Journal of Chromatography A, 1208 (2008) 8389

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Determination of macrolide antibiotics in meat and sh using pressurized liquid extraction and liquid chromatographymass spectrometry
Houda Berrada a, , Francesc Borrull b , Guillermina Font a , Rosa Maria Marc b
a b

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de Valncia, Avda Vicent Andres Estells s/n, 46100 Valncia, Burjassot, Spain Department of Analytical Chemistry and Organic Chemistry, Universitat Rovira i Virgili, Sescelades Campus, Marcell Domingo s/n, 43007 Tarragona, Spain

a r t i c l e

i n f o

a b s t r a c t
We developed a method for determining the quantities of seven macrolide antibiotics in meat and sh by using pressurized liquid extraction (PLE) and liquid chromatographymass spectrometry with electrospray ionization (LC(ESI)MS). The PLE was optimized with regard to solvents, temperature, pressure, extraction time and number of cycles. The optimum conditions were: methanol as the extraction solvent; a temperature of 80 C; a pressure of 1500 psi; an extraction time of 15 min; 2 cycles; a ush volume of 150% and a purge time of 300 s. All recoveries for macrolide antibiotics were over 77% at 200 g/kg, except for erythromycin, which was 58%. The repeatability and reproducibility on days in between, expressed as %RSD (n = 12), were lower than 10% and 12%, respectively. The quantication limits of all compounds were 25 g/kg of dry weight of animal muscle except for troleandomycin (50 g/kg). The method was applied to determine the pharmaceuticals in real samples taken from 18 meat and sh samples. The results showed that PLE is quantitative short time consuming technique, with use of smaller initial sample sizes. Greater specicity and selectivity in extraction and increased potential for automation were shown. 2008 Elsevier B.V. All rights reserved.

Article history: Received 29 May 2008 Received in revised form 11 August 2008 Accepted 14 August 2008 Available online 4 September 2008 Keywords: Macrolides Meat Fish Food LC(ESI)MS PLE

1. Introduction Antimicrobials are widely prescribed for both therapeutic and prophylactic reasons against microbial infections and also as growth promoting substances at sub-therapeutic levels in animal farms and aquaculture. Macrolides are a group of antibacterial compounds active against gram-positive and some gram-negative bacteria that are widely used in human and veterinary medicine. The incorrect use of these drugs can leave residues in food products and this can have undesirable effects on consumer health [1,2]. Recently, concern has been expressed that continuous sublethal levels of antibiotics in food have led to the emergence of harmful bacteria resistant to antibiotics. Some macrolides are now restricted to veterinary therapeutic use and maximum residue limits (MRLs) have been established for these substances in muscle tissue, fat, liver and kidneys. Therefore, the use of these antibiotics in foodstuffs is regulated by Council Directive 2377/90 EC [3], which describes the procedure for establishing MRLs for veterinary medicinal products in foodstuffs of animal origin.

Presented at the 7th Meeting of the Spanish Society of Chromatography and Related Techniques, Granada, Spain, 1719 October 2007. Corresponding author. Tel.: +34 96 3544958; fax: +34 96 3544954. E-mail address: houda.berrada@uv.es (H. Berrada). 0021-9673/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.107

Erythromycin, spiramycin, tilmicosin and tylosin are included at annex 1 as substances with MRL values. Josamycin was included at annex III as substances with provisional MRL values. Roxithromycin and troleandomycin have not a marketing authorization for use in food-producing animals in the European Union and no MRL values could be established for these substances. In order to assess the occurrence of these residues in food, sensitive analytical methods are required which enable us to determine multiple compounds simultaneously at quite low concentration levels. One important requirement of the extraction method was that it should be fast and robust enough to allow the rational analysis of a large number of eld samples. However, at the same time the method ought to allow the quantication of macrolides with sufcient sensitivity. The literature reports the determination of macrolides in urine [4], plasma [5], soil [6], animal tissues [7,8] and sludge [9,10] using such analytical techniques as liquid chromatography (LC) with ultraviolet (UV) or uorimetric detection, LCmass spectrometry (LCMS) and LCtandem mass spectrometry (LCMSMS) [11,12]. However, there are relatively few published multiresidue methods for determining the selected macrolides in animal tissues [13,14]. In a previous work, we used LC and a diode array detector (DAD) to successfully determine the macrolides in liver and kidney tissues from animals and we conrmed the results with electrospray mass spectrometry [8]. LCDAD was also used to conrm the identity of the residues according to the European Commission Decision

