Analytica Chimica Acta 645 (2009) 517

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Analytica Chimica Acta

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Review

A review of novel strategies of sample preparation for the determination of antibacterial residues in foodstuffs using liquid chromatography-based analytical methods
M.D. Marazuela a, , S. Bogialli b
a b

Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain Department of Chemistry, University of Rome La Sapienza, Piazza Aldo Moro, 5 00185 Rome, Italy

a r t i c l e

i n f o

a b s t r a c t
The determination of trace residues and contaminants in food has been of growing concern over the past few years. Residual antibacterials in food constitute a risk to human health, especially because they can contribute to the transmission of antibiotic-resistant pathogenic bacteria through the food chain. Therefore, to ensure food safety EU and USA regulatory agencies have established lists of forbidden or banned substances and tolerance levels for authorized veterinary drugs (e.g. antibacterials). In addition, the EU Commission Decision 2002/657/EC has set requirements about the performance of analytical methods for the determination of veterinary drug residues in food and feedstuffs. During the past years, the use of powerful mass spectrometric detectors in combination with innovative chromatographic technologies has solved many problems related to sensitivity and selectivity of this type of analysis. However sample preparation still remains as the bottleneck step, mainly in terms of analysis time and sources of error. This review covering research published between 2004 and 2008 intends to provide an update overview of the past ve years, on recent trends in sample preparation for the determination of antibacterial residues in foods, making special emphasis in on-line, high-throughput, multi-class methods and including several applications in detail. 2009 Elsevier B.V. All rights reserved.

Article history: Received 28 January 2009 Received in revised form 21 April 2009 Accepted 22 April 2009 Available online 3 May 2009 Keywords: Antibacterials Residue determination Food analysis Sample preparation Multi-class methods Liquid chromatography

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.1. Antibacterial residues in foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.2. Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.3. Limitations of the current analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Recent trends in sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.1. Off-line extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.1.1. Pressurized liquid extraction (PLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.1.2. Microwave-assisted extraction (MAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2. On/off-line clean-up techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.2.1. Novel SPE/SPME procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.2.2. Matrix solid phase dispersion (MSPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2.3. Restricted access materials (RAMs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.

Abbreviations: AGs, Aminoglycosides; AMPs, Amphenicols; -LCs, Beta-lactams; CAP, Chloramphenicol; CPs, Cephalosporins; CTC, Chlortetracycline; CIPRO, Ciprooxacin; DAP, Dapsone; ENRO, Enrooxacin; ERY A, Erythromycin A; FLU, Flumequine; FQs, Fluoroquinolones; IPhs, Polyether ionophores; LCs, Lincosamides; MCs, Macrolides; MON, Monensin; NEO, Neomycin; NFs, Nitrofuranes; NMZs, Nitroimidazoles; NOR, Noroxacin; OXO, Oxolinic acid; OTC, Oxytetracycline; PCs, Penicillins; Qs, Quinolones; STR, Streptomycin; SAs, Sulphonamides; TCs, Tetracyclines. Corresponding author. Tel.: +34 91 394 4322; fax: +34 91 394 4329. E-mail address: marazuela@quim.ucm.es (M.D. Marazuela). 0003-2670/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.04.031

M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517

3.

2.2.4. Turbulent ow chromatography (TFC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Molecularly imprinted polymers (MIPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Multi-class antibacterials determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12 13 13 15 16

1. Introduction 1.1. Antibacterial residues in foodstuffs In recent years, food safety has become an issue of concern, especially after the diverse food crises that have affected public health worldwide (e.g. bovine spongiform encephalopathy, dioxins, avian inuenza, and melamine). Therefore, in the eld of food safety, scientists and regulatory agencies need to identify any potential risks to consumers related to the consumption of food. Organic contaminants that might be present in food, whether from natural or anthropogenic origin, can be divided into four main categories, namely pesticides, persistent environmental chemicals, naturally occurring toxins and veterinary drugs. Among the veterinary drugs, the antibacterial agents are widely used for preventing and treating infectious diseases in farm animals. Antibacterials or antimicrobials comprise synthetic, semi-synthetic and natural compounds, being the latter ones strictly named as antibiotics. However, today the term antibiotic is often used for the whole class of drugs, as a synonymous of the more general term antibacterial. The application of antibacterials in veterinary medicine began in the 1950s with the use of oxytetracycline (OTC) and chlortetracycline (CTC) as feed additives. Nevertheless, the World Health Organization (WHO) and other international organisms have pointed out the risks associated to the overuse or misuse of antibacterial treatments, both in human medicine and veterinary practices [1]. For instance, the presence of antibacterial residues in foodstuffs can provoke undesirable effects, such as allergic reactions in some hypersensitive individuals. Moreover, of particular concern is that low-level doses of antibacterials for prolonged periods could result in the transmission of antimicrobial-resistant pathogenic bacteria from food to humans, rendering such treatments ineffective against them [2]. In addition to immediate adverse effects, the long-term effects due to the exposure of low levels of antibacterial residues are still unknown. Thus, in the European Union (EU), many efforts have been made to protect consumers health [3] and since 2001 measures are being adopted in the food safety area to reduce antimicrobial resistance. For example, EU legislation on animal nutrition bans the use of antibiotics for growth promotion in animal feed since January 2006 [4]. However, this inappropriate use of antimicrobial agents in food producing animals continues in North America, Australia [5] and many developing countries, where the antimicrobial resistance problems are particularly pressing [6]. Human migration and global food markets may facilitate the spread of antimicrobial resistance across almost any geographic boundary, so response to this problem demands concerted efforts from multiple sectors, both in developed and developing countries [7]. 1.2. Legislation The Codex Alimentarius is the global reference point for consumers, food producers and processors, national food control agencies and the international food trade [8]. The Codex Alimentarius food standards are internationally acknowledged as the best established measures to protect human health from risks arising from contaminants in foods. To include a certain substance in a list of forbidden or regulated compounds in foodstuffs, generally differ-

