INTRODUCTION

1. DETERMINATION OF PENICILLIN ANTIBIOTICS IN ANIMAL TISSUE BY ON-LINE SOLID PHASE EXTRACTION COUPLED TO LC/MS/MS N. Bellós1, L. Bonetto1, J. Hurtado de Mendoza1, J. Melero1, M. Sibum2 and F.A. Mocholí1 1SAILab. Argenters, 5. Parc Tecnològic del Vallès. 08290 Cerdanyola del Vallès. Barcelona. Spain. 2Spark Holland B.V. Bendienplein, 5. 7815 SM. Emmen. The Netherlands. e-mail: fm@sailab.es INTRODUCTION Through the last years, Liquid Chromatography tandem Mass Spectrometry (LC/MS) use have been growing on the quantitation of residues in food such as vegetables, meat, etc., versus other detection systems, because of the good sensitivity, linearity and robustness of this detection technique. To achieve good results in those conditions, and avoid instrumental problems, solid phase extraction (SPE) has become the typical election. On-Line SPE has been commonly used, mainly, with liquid samples, where, not only is important to remove undesirable compounds, but also the possibility to inject more sample to get better sensitivity. In this work all those aspects have been important to develop a simple method to analyze residues of six penicillins like amoxicillin, ampicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G and penicillin V in pork muscle, arriving to concentrations up to 5 µg/Kg. Complete extraction method is summarized in figure 6. There, all steps included in the method can be observed. INSTRUMENTAL Fig. 6 Sample extraction procedure 4g SAMPLE This Part II and Part III are all involved in the same instrumental procedure. They are separated here in order to be better explained. SOLVATION 2 ml Methanol 20 ml SOLVENT EXTRACTION ACN:H2O (9:1) EQUILIBRATION 2 ml Water (shown in figure 3) Part III. Chromatographic analysis Chromatography was carried out with a Phenomenex Sinergy MAX 150mm, 4.6mm, 3 mm column, at 1 ml/min flow, using 2 mM ammonia formiate, 0.1% formic acid in water and acetonitrile as mobile phases. Mass Spectrometry detection was performed by an Applied Biosystems 3200 QTrapTM system. Analytical conditions in Turbo V source has been 10 psi curtain gas (N2), negative ionization, 40 psi ionization gas (air), 70 psi turbo gas (air). HOMOGENIZATION SAMPLE FROM PART I 1 ml CENTRIFUGATION SAMPLE EXTRACTION 2.5 ml Water (shown in Fig. 4) FILTRATION 0.45 UM RESULTS AND DISCUSSION Extraction from the matrix has been carried out with organic solvent, but diluted with aqueous solvent to match with the solvent allowed by the XLC cartridge. Scheme in figure 1 show the optimized extraction. Dilution solvent has been McIlvaine buffer. As this buffer contains phosphate is important to wash efficiently from the cartridge. Injecting 1 ml of sample allow to have a very high amount of sample in column, but allow also to dilute matrix up to 1:100 Chromatographic method has been tested with matrix prepared standards to maintain response against standards in solvent. Then, matrix effect is minimized, either in XLC part (cartridge is eluted enough time) and also as ionic suppression in the mass spectrometer source. Separation achieved with the conditions described above is shown in figure 7. WASH Symbiosis Pico XLC System 3200 QTrapTM LC/MS/MS System DILUTION 1:20 McILVAINE BUFFER pH7.6 Nafcillin ELUTION TO THE COLUMN VIAL TO INJECT Oxacillin Ampicillin Cloxacillin Part I. Extraction from the tissue First part of the method is to extract target analytes from the animal tissue. Procedure used is shown in Fig 1. Minced previously meat is weighted and mixed with extraction solvent, homogenization with Ultraturrax like apparatus and centrifugation is enough to extract antibiotics from matrix. Last part of the extraction procedures is only dilution with McIlvaine buffer. Penicillin V Figure 1. Sample extraction procedure Dicloxacillin Penicillin G Amoxicillin Fig. 7. 1 µg/l XLC/MS/MS chromatogram Total chromatographic analysis last 12 min to elute completely all matrix and background compounds. That include SPE preparation time. Validation This method show very good linearity response in the range studied as can be seen in figure 8 and Table I (r2). Linearity (correlation coefficients) observed are over 0.99 for all compounds. Detection and quantitation limits are shown in Table II. Almost all quantitation limits are below the limit fixed previously, all detection limits meet the specification established Characteristics of target analytes focus to use organic extraction solvent in contact with meat, but there are two hydrophilic compounds, so need water for extraction, Amoxicillin and Ampicillin. It is important also that Amoxicillin is more stable in basic conditions, so that is the reason to put a basic solvent as dilution solution. Let’s note that total dilution applied to the sample is 1:100. Table I. Correlation coefficients and detection and quantitation limits Fig. 2. Optimization of SPE Cartridge type Fig. 3. Optimization of SPE Cartridge Equilibration volume (ml) Part II. Clean-up. On-line solid phase extraction In this method On-Line Solid Phase Extraction (XLC) has been used as a clean-up step prior to the LC/MS analysis. Symbiosis Pico system include both on-line extraction device and HPLC system, and can be used as HPLC only or XLC, changing from the software. Hysphere C8HD 7µm (C8), Hysphere C18HD 8µm (C18) and Hysphere Resin GP 10µm (GP) cartridges has been tested. Added to that, Equilibration volume, Extraction volume and Elution time has been optimized. development of the four extraction method parameters are shown in figures 2, 3, 4 and 5 respectively. Fig. 8. Calibration curves for all compounds studied Precision and accuracy are show in figures 9 and 10 respectively. Good precision, both intraday and interday, can be observed, below 10%, for all compounds except amoxicillin, and this show results not much higher that 25%. Amoxicillin show recovery (accuracy) over 70% while the others are higher, over 80%) Fig. 4. Optimization of SPE Extraction volume (ml) Fig. 5. Optimization of SPE Cartridge elution time (min) Fig. 9. Precision (RSD, %) intraday and interday Fig. 10. Overall accuracy (%) CONCLUSIONS Method developed in this work has been tested that gives very good results analyzing antibiotics (penicillins) residues in animal tissue such as pork meat. Sensitivity achieved injecting 1 ml and with this conditions give the alternative to dilute the sample up to 1:100, eliminating almost completely the matrix effect. Linearity accomplished is very good in the range 0.05 to 5.00 µg/l injected. Taking dilution, from 5 to 500 µg/Kg. In sample, detection limit (L.D.) and quantitation limit fit well with the requirements of the method for all the compounds. Also for precision and accuracy On-line SPE/LC/MS (XLC/MS) has been checked as a proven technique to analyze penicillins in pork meat giving simplicity and very good results. Both three cartridges tested give good response for all compounds, shown in figure 2. except amoxicillin and ampicillin that give no response with C8 and GP cartridges. Cartridges are solvated with 2ml of methanol and equilibration volume is optimized in order to better adsorb the analytes (figure 3). As injection volume is 1 ml of sample, extraction volume must be optimized. This solvent is used with two purposes, carry the sample from loop in the autosampler and wash matrix from the cartridges, that can be seen in figure 4. Equilibration, extraction and wash were done with water. Different pH water solvents were checked but no difference were observed. 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