84

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389

2002/657/EC [15]. An aqueous extraction of these antibiotics from liver and kidney using EDTA-McIlvaines buffer before cleaning these by SPE was used [8]. Another study using 0.2% metaphosphoric acidmethanol to extract antibiotics from meat and sh has also been reported [13]. However, meat is protein rich matrix that is important for those antibiotics that bind easily to proteins. Antibacterialprotein binding must be weakened by diluting the sample with a saline dilution, protein denaturation or enzymatic and chemical hydrolysis of the drugprotein complexes [16]. The quantitative determination of total antibiotics required a digestive step, which was time consuming and necessary for meat products to free protein-bound analytes. This step was delicate, with poor potential for automation. Efforts have been directed to attain high-throughput methods able to extract a large number of samples in a short time. Pressurized liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction) is investigated here as an alternative technique to avoid a digestive step prior to solid-phase extraction to free bound protein [17]. PLE is a rather new technique that uses solvent at a relatively high pressure and temperature without their critical point being reached. This improves efciency compared to extractions at room temperature and atmospheric pressure [18,19]. Recently, Gbel et al. have reported extracting macrolides by using rapid and simple PLE procedures with a mixture of water and methanol as the extracting solvent in sewage sludge [9]. In the same way, Schlsener et al. [19], Jacobsen et al. [6] and Nieto et al. [10] reported using PLE with mixtures of methanol and buffered water for macrolides concentrated in soils and sludges. Other studies have reported using PLE to extract other pharmaceutical compounds from let tissues, such as sulfonamides in meat [20] and paroxetine and uoxetine in sh [21]. All groups remarked on the technologys benets in providing rapid and reliable analysis. Optimization of the extraction process generally begins by choosing an appropriate extraction solvent. Often, the same solvent used for conventional extractions is initially tested [17]. Other experimental parameters of the extraction are temperature, pressure, static time, and cell size [2225]. The present study focuses on developing a robust, simple and practical method capable of simultaneously extracting and determining seven macrolide antibiotics which belong to different macrolide subgroups in meat and sh. The macrolide antibiotics selected were erythromycin, josamycin, roxithromycin, spiramycin, tilmicosin, troleandomycin and tylosin. To the best of our knowledge, no studies have been conducted into extracting these macrolide antibiotics from a biological matrix using a PLE. 2. Experimental 2.1. Materials and reagents Commercial macrolide standards of erythromycin, tylosin hemitartrate and spiramycin were supplied by Riedel-de Han (Seelze, Germany); josamycin, roxithromycin, troleandomycin and tilmicosin were purchased from SigmaAldrich (Madrid, Spain). Standard stock solutions of individual macrolides (1 mg/ml) were prepared by dissolving 25 mg in 25 ml of methanol. These solutions were stored in dark glass bottles at 4 C and were stable for at least 3 months [8]. A nal standard mixture of macrolides was prepared each week. These solutions were also stored at 4 C. Ultra pure water was obtained with a Milli-Q water purication system (18.2 M cm) (Millipore, Bedford, MA, EEUU), acetone, acetonitrile and methanol (HPLC-grade) were from SDS (Peypin,