ent committees of experts take into account the following criteria [9]: (a) The compound has been often detected in at least one commodity at a signicant concentration by reliable analysis. (b) The compound has proved or is suspected to be toxicologically adverse to human or animal health at the concentration observed in the foodstuff. (c) The foodstuff signicantly contributes to the total intake (e.g. Threshold Daily Intake, TDI or Acceptable Daily Intake, ADI) of the contaminant of interest established from toxicological data. On the other hand, the Food and Drug Administration (FDA) in USA and the European Commission have established lists of tolerance levels, also known as Maximum Residue Limits (MRLs), for different food contaminants in a certain number of raw foods, on the basis of toxicological data, acceptable daily intake values and the performance of the present day analytical technology. Within the EU, the main document stating the MRLs of authorized veterinary drugs (Group B compounds [10]) in foodstuffs of animal origin is Council Regulation 2377/90/EC, last amended by Commission Regulation 542/2008/EC [11], whereas in the USA is the Code of Federal Regulation set by the FDA [12]. Other helpful databases are available on the websites of Government Agencies, such as the Food and Agriculture Organization (FAO) of the United Nations [13] or the United States Department of Agriculture (USDA) [14]. According to European Commission Decision 2002/657/EC [15], the analytical methods for the control of veterinary drug residues in animal food products can be clearly distinguished between screening and conrmatory methods. Screening methods, mainly consisting in bioassays, are often sensitive enough, cheaper and faster than conrmatory ones, although sometimes lack of specicity. A reasonable percentage of false positives results is acceptable, as they will be further submitted for conrmatory analysis but screening methods must avoid or reduce to a minimum the number of false negative results (non-compliant) because they will not be further analysed [16]. Conversely, conrmatory methods, mainly based on liquid chromatography combined with tandem mass spectrometry (LCMS2 ) are required for unequivocal identication and, if necessary, quantication of the analyte of interest. Only after a conrmatory analysis, a suspected contaminated sample will be declared non-compliant. Although Commission Decision 2002/657/EC still accepts detection techniques like diode-array (DAD) or uorimetric detection (FLD) as possible conrmatory techniques; nevertheless from the practical point of view conrmation of antibacterial residues in food is performed by LCMS techniques since they can provide information about the chemical structure of the analyte. Mass spectrometric detection can be carried out by recording full mass spectra (full-scan mode), or by selected ion monitoring (SIM) and multiple reaction monitoring (MRM) (e.g. in single and triple quadrupole, respectively). Furthermore, Decision 2002/657/EC introduces the concepts of identication points (IPs) and tolerated ion intensities ratios for conrmatory methods. Thus, for banned or unauthorized substances included in the Group A of Directive 96/23/EC [10] a minimum of four IPs (in correct ratios) is required, whereas for the conrmation of substances listed in Group B, a minimum of three IPs is necessary. The number of IPs earned

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depends on the identication power of the basic MS techniques used. Moreover, EU Ofcial laboratories that perform the analysis of food and feed samples taken during ofcial controls should be accredited, in accordance to the European ISO/IEC 17025 standard (the latest version was published in 2005, ISO 17025:2005), on General requirements for the competence of testing and calibration laboratories taking into account criteria for different testing methods laid down in Community feed and food law. The accreditation of EU Ofcial laboratories may relate to individual tests or groups of tests. In contrast to the EU, regulatory laboratories of the FDA rely only on standardized analytical methods to conrm the identity and/or quantity of veterinary drugs residues in foodstuffs. However, an ofcial standardized method is a result of a long and complicated process. So, the EU regulatory laboratories can take advantage of constant improvement of the recent developed analytical methods. Following the criteria dened in Decision 2002/657/EC, all analytical methods for residue control in animal products should be validated in terms of selectivity/specicity, ruggedness/stability, trueness/accuracy, precision and linearity [15]. The denitions of the analytical limits are now based on the decision limit (CC ) and detection capability (CC ), replacing the formerly used limits of detection (LODs) and limits of quantication (LOQs). In addition, a new regulatory limit was established, the minimum required performance limit (MRPL), which is the minimum content of an analyte in a sample that has to be detected and conrmed. This limit harmonizes the minimum performance characteristics, when establishing and validating methods for prohibited substances or those for which no MRLs have been established. Finally, a rapid alert system covering food and feed has been established within the EU for the notication of a direct or indirect risk to human health deriving from contaminated food or feed, either of Community origin or imported from a third country [3]. When lots of incurred samples are individuated by National Reference Laboratories, a notication is sent to Control Authorities to conscate the suspected batches. Nowadays, among the antibacterials, strong attention is focused to unauthorized or banned substances, such as chloramphenicol (CAP) and nitrofurans (NFs), and their metabolites in EU [17], while in the USA the FDA recommends food producers to avoid contaminations of penicillins (PCs) in food that could cause allergic reactions to USA citizens [18]. 1.3. Limitations of the current analytical methods LCMS2 using triple quadrupole (LC-QqQMS), in the MRM mode is at the moment the top analytical methodology for simultaneous, unambiguous identication and quantication of antibacterial residues in foodstuffs [19]. Although it is a very sensitive and selective technique, there is a technical limit for the number of target compounds that can be monitored by MRM-type experiments, hindering the utility of this approach. So, for multi-residue determination there is an emerging trend to the use of accurate mass full-scan MS techniques (e.g. time of ight mass spectrometry, TOF-MS) that allow simultaneous determination of hundred of different compounds in complex matrices in a single analysis [20]. This is especially useful for routine laboratories that have to conrm the presence of a large number of veterinary drugs in the food commodities. Regardless the excellent performance of the QqQ and TOF-MS analysers, there are still some limitations in the majority of the current LC methods, mainly due to sample preparation or matrix effects. Sample preparation, is one of the key issues in food analysis, because it can be a source of inaccuracy, as well as a limitation for the development of high-throughput methods [21]. The complex

