation of the eluates, the redisolution of the residues was optimised employing a 21 volume of 500 μL of water or a mixture of water/acetonitrile (70:30, v/v). 22

In general, the lowest recoveries of the analytes were obtained when residues were 23 redissolved in water/acetonitrile mixture in all extraction solvents tested 24 (water/acetonitrile, buffer phosphate pH 3.4/acetonitrile), even the antibiotics AMP, 25 CFX and CFL did not appear in the chromatogram, probably due to the greater polarity 26 of these analytes with respect to the rest. Therefore, water was considered the best 27 redisolution solvent and used for further experiments. 28

The results reported in Table 1 show that, in general, the presence of phosphate 29 enhances the antibiotic recoveries. The use of phosphate buffer in combination with 30 acetonitrile (30:70, v/v) provided the best recoveries for all compounds within the range 31 79-96 %. Therefore, phosphate buffer pH 3.4/acetonitrile (30:70, v/v) was selected as 32 SPE extraction solvent. 33

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3. 3. Analytical performance and application 35

Representative chromatograms obtained at 210 nm of a blank milk sample and milk 36 sample spiked at 6 μgkg-1 concentration level are shown in Figure 2. Non-interfering 37 peaks appeared on the chromatogram of the spiked sample at β-lactam retention 38 times. 39

The linearity of the proposed method was studied for all the test antibiotics under 40 optimised conditions. Calibrations curves were obtained by plotting the peak area 41 against increasing concentrations of the nine β-lactams on the spiked milk (range 2-20 42 μgkg-1). For each point on the calibration plot, 10 injections were performed. Linear 43 regression data, reported in Table 2, showed good linearity for all the analytes with R2 44 coefficients higher than 0.9931. 45

The precision of the method, calculated from the intra-day and inter-day variability on 46 recovery measurements, was studied for the antibiotics at 2 μgkg-1 and 6 μgkg-1 47 concentration levels using a mixed model that takes into account the initial antibiotic 48 concentrations of milk samples and the day the analysis was made. The model is 49 based on the following expression: yiijk=μ+conceni+dayj+eijk, where yiijk is the recovery 50

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value for the antibiotic, μ is the antibiotic global mean, conceni is the initial antibiotic 1 concentration present on sample, dayj is the random effect of the day on which 2 antibiotic recovery values were measured (4 levels) and eijk is the error (n=16) [37, 38]. 3 Obtained mean values for all analytes ranged from 79 to 96 μgkg-1 with relative 4 standard deviations (RSD) between 0.5-4.9 % (Table 3). 5

The limits of detection (LOD) and limits of quantification (LOQ) for the β-lactam 6 antibiotics were calculated by the method proposed by Miller et al. [38]. As reported in 7 Table 2, the LOQ for the studied compounds ranged from 3.4 μgkg-1 to 8.6 μgkg-1, 8 which are lower than the MRL established by the EU for the studied β-lactams in ewe 9 milk [14]. This makes the method suitable for performing routine analyses. 10

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4. Conclusion 12

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A sensitive and reliable method using liquid chromatography with UV-diode array 14 detection (LC-DAD) has been developed for the simultaneous determination of nine β-15 lactam antibiotics: ampicillin (AMP), benzylpenicillin (PEG), cephalexin (CFX), cefazolin 16 (CFL), cefoperazone (CFP), cloxacillin (CLO), dicloxacillin (DCL), oxacillin (OXA), and 17 phenoxymethylpenicillin (PEV) in Manchega ewe milk. In this study, an analytical 18 method that could simultaneously isolate a wide range of β-lactam residues in milk was 19 established. The simple sample treatment, based on a protein precipitation step 20 followed by a solid-phase extraction clean-up with a significant reduction in sample 21 analysis time, allowed purification of the analytes, which yielded extracts ready for fast 22 chromatographic identification and quantification (10 min). 23

The developed method provided good performance and satisfactory recovery, which 24 suggest that it could be easily applied to the routine analysis of β-lactams in ewe milk 25 samples at ppb levels. Furthermore, to our knowledge, this is the first instance in which 26 an analytical procedure for the simultaneous determination of these nine β-lactams was 27 fully applied to ewe milk samples at concentrations approaching the MRLs established 28 under current regulations. 29

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Acknowledgements 31

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The authors wish to thank the Spanish Ministry of Science and Innovation (project 33 AGL2009-12589) and the Comunidad de Castilla-La Mancha (project PAC 06-0037) for 34 financial support of this study, and also Dr. Manuel Ramón and Adrian Burton for the 35 revision of the manuscript. 36

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[33] Becker, M., Zittlau, E., & Petz, M. (2004). Residue analysis of 15 penicillins and 14 cephalosporins in bovine muscle, kidney and milk by liquid chromatography–tandem 15 mass spectrometry. Analytica Chimica Acta, 520,19-32. 16

[34] Granelli, K., & Branzell, C. (2007). Rapid multi-residue screening of antibiotics in 17 muscle and kidney by liquid chromatography-electrospray ionization–tandem mass 18 spectrometry. Analytica Chimica Acta, 586, 289-295. 19

[35] Coantic, S., Mouysset, D., Mignani, S., Tabart, M., & Stella, L. (2007). 20 Stereoselective synthesis of trans-disubstituted-β-lactams from N-phenylsulfenylimines. 21 Tetrahedron Letters, 48, 4301-4303. 22

[36] García-Mayor, M.A., Garcinuño, R.M., Fernández-Hernando, P., Durand-Alegría, 23 J.S. (2006). Liquid chromatography–UV diode-array detection method for multi-residue 24 determination of macrolide antibiotics in sheep's milk. Journal of Chromatography A, 25 1122, 76-83. 26

[37] Long, G.L., & Winefordner, J.D. (1983). Limit of detection. A closer look at the 27 IUPAC definition. Analytical Chemistry, 55, 712A-724A. 28

[38] Miller, J., & Miller, J.C. (2002). Estadística y Quimiometría para Química Analítica. 29 (4th ed.). Madrid: Pearson Education S.A. 30

12

FIGURE AND TABLE CAPTIONS

Figure 1. β-lactam extraction procedure.