France), nitrogen was from Carburos Metlicos (Tarragona, Spain) and phosphoric acid (97%) was from Merck (Darmstadt, Germany). 2.2. Sample pretreatment Beef, chicken, pork, sea bream and trout lets were collected from different supermarkets in the city of Tarragona (Spain). Samples, around 500 g, were shipped to our laboratory under cool conditions in a portable refrigerator and were frozen at 30 C upon reception. Samples were lyophilized before being analyzed by the freeze dry system (Labconco, MO, USA). Then they were homogenized using a mortar and pestle and sieved to obtain particles with a diameter of less than 125 m. To optimize the method, a 5 g lyophilized muscle sample was spiked with all compounds, which were dissolved in 10 ml acetone. After spiking, the samples were shaken intensively so that the compounds spread throughout the spiking solution in the sample and were in sufcient contact with the matrix. They were then evaporated to dryness at room temperature. 2.3. PLE extraction Lyophilized muscle samples were extracted using an ASE 200 PLE system (Dionex, Sunnyvale, CA, USA). A total of 5 g of the pretreated muscle was thoroughly mixed with 7 g of aluminum oxide and the mixture was put into a 33 ml stainless steel extraction cell. This cell, lled with aluminum oxide, was positioned in the PLE system connected to a four bottle solvent controller. Nitrogen at a pressure of 10 bars was supplied to assist the pneumatic system and to purge the extraction cells. The aluminum oxide had been heated at 120 C in the oven for 24 h before use. The extracting solvent was 100% methanol. The operating conditions were as follows: extraction temperature, 80 C; extraction pressure, 1500 psi; preheating period, 5 min; static extraction, 15 min; nal extraction volume, 40 ml; ush volume 150% of the cell volume; nitrogen purge, 300 s; and number of extraction cycles, 2. Each PLE extract was concentrated to about 1 ml in a Bchi R200 (Labortechnik, Flawil, Switzerland) rotary evaporator set at 40 C and 250 mbar in 50 ml round-bottomed asks. Then, the extract was transferred to a 15 ml conical tube and the round-bottomed ask was rinsed twice with 0.5 ml of methanol and evaporated to dryness using a multi-sample Turbovap LV Evaporator (Zymark, Hoptkinton, USA) provided with a nitrogen stream and a water bath at 50 C. After solvent evaporation it was reconstituted in 0.5 ml of methanol. The extract was ltered through a 0.45 m nylon lter (Teknokroma, Barcelona, Spain), and then analyzed by LC(ESI)MS. 2.4. Chromatographic analysis The chromatographic system was an HP 1100 series (Agilent technologies, Waldbronn, Germany) equipped with an automatic injector, a degasser system and a single quadrupole mass detector with electrospray ionization (ESI). The chromatographic column was a Kromasil 100 C18 (25.0 cm 0.46 cm, 5- m particle size) (Teknokroma). A binary mobile phase with a gradient elution was used. Solvent A was Milli-Q water with 1% acetic acid (pH 2.8) and solvent B was acetonitrile. The gradient was 20% B, and was increased to 60% in 25 min, to 100% in 35 min, held at 100% for 2 min and then returned to the initial composition in 4 min. The ow-rate was 1 ml/min, and the column temperature was 35 C. The injection volume was 50 l and the compounds studied eluted within 20 min. The mass spectrometer simultaneously acquired data in full-scan and under selected ion monitoring (SIM).

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389 Table 1 Method validation parameters for the entire beef analytical method PLELC(ESI)MS Antibiotic Erythromycin A Josamycin Roxithromycin Spiramycin Tilmicosin Troleandomycin Tylosin
a b c

85

Linear range ( g/kg) 25400 25400 25400 25400 25400 50400 25400

r2 0.990 0.993 0.992 0.994 0.992 0.992 0.993

Ions 735(100), 576(60), 158(55) 829(100), 174(15) 838(100), 414(80), 679(35) 422(100), 174(35), 843(15) 435(100), 869(20), 174(18) 772(100), 435(10), 814(10) 916(100), 174(15), 772(10)

LOD ( g/kg) 41 27 35 20 23 51 18

MRLsa ( g/kg) 200 200, 250b 50, 75c 100

Maximum residue limits established for all food producing species in annexes IIV of Council Regulations 2377/90. At porcine muscle. At poultry muscle.

The average conditions selected for the optimum performance of the ESI interface in the positive mode were: nebulizer pressure 40 psi, drying gas ow-rate 12 l/min, drying gas temperature 350 C and capillary voltage 3500 V. Different fragmentation voltages were studied to nd spectra with three ions for most of the studied compounds. Fragmentation voltages were dened individually and the values were: 75 V for tilmicosin and troleandomycin and 125 V for erythromycin, josamycin, roxithromycin, spiramycin and tylosin. The ions selected for quantifying the samples are shown in Table 1. The most abundant ion was used for quantication and the second and third ones were used for conrming the results except in the case of josamycin, which has spectra with only two ions. 2.5. Method validation The whole PLE procedure was optimized with beef meat. Carefully checked lyophilized muscle samples were used as blanks before they were spiked with 200 g/kg of each compound for the PLE. To validate the analytical method, beef meat spiked at ve different concentrations 25.0 (50.0 g/kg for troleandomycin), 100.0, 200.0, 300.0 and 400.0 g/kg was used for matrix-matched calibration standard curves (Table 1). These spiked samples containing macrolide standards were processed through the complete extraction procedure. To make sure no interfering substances were present around the retention time of analytes, we analyzed three meat samples from ve different species (beef, chicken, pig, trout and sea bream sh). In all of them, 15 let samples were analyzed. Repeatability and recovery were assessed by performing tests on blank samples of beef. Six aliquots were fortied at 50, 100 and 200 g/kg. These levels are around the MRLs set by the EU, which is more or less equivalent to MRL/2, MRL, and 2 MRL established levels. As mentioned above, there is no directive in existence regulating tolerance levels of josamycin, roxithromycin and troleandomycin in meat. In this case, analyte recoveries were estimated by following the same procedure as reported above, i.e., at 50, 100 and 200 g/kg. We analyzed each level three times to nd the mean concentration and the relative standard deviation (%) of the fortied samples.