food matrices and the different physico-chemical characteristics of the antibacterial families make difcult the development of analytical methods appropriate for a great variety of antibacterial/food commodity combinations. In general, most analytical methods described in the literature, target a single class of antibacterials. A way to improve cost-effectiveness is to perform multi-class determination, thus increasing the number of antibacterials that can be determined by a single procedure or from a single portion of test material. Therefore, generic sample preparation procedures are necessary to simultaneously extract a broad range of antibacterials from different food matrices, down to their MLR or MRPL levels. On the other hand, quantication is sometimes a very difcult issue because of matrix interferences. Food sample extracts produce very often matrix effects in LCMS methods, using electrospray (ESI) or atmospheric pressure chemical ionization (APCI) sources [22]. The most common matrix effect is the enhancement or suppression of the analyte signal, induced by the co-extracted and co-eluted matrix compounds. These unwelcome effects can severely affect methods accuracy and precision. Several strategies can be used for the elimination and/or compensation of matrix effects in LCMS methods, as for instance, the use of either matrix-matched standards and/or isotopic labelled internal standards during method validation [23]. Nevertheless, calibration with matrix-matched standards can be very time-consuming and isotopic labelled internal standards are expensive and not always commercially available. Recently, Cappiello et al. [24] have suggested the use of an efcient LCMS interface based on direct electron ionization (direct-EI) to overcome matrix effects. In this type of interface, the sample extracts are directly introduced into the ionization source kept under high vacuum conditions, followed by a complete and rapid conversion to the gas phase. Once in the gas phase, the analyte is ionized through the interaction with the electron beam. Thus, all the steps leading to analyte ionization in the direct-EI interface are inuenced, neither by the matrix components nor by the mobile phase composition. Although direct-EI works reasonably well for small-medium molecular weight compounds, nevertheless ionization efciency in ESI is far more sensitive than EI for certain compounds. Additionally, to improve validation of analytical methods for antibacterial residues in foodstuffs, there is a need for new certied reference materials (CRMs) and matrix blank materials [25]. Preparation of CRMs is undoubtedly a difcult and time-consuming task, especially when many different analyte/matrix combinations are required, but the number of the currently available CRMs is quite limited. The Institute for Reference Materials and Measurements [26] provides several CRMs, and blank matrix materials for a few antibacterial/food commodities, such as OTC/milk powder, CAP/pork muscle, nitroimidazoles (NMZs)/pork muscle, CTC/pork tissues and umequine (FLU) and oxolinic acid (OXO)/salmon tissue, but this number is clearly insufcient. Consequently, the preparation of multi-analyte CRMs, as one single material, would be necessary for the validation of multi-class antibacterial analytical methods. Last, but not least, most of the reported analytical methods for antibacterial residues, are focused to those target animal species and derived foodstuffs included in Council Regulation 1353/2007/EC [11]. However, other processed foods not included in 1353/2007/EC (e.g. infant foods) represent a demanding research eld, especially after the latest food crisis in China, so probably legislation might be revised to face new challenges. In fact, the European Commission decided to set up MRL levels for pesticides and mycotoxins in different infant foods [27,28], so it is likely that in near future legislation could be extended to other contaminants and/or residues (e.g. veterinary drugs) to protect childrens health.

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Up to our knowledge only a few analytical methods for antibacterial residues determination in infant foods have been reported in the literature [2931] so much effort should be made in this particular area. 2. Recent trends in sample preparation Adequate sample preparation is a key aspect of any analytical procedure. Although many classical extraction methods for food analysis are still in use, recent trends in sample preparation have evolved towards: Use of smaller sample sizes. Reduction or elimination of organic solvents. Generic extraction procedures for multi-class compounds. Potential for automation and/or high-throughput determination.