Figure 2. Chromatograms obtained at 210 nm corresponding to: (A) Milk sample

spiked with mixture of β-lactams at 6 μgkg-1 concentration level using LC-DAD under

optimum conditions. Peak identification: 1.AMP, 2. CFX, 3. CFL, 4. CFP, 5. PEG, 6.

PEV, 7. OXA, 8. CLO, 9. DCL; (B) Treated blank milk sample.

TABLES Table 1. Recovery* study for each β-lactam antibiotic using different eluent and

redisolution solvents.

Table 2. Analytical performance of the developed method.

Table 3. Intra- and inter-day, precision and accuracy of the method at two spiking

levels (all means are the average of at least four replicates.

Table 1. Recovery* study for each β-lactam antibiotic using different eluent and redisolution solvents

Antibiotic

Eluent solvents Eluent solvents

water/

ACN

30:70

BP/ACN

30:70

BP/ACN

20:80

BP/

MeOH/

ACN

20:20:60

water/

ACN

30:70

BP/ACN

30:70

BP/ACN

20:80

BP/

MeOH/

ACN

20:20:60

Redisolution solvent: water Redisolution solvent: water/ACN (70:30)

Ampicillin 20 90 33 21 / / / /

Cephalexin 21 80 35 23 / / / /

Cefazolin 24 96 60 27 / / / /

Cefoperazone 26 79 39 29 12 12 10 16

Benzylpenicillin 22 94 30 26 27 27 21 36

Phenoxymethyl

penicillin 55 93 40 60 110 110 79 118

Oxacillin 18 84 32 21 44 44 35 60

Cloxacillin 16 96 30 18 40 40 32 52

Dicloxacillin 16 83 25 19 40 40 30 48

Buffer Phosphate (BP); Methanol (MeOH); Acetonitrile (ACN) * Recovery data in %

Table 2. Analytical performance of the developed method.

Antibiotic Regression equation R2 LOD (μgkg-1) LOQ (μgkg-1) MRL (μgkg-1)

Ampicillin y = 18.559x – 27.426 0.9996 2.8 3.7 4

Cephalexin y = 24.356x – 27.218 0.9978 4.0 8.6 /

Cefazolin y = 27.815x – 29.859 0.9995 2.5 3.4 50

Cefoperazone y = 16.188x – 5.5712 0.9931 2.8 6.2 /

Benzylpenicillin y = 24.67x – 40.056 0.9994 3.1 4.0 4

Phenoxymethylpenicillin y = 16.911x – 19.635 0.9989 4.0 8.4 /

Oxacillin y = 28.589x – 10.526 0.9992 2.1 3.9 30

Cloxacillin y = 29.62x – 26.88 0.9988 2.5 3.9 30

Dicloxacillin y = 33.279x – 42.476 0.9991 2.8 4.0 30

Table 3. Intra- and inter-day precision and accuracy of the method at two spiking levels (all means are the average of at least four replicates).

Antibiotic Spiking level

(μgkg-1)

Intra-day recovery

(%) RSD (%)

Inter-day recovery

(%) RSD (%)

Ampicillin 2

6

90

92

1.5

0.8

89

93

2.9

1.8

Cephalexin 2

6

79

83

2.2

1.6

80

81

3.8

2.9

Cefazolin 2

6

96

98

3.4

2.6

93

97

4.9

1.9

Cefoperazone 2

6

77

79

2.7

1.1

78

80

3.9

2.6

Benzylpenicillin 2

6

94

97

3.5

2.0

93

96

3.3

2.9

Phenoxymethyl

penicillin

2

6

92

94

2.3

1.2

91

95

3.8

2.7

Oxacillin 2

6

84

87

2.6

1.4

86

88

3.7

2.5

Cloxacillin 2

6

95

99

3.7

2.4

91

98

4.4

2.5

Dicloxacillin 2

6

83

89

1.1

0.5

82

90

3.4

2.1

Figure 1

Spiked milk5 mL

Homogenization- Vortex 1 min

- 10 min room temperature

Acetonitrile 5 mL

Centrifugation- 20 min

- 3.500 rpm

- 4ºC

Aqueous phase

Solid phase

(Proteins)

Filtration

0,45 μL membrane filter

SPE

Eluted analytes

N2 stream

Residue

Dissolve with 500 μL water

HPLC-DAD

100 μL

Spiked milk5 mL

Homogenization- Vortex 1 min

- 10 min room temperature

Acetonitrile 5 mL

Centrifugation- 20 min

- 3.500 rpm

- 4ºC

Aqueous phase

Solid phase

(Proteins)

Filtration

0,45 μL membrane filter

SPE

Eluted analytes

N2 stream

Residue

Dissolve with 500 μL water

HPLC-DAD

100 μL

Figure 2

2 4 6 8 10 0

0

500

1000

1500

2000

2500

min

mAU

1 2 3

4 5 6 7 8 9

A

2 4 6 8 10 0

0

500

1000

1500

2000

2500

min

mAU

B

Highlights

- A method for simultaneous determination of nine β-lactams in ewe milk

using HPLC-DAD is described

- Sample treatment allowed extracts ready for fast identification and quantification

- Analyte recoveries ranged from 79 to 96 % with relative standard

deviations between 0.5-4.9 %.

- LOQs were 3.4-8.6 μgkg-1, lower than MRLs established by the European Union

- A powerful and popular tool for confirmation of antibiotics in small milk industries is described