These spiked samples were also extracted and injected on different days to nd the inter-day precisions (n = 4 3). 3. Results and discussion 3.1. PLE optimization The initial conditions for optimizing the PLE were taken from a previous study which determined some of the studied macrolides, among other pharmaceuticals, in sewage sludge samples [10]. These conditions were: a solvent that was made up of water/methanol (50:50, v/v), 1500 psi, 100 C, 2 cycles, extraction time 15 min, 300 s of purge time, 5 g of dry sample and nally 150% of ush volume. The rst challenge when developing the PLE method is choosing an appropriate extraction solvent [2628]. Acidic water at pH 3.6, methanol, acetonitrile and mixtures of methanol and water were tested to choose the best extracting solvent. Pure organic solvents, such as acetonitrile or methanol, as well as aqueous phosphoric acid/methanol mixtures, were able to extract the studied macrolides from beef meat to a certain degree (Table 2). Acidic water itself showed extraction efciencies of around 55% for most of the macrolides except for tylosin, which showed 77%. When mixtures of water with methanol were used, lower extraction efciencies (on average 20% lower) were observed for the compounds compared with single methanol as the extracting solvent. Similar conclusions for some macrolides in sewage sludge were made by Gbel et al. [9]. Based on results presented in Table 2, methanol was chosen as the solvent for extracting macrolides from beef muscle since it recovered over 75% of them, except for erythromycin. These results are comparable with those obtained using other techniques such as liquidliquid extraction, matrix solid-phase dispersion and solid-phase microextraction, whose recovery values were from 60 to 120% [11], although a larger volume of solvents, and in some cases, several solvents, must be used. According to Lou et al. [27] time, temperature and pressure are the most important variables that can affect PLE efciency. In order to see if extraction could be improved, we decided to use methanol in various experiments, changing the temperature (40, 60, 80, 100 and 120 C) and pressure (500, 1000, 1500, 2000 and 2500 psi).

Table 2 Solvent inuence on the extraction recovery of studied macrolides from beef meat (n = 3) Extraction solvent % Recovery (% RSD) Erythromycin A Acetonitrile Methanol Methanol/water (3:1) Methanol/water (1:1) Methanol/water (1:3) Water pH 3.6 32 (15) 58 (8) 51 (8) 50 (6) 47 (11) 42 (6) Josamycin 54 (11) 77 (6) 72 (9) 67 (11) 53 (15) 43 (7) Roxithromycin 48 (10) 78 (6) 69 (6) 59 (10) 54 (13) 50 (10) Spiramycin 60 (9) 80 (5) 74 (9) 59 (7) 53 (9) 48 (12) Tilmicosin 54 (12) 82 (5) 75 (7) 69 (12) 63 (15) 56 (10) Troleandomycin 48 (13) 74 (10) 67 (15) 63 (16) 61 (13) 57 (9) Tylosin 72 (8) 90 (6) 83 (8) 79 (7) 77 (12) 78 (15)

86

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389

Fig. 2. Recoveries obtained by successive extractions. For experimental conditions, see text.