2.1. Off-line extraction techniques 2.1.1. Pressurized liquid extraction (PLE) The use of automated extraction techniques leads to a reduction in uncertainty. Automated methods are generally more reproducible than manual ones and will also decrease the time spent on sample preparation, which is often the bottleneck in analysis. Pressurized liquid extraction (PLE) is becoming increasingly important as sample preparation technique in food analysis, combining the benets of high-throughput, automation and low solvent consumption, although expensive lab-equipment is required [32,33]. Several authors have demonstrated the feasibility of using PLE for sample preparation in the determination of antibacterial residues in different types of foods (Table 1). One example is the application of PLE to the extraction of quinolones (Qs) from egg samples [34]. Whole eggs were rstly homogenized and then, aliquots were mixed with a dispersing agent (diatomaceous earth) before extraction by PLE. The optimised PLE conditions consisted of using phosphate buffer pH 3.0/ACN (50:50, v/v) as extraction solvent, at 1500 psi, 70 C and three static cycles of 5 min each. Under these conditions recoveries ranged between 67 and 90% and the obtained extracts did not require further clean-up. The PLE-LC-FLD method has been successfully applied to the determination of enrooxacin (ENRO) and its metabolite ciprooxacin (CIPRO) in incurred eggs from ENROtreated hens, showing that the method could be useful for screening contamination or illegal use of Qs in laying hens. PLE has also been applied to the extraction of macrolides (MCs) from meat and sh samples before LCMS2 determination [35]. In this case, lyophilized samples were extracted using methanol, as extraction solvent at 1500 psi, 80 C and applying two static cycles of 15 min. The 40 mL extracts were evaporated to dryness and nally reconstituted in 0.5 mL before LC determination. No interferences were found at the retention times of the MCs, which allows direct injection of the extracts without the need of further clean-up. But, relatively low recoveries were obtained for some of the analysed compounds, e.g. erythromycin A (ERY A) with only 58% recoveries at the MRL level. On the other hand, the use of water as extraction solvent for PLE generally referred as pressurized hot water extraction (PHWE) has gained interest during the last years [33]. PHWE is cheaper, cleaner and more environmentally friendly than conventional PLE. Moreover, the dielectric constant (polarity) of water can be significantly reduced with increasing temperature, so under pressure, heated water can behave like an organic solvent, thus making more selective the extraction of moderately polar compounds. Thus, at 100200 C, water can act as a medium/non-polar solvent. How-

ever, due to the high temperatures involved, the thermal stability of the analytes must always be checked before extraction. For instance, degradation of MCs has been observed at temperatures above 100 C [36]. Following this principle, Gentili et al. have developed a PHWELCMS2 method for the determination of 14 sulphonamides (SAs) in raw meat and infant foods [29]. The samples were blended with a C18 dispersing agent and extracted with 10 mL of water at 160 C, 1400 psi and applying one static cycle (5 min). After cooling the extracts at 18 C for 1 h, in order to allow matrix components to precipitate, the supernatants can be directly injected into the LCMS2 , without any further clean-up. When applying the method to the analysis of real samples, two positive infant food samples were detected that corresponded to veal meat- and ham-based formulations. From the analysis of raw meats, residues of SAs were found in two samples, corresponding to veal and pig, respectively, although the levels did not exceed the MRLs set by the EU. Recently, Carretero et al. have described a multi-class method for the simultaneous determination of 31 antibacterials in meat samples by PHWE-LCMS2 [37]. Meat samples were previously homogenized and blended with EDTA-washed sand and nally extracted at 1500 psi, 70 C and applying one extraction cycle (10 min). A drawback of the method is the big volumes of extract (40 mL) obtained that required evaporation to reduce the extract volume to 10 mL and thus, increase sensitivity. Considering that water is the solvent of choice, this evaporation step takes time, which on the other hand, increases considerably the time required for sample preparation. The proposed methodology has been applied to the analysis of 152 samples of cattle and pig tissues, resulting that in the 15% of the analysis was detected the presence of Qs, tetracyclines (TCs) and SAs, although at concentrations below the MRLs.

2.1.2. Microwave-assisted extraction (MAE) MAE is a process of using microwave energy to heat a solvent in contact with a sample, in order to partition analytes from the sample matrix into the solvent [38]. Although MAE is now established as a routine, well-developed method for sample preparation in environmental analysis (soils, sediments, etc.), only two papers have been found in the literature for the application of MAE to the extraction of antibacterials from solid foodstuffs [39,40]. This is generally due to the limited diffusion of the solvent in samples containing more than 30% of water (as it is the case of food samples), resulting in low analyte recovery. This problem can be circumvented by prior drying of samples by lyophilization. In the rst work the authors have compared traditional solvent extraction by vortex with MAE for the determination of salinomycin in eggs and chicken tissue by LCUV, with post-column derivatization [39]. In this case, a mixture of ethanol/2-propanol (88:12, v/v) was used for MAE extraction. The results demonstrated that MAE provides extraction efciency comparable to conventional vortex extraction with acetone, having additional advantages, such as the smaller sample sizes, reduced organic solvent and shorter extraction times. In another example, Hermo et al. have applied an openfocused microwave system for the extraction of nine Qs from pig muscle [40]. As extraction solvent they used a mixture of 0.3% mphosphoric acid:ACN (75:25, v/v) and the system was programmed with 40% of the microwave power for 4 min, in order to minimize the amount of co-extracted matrix compounds. This procedure was compared with an alternative solvent extraction and SPE clean-up method, giving similar accuracy values for all analytes, except for the most acidic ones. Moreover, matrix dissolution was more efcient with the application of MAE, leading to cleaner extracts and in consequence, to lower LODs and LOQs.