3.2. Method validation The water losses in samples during drying were determined by lyophilisation and this information was taken account to express obtained results at whole (wet) samples. The effect of lyophylisation was also checked and the recoveries were calculated by comparing the peak area obtained from meat samples spiked at 200 g/kg before and after the sample preparation. The data from both pretreatment procedures were compared with a t-test for both compounds. They were not signicantly different for a condence interval of 95%. All research was done on beef meat for validative purposes. The linear range was calculated by matrix-matched calibration standard curves and linearity was good, between 25 and 400 g/kg for most of the compounds with the determining coefcient values (r2 ) above 0.99, except for troleandomycin, which showed good linearity between 50 and 400 g/kg. The recovery and repeatability of the LC(ESI)MS method was evaluated by spiking six blank samples of beef meat in triplicate with 50, 100 and 200 g/kg of each macrolide. The values were higher than 78% at 200 g/kg and all %RSDs were below 15% (n = 3) except for erythromycin A, whose average recovery was 58% and whose RSD was 12%. For inter-day assays, recovery data were also satisfactory. The inter-day repeatability was determined in triplicate on 4 successive days. The analytical results are summarized in Table 3. When we analyzed samples spiked with three different concentrations (50, 100 and 200 g/kg), recoveries were higher than 68%. To evaluate the ionic suppression effect, data obtained from spiked samples and those obtained from standards at the same concentration as the spike were compared with a t-test. They were not signicantly different for a condence interval of 95%. Ionic suppression was not observed and the experiments proved that recoveries were independent of applied fortication levels. The whole procedure was applied to 15 blank samples of different origins (beef, chicken, pig, sea bream and trout) to verify the specicity of the method and it was shown that no interference was detected around the retention time of the seven analytes in any of the samples analyzed. The recovery values of the macrolides extracted from different matrices were similar (the differences found were less than 15%), indicating that the matrix effects of the origin meat were minimal (Table 4). The method allows recoveries comparable to the most commonly applied extraction methods [8,11,13], without the need for further clean-ups or for increasing throughput due to the high PLE automation grade. The specied PLE extraction procedures generated interferencefree chromatograms at the retention times of the macrolides

Fig. 1. (A) Effect of the temperature on the extraction efciency. (B) Effect of the pressure on the extraction efciency. For experimental conditions, see text.

As temperatures increase, interactions between analytes and matrix components are weakened and viscosity and surface tension are decreased. As can be seen in Fig. 1a, the best results were obtained at 80 C, with recoveries ranging from 58 to 90% and %RSDs from 4 to 12% (n = 3). At 100 C the color, cloudy suspension and %RSDs increased because compounds of high molecular mass were co-extracted. We used methanol in a PLE at different pressures to extract macrolides from beef muscle. As Fig. 1b shows, the highest recovery of macrolides was obtained at 1500 psi. Good recoveries are also obtained at low pressure, close to 500 psi, which subsequent analysis shows is the lowest pressure possible. However, the system becomes unstable (overlled collections vials) because of difculties in maintaining the set pressure. High pressure during extraction keeps the solvent in a liquid state when working at temperatures at or above boiling point. Extraction is also more efcient because it forces the solvent into areas where it would not normally go under atmospheric conditions [28,29]. Lengthy exposure to solvents allows the matrix to swell, thus improving the penetration of the solvent into the sample interstices and the contact of the solvent with the analytes [30]. The number of cycles was optimized and four consecutive extractions from the same sample were made. Signicant amounts of analytes were found in the second extract but the recoveries for all the compounds were considered negligible in the third cycle, as Fig. 2 shows. For this reason, two cycles were considered optimum and this allowed us to introduce fresh solvent and maintain a favorable balance between solvent and sample. We checked the percentage of ush from 150% down to 50% to see if this increased the preconcentration factor. However, recoveries were higher with 150% ush volume and so this was chosen as the optimum.

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389 Table 3 Inter- and intra-day recovery of the method at three levels of spiking beef meat (n = 3) Antibiotic Erythromycin A Spiking level ( g/kg) 50 100 200 50 100 200 50 100 200 50 100 200 50 100 200 50 100 200 50 100 200 Intra-day recovery (%) 48 54 58 69 74 77 69 73 78 72 84 82 73 78 82 71 80 84 72 83 90 RSD (%) 13 11 8 9 7 6 10 6 6 10 7 5 7 7 5 11 8 4 11 7 4 Inter-day recovery (%) 45 59 57 65 72 74 65 69 69 77 81 80 67 74 76 68 73 74 76 80 87

87

RSD (%) 15 14 12 11 10 8 13 12 9 12 8 7 14 10 11 15 11 7 14 11 7

Josamycin

Roxithromycin

Spiramycin

Tilmicosin

Troleandomycin

Tylosin

Table 4 Recoveries and %RSD (n = 3) of macrolides from various meat and sh matrices at fortication level of 200 g/kg Antibiotic Erythromycin A Josamycin Roxithromycin Spiramycin Tilmicosin Troleandomycin Tylosin Bovine meat 58 (8) 77 (6) 78 (6) 82 (5) 82 (5) 74 (4) 90 (4) Porcine meat 63 (6) 84 (4) 81 (9) 75 (8) 83 (5) 72 (6) 88 (5) Poultry meat 59 (9) 82 (5) 86 (5) 80 (5) 85 (4) 70 (6) 86 (4) Sea bream sh 68 (6) 90 (5) 83 (6) 84 (5) 87 (3) 75 (7) 84 (4) Truite Fish 66 (7) 91 (4) 85 (5) 82 (6) 79 (3) 69 (6) 90 (5)

studied and Fig. 3 shows LC(ESI)MS chromatograms of spiked beef meat samples at 100 g/kg. The detection limits were experimentally calculated from the analysis of beef samples spiked with a standard mixture of the analytes at serially diluted concentrations as the minimum concentration of an analyte. These were given a signal to noise ratio of 3, as set in the instruments software package. The detection limits were lower than 15 g/kg for all compounds and the quantication limits, considered as being at the lower point of the linear range, were 25 g/kg for most compounds. This showed that the method could be useful for determining macrolide residues in contaminated meat and sh tissues. The analytical limits CC and CC were determined as required by Commission Decision 2002/657/EC. Table 5 shows the CC and CC with an error of 5%, considering the experimental standard deviation of within-laboratory reproducibility at the adequate contamination level. In the case of erythromycin, spiramicin, tylosin and tilmicosin, which have an established MRL, CC and CC were calculated by
Table 5 CC and CC values for studied macrolides in beef meat Antibiotic Erythromycin A Josamycin Roxithromycin Spiramycin Tilmicosin Troleandomycin Tylosin CC ( g/kg) 208 6 8 204 53 12 102 CC ( g/kg) 211 15 17 206 54 35 104

analyzing 20 blank beef meat, all fortied with the analyte at the maximum permitted limit according to the EU criteria. However, since josamycin, roxithromycin, and troleandomycin are not licensed for use in veterinary products, it does no have an established MRL and as a consequence, food for human consumption should be free from. CC was calculated for these macrolides by analyzing 20 blanks to be able to calculate the signal to noise ratio at the time window in which the analyte is expected. Three times to the signal to noise ratio can be used as decision limit and CC was calculated by analyzing 20 blanks spiked at the decision limit. The values of the decision limit plus 1.64 times the standard deviation of the within-laboratory reproducibility of the measured content equals the detection capability ( = 5%). 3.3. Method application This method has been used to detect the presence of macrolides in raw meat and sh bought from the market and from butchers shops in Tarragona. Four pieces of beef, four of chicken, four of pig, three trout lets and three sea bream lets were purchased to investigate the occurrence of macrolides. Among 18 analyzed food samples, three sea bream samples were positive to LCMS. Only erythromycin A was found at 87, 69 and 58 g/kg with RSDs (%) (n = 3) of 12, 15 and 16%, respectively. Fig. 4 shows the extracted ion chromatogram of a sea bream sample collected in September 2006. The total level of residues found did not exceed 100 g/kg, the set MRL. However the presence of erythromycin A in the edible lets, a compound that should only be administered by injection or in feed, involves the routine control of the antibiotics in food.

88

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389

Fig. 3. SIM chromatograms corresponding to an extract of a meat sample with the compounds added at 100 g/kg.

Fig. 4. SIM chromatogram corresponding to an extract of a sea bream let sample where erythromycin was found at 87 g/kg.

4. Conclusions This work described for the rst time how PLE was used to extract seven macrolide antibiotics from meat and sh lets and how these were simultaneously and efciently determined using LC(ESI)MS. The method was precise with good recovery and low quantication limits. Samples were lyophilized before analysis, which gave clean extracts and avoided further clean-ups being needed. The limits of quantication were lower than the MRL and the efciency of PLE was also comparable to that of conventional techniques. Acknowledgements The authors wish to thank the Ministerio de Ciencia y Tecnologa (Spain) for funding this study through the Project AGL