M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517 Table 1 Overview of off-line extraction techniques for LC determination of antibacterials residues in foodstuffs. Drug class FQs Matrix Eggs Extraction technique PLE Extraction conditions Recoveries Advantages/drawbacks No clean-up is required after PLE extraction, just cooling the extracts at 4 C for 1520 min to facilitate precipitation of proteins and other matrix components. Low organic solvent consumption High organic solvent consumption. Extracts (40 mL) need evaporation before LC determination. Low throughput (extraction time > 30 min/sample + evaporation of extracts) Environmentally friendly and inexpensive solvent. High sample throughput (extraction time: 5 min/sample) Allows multi-class determination. Extracts (40 mL) need evaporation to reach adequate sensitivity. Evaporation of aqueous extracts increases considerably sample preparation time Low organic solvent consumption and very short extraction times. High sample throughput. Determination of a single analyte Short extraction times. High sample throughput. Efcient matrix dissolution that avoids further clean-up of the extracts Ref. [34]

Phosphate buffer 50 mM pH 3.0/ACN (50:50, v/v), 6790% 1500 psi, 70 C, ush volume 50%, static time 5 min, 3 cycles, 11 mL extraction cells Methanol, 1500 psi, 80 C, ush volume 150%, static 7790%, time 15 min, 2 cycles, 33 mL extraction cells ERY (58%)

MCs

Meat, sh muscle

PLE

[35]

SAs

Meat, infant foods Meat

PHWE

H2 O, 1400 psi, 160 C, static time 5 min, 1 cycle, 5 mL extraction cells H2 O, 1500 psi, 70 C, ush volume 60%, static time 10 min, 1 cycle, 22 mL extraction cells

70101%

[29]

SAs, Qs, MCs, TCs, LCs, -LCs

PHWE

7599%

[37]

SAL

Eggs, muscle

MAE

Extraction with 17 mL of ethanol/2-propanol (88:12, v/v) at maximum microwave power for 9 s Double extraction with 25 + 10 mL of 0.3% m-phosphoric acid:ACN (75:25, v/v), 40% microwave power for 4 min

8898%

[39]

Qs

Muscle

MAE

82104%

[40]

Acronyms: -LCs: -lactams; ERY: erythromycin; FQs: uoroquinolones; LCs: lincosamides; MCs: macrolides; MAE: microwave-assisted extraction; PLE: pressurized liquid extraction; PHWE: pressurized hot water extraction; Qs: quinolones; SAL: salinomycin; SAs: sulphonamides; TCs: tetracyclines.

2.2. On/off-line clean-up techniques Sample pretreatment procedures adopted in most analytical methodologies for determining antibacterial residues in food are based on solvent extraction, followed by clean-up by solid phase extraction (SPE). Nowadays, the analyst can choose different SPE formats, such as cartridges, disks, 96-well plates or in-tube capillary columns. Batch-to-batch reproducibility, followed by labor time and cost are perceived as the most important parameters for selecting an SPE format and material [41]. SPE disks have the advantage to admit higher ow rates, but this topic is important mainly in environmental analysis. The 96well SPE plates were introduced in 1996 with the aim of increasing sample throughput and reduce the volume required for quantitative elution of the analytes. Its use has been mostly limited to the pharmaceutical industry (Table 2). Solid phase microextraction (SPME), coupled on-line to GC, is a well-established and diffused technique [42], by opposite SPME coupled to LC is still a relatively new technique, since it was put forward by Eisert and Pawliszyn [43]. This review is focused on recent improvements of clean-up techniques with special emphasis on automated, on-line systems or innovative materials/formats. Sorbents for on/off-line SPE comprise a large variety of media including traditional reversed-phase and mixed-mode ion-exchange phases, restricted access materials (RAMs), monolithic materials, carbon nanotubes (CNs), large particles for turbulent ow chromatography (TFC) and molecularly imprinted polymers (MIPs). Some reviews covering in detail some of these specic applications are available [4446].

2.2.1. Novel SPE/SPME procedures Starting by off-line sample preparation, Rubies et al. have reported the use of Oasis HLB 96-well plates for the determination of Qs in meat samples [47]. Sample pretreatment consisted of a simple ultrasonic extraction with pure water, centrifugation and direct loading in the cartridges without additional treatments. In addition, LC separation has been speed up with the use of a short chromatographic column (50 mm), so the Qs were eluted in less than 12 min. The proposed methodology enabled the analysis of up 24 samples per working day.