2006-04438/ALI. H. Berrada also thanks the Universitat de Valncia for a mobility grant. References
[1] T. Heberer, Toxicol. Lett. 131 (2002) 5. [2] S. Omura (Ed.), Macrolide Antibiotics: Chemistry, Biology and Practice, second ed., Academic Press, Orlando, FL, 2002. [3] Council Directive 2377/90 and the later modications 1570/98, 2593/99, 2338/2000 and 1181/2002, Commission of the European Communities, Brussels, 1990, 1998, 1999, 2000, 2002. [4] M.J. Gonzlez de la Huebra, G. Bordin, A.R. Rodrguez, Anal. Chim. Acta 517 (2004) 53. [5] F. Kees, S. Spangler, M. Wellenhofer, J. Chromatogr. A 812 (1998) 287. [6] A.M. Jacobsen, B. Halling-Srensen, F. Ingerslev, S.H. Hansen, J. Chromatogr. A 1038 (2004) 157. [7] M. Horie, K. Saito, R. Ishii, T. Yoshida, Y. Haramaki, H. Nakazawa, J. Chromatogr. A 812 (1998) 295. [8] H. Berrada, J.C. Molt, F. Borrull, G. Font, R.M. Marc, J. Chromatogr. A 1157 (2007) 281.

H. Berrada et al. / J. Chromatogr. A 1208 (2008) 8389 [9] A. Gbel, A. Thomsen, C.S. McArdell, A.C. Alder, W. Giger, N. Thei, D. Lfer, T.A. Ternes, J. Chromatogr. A 1085 (2005) 179. [10] A. Nieto, F. Borrull, R.M. Marc, E. Pocurull, J. Chromatogr. A 1174 (2007) 125. [11] M.J. Gonzlez de la Huebra, U. Vincent, J. Pharm. Biomed. Anal. 39 (2005) 376. [12] M.J. Gonzlez de la Huebra, U. Vincent, C.V. Holst, J. Pharm. Biomed. Anal. 43 (2007) 1628. [13] M. Horie, H. Takegami, K. Toya, H. Nakazawa, Anal. Chim. Acta 492 (2003) 187. [14] M.A. Garca-Mayor, R.M. Garcinuno, P. Fernndez-Hernando, J.S. DurandAlegra, J. Chromatogr. A 1122 (2006) 76. [15] EC Decision 2002/657, Off. J. Eur. Commun. L221 (2002) 8. [16] K. Ridgway, S.P.D. Lalljie, R.M. Smith, J. Chromatogr. A 1153 (2007) 36. [17] R. Carabias-Martnez, E. Rodrguez-Gonzalo, P. Revilla-Ruiz, J. HernndezMndez, J. Chromatogr. A 1089 (2005) 1. [18] J.A. Mendiola, M. Herrero, A. Cifuentes, E. Ibanez, J. Chromatogr. A 1152 (2007) 234. [19] M.P. Schlsener, M. Spiteller, K. Bester, J. Chromatogr. A 1003 (2003) 21. [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]

89

E.L. McClure, C.S. Wong, J. Chromatogr. A 1169 (2007) 53. S. Chu, C.D. Metcalfe, J. Chromatogr. A 1163 (2007) 112. G. Font, A. Juan, Y. Pic, J. Chromatogr. A 1159 (2007) 233. K. Adou, W.R. Bontoyan, P.J. Sweeney, J. Agric. Food Chem. 49 (2001) 4153. A. Gentili, D. Perret, S. Marcheses, M. Sergi, C. Olmi, R. Curini, J. Agric. Food Chem. 52 (2004) 4614. O.P. Heemken, N. Theobald, B.W. Wenclawiak, Anal. Chem. 69 (1997) 2171. R.M. Alonso-Salces, E. Korta, A. Barranco, L.A. Berrueta, B. Gallo, F. Vicente, J. Agric. Food Chem. 49 (2001) 3761. X.W. Lou, H.G. Janssen, C.A. Cramers, Anal. Chem. 69 (1997) 1598. J. Gan, S.K. Ppiernik, W.C. Koskinen, S.D. Yates, Environ. Sci. Technol. 33 (1999) 3249. H. Giergielewicz-Mozajska, L. Da Browski, J. Namiesnik, Crit. Rev. Anal. Chem. 31 (2001) 149. B.E. Richter, B.A. Jones, J.L. Ezzel, N.L. Porter, N. Avdalovic, C. Pohl, Anal. Chem. 68 (1996) 1033.