Dispersive-SPE (d-SPE) is a technique that simplies and speeds up sample clean-up compared to conventional cartridge-based SPE [48]. It involves thoroughly shaking of the sample extract with a sorbent that removes matrix interferences but does not retain the analytes. For instance, C18 sorbents remove highly lipophilic compounds and others like amino- or carbon-based phases are employed mainly for the removal of fatty acids and pigments, respectively. Fagerquist et al. have improved a previous analytical method replacing conventional SPE by d-SPE, for the determination of 10 beta-lactams ( -LCs) in bovine kidney samples [49,50]. The method involves solvent extraction/deproteinization with H2 O/ACN (20:80, v/v), followed by d-SPE clean-up with C18 sorbent and nal LCMS2 determination. Taking into account the simplicity of this protocol, accuracy was satisfactory with recoveries ranging from 87% to 103% and RSDs < 16%. Moreover, d-SPE allowed increasing by a factor of 3 or 4 times, the number of samples that can be prepared by day. The same scheme has been adopted for the determination of several antibacterials (Qs and ERY A), fungicides and parasiticides in salmon tissue [51]. In this case, samples were extracted with acidic ACN and afterwards, d-SPE was carried out with a Bondesil-NH2 sorbent, before LCTOF-MS determination. Excellent recoveries were obtained with the exception of ENRO (40%), although matrix suppression effects were observed for practically all target compounds. Concerning on-line SPE procedures a very interesting application is the screening of aminoglycoside (AGs) antibiotics in milk by automated on-line SPE in a short C18 capillary column coupled to an MS2 detector [52]. According to this procedure, milk samples (10 L) were deproteinated with heptauorobutyric acid (HFBA) and afterwards, extracts were directly injected into the capillary SPE column that retains the analytes by ion-pair interactions. Finally, a switching valve device enables direct elution of the antibiotics with methanol to the nanospray source. The method has been successfully applied to the determination of neomycin (NEO) in spiked milk samples, with recoveries >90% in all cases. In another work, Goto et al. have described a method for simultaneous determination of PCs and TCs in different animal tissues [53]. It involves on-line sample preconcentration on a short (1.5 cm) C18 column coupled to LCMS2 determination. Prior to on-line SPE, the samples were homogenized and extracted with water and then, extracts were puried by ultraltration. The overall recover-

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ies ranged from 70 to 115% with RSDs between 0.7 and 14.8% and sample throughput was of 3 samples per hour. The method is considered to be satisfactory for the rapid screening of PCs and TCs antibiotics in meat. An on-line SPE method using multi-walled carbon nanotubes (MWNTs) as sorbent material coupled to LC-UV has allowed simultaneous determination of 10 SAs in eggs and pork meat [54]. Sample pretreatment required the addition of ferrite potassium cyanide/zinc sulfate and heating at 75 C during 5 min. After ltration, addition of ACN, sonication and again ltration, the extracts were loaded into the on-line MWNTs-SPE column at 4.5 mL min1 for 23 min. Then, the antibacterials were eluted in the back-ush mode towards the analytical column by switching the LC valve from load to inject position. In this way, a complete cycle was performed in 35 min and enrichment factors of up to 160 were achieved. Among the main factors affecting on-line MWNTs-SPE were sample acidity, loading ow rate and time, concentration and ow rate of the eluent. The results obtained with the proposed clean-up method were compared to those provided by Oasis HLB SPE columns. In all cases, MWNTs-SPE performed equally well or even better than Oasis HLB SPE, obtaining higher enrichment factors and lower LODs. SPME using either bres or in-tube devices have also been applied for clean-up purposes. The main drawbacks of SPME using bres are its limited capacity and the difculty of coupling to LC for on-line applications [55]. However, the in-tube SPME format may solve the above-mentioned problems, since it is based on a capillary instead of a bre. Then, the length and thickness of the column are more easily tuneable and straightforward connected to LC. A way of increasing the thickness of the capillary and thus, improving the extraction efciency, is to use capillaries coated with monolithic sorbents of different functionalities. Fengs research group have prepared monolithic capillaries based on poly(methacrylic acid-ethylene glycol dimethacrylate) for in-tube SPME-LC, applied to the determination of SAs in milk and eggs [56,57], TCs in sh muscle [58] and uoroquinolones (FQs) in eggs [59]. SAs and FQs were extracted from their respective matrices with ethanol and/or phosphate buffer solution (pH 3.04.1) [57,59] whereas extraction of TCs was achieved with a 0.01 M EDTAMcIlvaine buffer solution (pH 4.0) [58]. The target compounds are adsorbed onto the monolithic phase through hydrophobic interactions, while adsorption of proteins and fat does not occur due to the presence of a hydrophilic pendant group (carboxylic acid). So no precipitating protein and removing fat steps are required prior to SPME. However, authors recommend including a rinsing step before elution to remove residual proteins and fat, in order to avoid contamination or clogging of the analytical column. Once retained, the analytes were eluted with the optimised mobile phase in each case: MeOH/phosphate buffer (pH 3.0) (30:70, v/v) [57], MeOH/ACN/0.02 M oxalic acid (pH 3.0) (20:20:60, v/v/v) [58] and phosphate buffer (pH 2.1)/MeOH/ACN (72:20:8, v/v/v) [59], respectively. The procedure is rapid, being possible to perform elution and separation of the analytes in about 2035 min. 2.2.2. Matrix solid phase dispersion (MSPD) In 1989, Barker et al. introduced the MSPD, as a sample preparation technique for solid or semi-solid samples [60]. Actually, MSPD can combine the procedures of homogenization, disruption, extraction and clean-up into one simple process. In fact, it is a sample preparation strategy that consists of a manual blending of samples with a bulk dispersing agent, to produce complete disruption of the original matrix structure, thus providing an enhanced surface area for subsequent sample extraction. Usually, the blended material is then transferred and packed into a column to perform sequential extraction and eventual clean-up with an appropriate solvent or a sequence of solvents. The MSPD is prone to use in combination with

automated extraction or clean-up systems (e.g. PLE see Section 2.1.1 or SPE), either on-line or off-line. Some miniaturized MSPD applications have also been described [61]. But to the best of our knowledge no papers dealing with the on-line coupling of MSPD to LC or GC have been already published. Recent reviews on this particular topic suggest that MSPD has attracted many researchers in environmental, clinical and food analysis [6264]. Table 2 lists some MSPD-based methods found in the literature for the determination of antibacterial residues in foods. Different bulk materials have been used as matrix dispersing agents, being C18 - and C8 -bonded silica the most popular by far. Zou et al. have described an MSPD procedure for the extraction of eight SAs from honey samples using C18 as solid support [65]. After the MSPD, the SAs were derivatized with 9uorenylmethyl-chloroformate FMOC-Cl and derivatives required further purication by SPE with silica gel, prior to LC-UV determination. The average recoveries for most SAs were >70%. By opposite, other polar sorbents, such as silica gel, CN or NH2 -bonded silica resulted in a strong absorption of the polar SAs, providing very low recoveries. As an alternative to the classical C18 or C8 apolar-bonded phases, other authors have proposed the use normal-phase supports to improve the isolation of more polar compounds, as well as to perform extraction and clean-up in a single step, prior to reversedphase LC determination. Following this trend, Kishida [66] has recently developed a simple method for the determination of six SAs in meat samples, using normal-phase MSPD with alumina NS and a 70% (v/v) ethanol solution as extraction solvent, followed by the evaporation of the extracts and LCMS/DAD determination. Average recoveries were >90% in all cases and the LOQs were well below the MRLs established by the EU. Probably, one of the most interesting MSPD-based techniques is MSPD with hot water extraction. Bogialli et al. have published several papers during the last ve years using this sample preparation technique for the determination of different antibacterials in a great variety of foodstuffs [6775]. In all cases, a home-made like-PLE apparatus was employed with the advantage of requiring smaller solvent volumes (<7 mL) than conventional PLE systems. Crystobalite, a thermally puried sand treated sometimes with EDTA [68,69,72] or hydrazine sulfate [73] before blending, has been used as dispersing agent. Water temperature was always the most critical parameter to effectively extract the antimicrobials without thermal degradation of the compounds and with minimal co-extraction of matrix compounds. Optimal extraction temperatures were in the range 65120 C (see Table 2). Furthermore, in some particular cases the use of acidied water or the addition of methanol increased the extraction yield of some compounds, such as MCs in milk [71], SAs in cheese [70] and Qs in eggs [75]. Thus, in the optimal extraction conditions, typical recoveries ranged from 70 to 102%. Moreover, the extracts required little manipulation (pH adjustment and ltration) before LC injection and volumes as large as 100200 L can be injected without producing any peak distortions. Although this procedure seems to be a very straightforward approach to one-step sample preparation, some improvements are still desirable. For instance, the automation of the home-made apparatus is mandatory for high-throughput analyses (Table 3). Yan et al. have used molecularly imprinted polymers (MIPs), as selective dispersing media for sample clean-up (MI-MSPD) in the determination of Qs in eggs and swine muscle samples by LC-FLD [76]. The use of MIPs enhances the selectivity of the MSPD procedure, allowing recoveries of the target Qs above 85% without observing matrix interferences in the nal determination. Thus, extraction and clean-up can be performed in a single step, which simplies sample preparation and reduces time and cost of the analysis. Moreover, the synthesized MIPs have shown good spe-

M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517 Table 2 Overview of on/off-line clean-up procedures for LC determination of antibacterials residues in foodstuffs. Drug class Qs, FQs Matrix Muscle Sample pretreatment Ultrasonic extraction with H2 O and centrifugation Extraction and deproteinization with H2 O/ACN (1:4, v/v) Extraction with ACN/NaCl Clean-up 96-Well plates, Oasis HLB d-SPE (C18 ) Mode Off-line Advantages/drawbacks Simple and rapid sample treatment. High throughput: 24 samples/day. Very low recoveries: 3055%. Suitable only for screening purposes Average recoveries: 87103%. Rapid and simple clean-up. Suitable for conrmation and quantication at concentrations below MRLs Recoveries > 80%, except ENRO (40%). Moderately efcient clean-up. Signal suppression (20%) for ENRO Rapid and straightforward procedure: minimal sample treatment and direct enrichment onto the capillary column. Short analysis times: 10 min/sample. Requires very small sample volumes (10 L). Determination of a single analyte: NEO Simultaneous determination of two antibiotic classes. Overall recoveries: 70115%. Low throughput: 2 samples/h Higher enrichment factors (up to 160), better precision and lower LODs than C18 . High sample loading ow rates: 4.5 mL min1 . Determination within 35 min Enrichment factors in the range 519. Very little volumes (50 L) of ACN are used during the desorption step. 50 extraction/desorption cycles can be performed with the same column. Determination of SAs is affected by matrix interferences to some extend High stability of the polymeric capillary column (over 2 months). Enrichment factors in the range 826. No organic solvents are used High extraction efciency. Enrichment factors in the range 6885. Adsorption/elution of FQs can be achieved in 10 and 7 min, respectively. No organic solvents are used Allows direct injection of milk samples with excellent recoveries (80100%). Large number of injections (400) with the same RAM column. Overall analysis time < 30 min/sample Low recovery rates for OTC (50%) and TC (67%). No information about reusability of the RAM column. Overall analysis time: 25 min/sample More than 400 injections (Honey extracts). Moderately efcient clean-up for honey samples (honey-dependent-matrix effects). Overall analysis time: 4 and 18 min for tissue and honey samples, respectively. High solvent consumption Minimal sample treatment. High selectivity and excellent recoveries (98102%). Excellent sensitivity: LODs 10-fold <MRPL Inexpensive sample preparation procedure. Poor selectivity towards Qs Acceptable recoveries: 7294%. Adsorption and desorption of TCs can be achieved in 30 and 10 min, respectively. MIP-coated bres can be reused for more than 100 extractions Ref. [47]

11

-LCs

Bovine kidney

Off-line

[49,50]

Qs, ERY

Fish muscle

d-SPE (Bondesil-NH2 ) Capillary C18 SPE

Off-line

[51]

AGs

Milk

Homogenization and deproteinization with HFBA

Automate on-line

[52]

PCs, TCs

Animal tissues

Homogenization, extraction with H2 O and ultraltration Ferrite potassium cyanide/zinc sulphate, followed by ultrasonic extraction with ACN Extraction with phosphate buffer (pH 3.0) and centrifugation

C18 SPE column

On-line

[53]

SAs

Eggs, muscle

MWNTs-SPE

On-line

[54]

SAs

Milk, eggs

PMME

Automate on-line

[57]

TCs

Fish muscle

Extraction with 0.01 M EDTAMcIlvaine buffer (pH 4.0) and centrifugation Extraction with phosphate buffer (pH 4.1) and centrifugation

PMME

Automate on-line Automate on-line

[58]

FQs

Eggs

PMME

[59]

CPs

Milk

BSAphenyl-RAM

Automate on-line

[78]

TCs

Milk

Extraction with EDTAMcIlvaine buffer and centrifugation. Dilution with H2 O and ltration (honey). Extraction with ACN/H2 O/0.01% formic acid (tissues)

ADSC8 -RAM

Automate on-line Automate on-line

[79]

Qs, FQs

Honey, tissues

TFC

[80,81]

CAP

Milk

Centrifugation

MISPE

Off-line

[90]

Qs, FQs TCs

Baby foods Chicken muscle, milk

Ultrasonic extraction with MeOH Extraction with citrate buffer (pH 5.0)/ethyl acetate (muscle). Milk deproteinization with HCl/ACN

MISPE MIP-SPME

Off-line On-line

[31] [94]

Acronyms: ADS-RAM: alkyldiolsilica-restricted access material; -LCs: -lactams; BSA-RAM: bovine serum albumin-restricted access material; CPs: cephalosporins; CAP: chloramphenicol; d-SPE: dispersive-SPE; ENRO: enrooxacin; ERY: erythromycin; FQs: uoroquinolones; HFBA: heptauorobutyric acid; LODs: limits of detection; MISPE: molecularly imprinted solid phase extraction; MIP-SPME: solid phase microextraction based on MIP-coated bers; MRPL: minimum required performance limit; MWNTs-SPE: multi-walled carbon nanotubes-solid phase extraction; NEO: neomycin; OTC: oxytetracycline; PCs: penicillins; PMME: polymer monolith microextraction; Qs: quinolones; SAs: sulphonamides; SPME: solid phase microextraction; TCs: tetracyclines; TFC: turbulent ow chromatography.

cic recognition of the Qs in aqueous media, which is especially important for nal application to complex matrices, such as food samples. 2.2.3. Restricted access materials (RAMs) RAM sorbents are porous chromatographic supports partially based on size-exclusion mechanisms, that have been specically developed for protein removal [77]. While macromolecules are excluded from the stationary phase and eluted with the mobile phase, the small molecules are able to permeate through the pores

of the RAM sorbent and interact with it by diverse mechanisms. RAMs are frequently used as pre-columns in column-switching LC systems, using two pumps and a selection valve with a synchronization unit. These systems allow automate on-line protein removal and analyte preconcentration on the RAM pre-column, and afterwards separation of the target compounds in the analytical column, avoiding or reducing sample pretreatment. Certain precautions should be followed when using RAM columns, for instance the pH and composition of the loading mobile phase. In practice, no more than 20% of organic solvent should be

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Table 3 Overview of MSPD-based procedures coupled to LC determination of antibacterials residues in foodstuffs. Drug class SAs Matrix Honey Dispersant C18 Extraction/clean-up Ethyl acetate, derivatization and clean-up with silica SPE cartridges 70% (v/v) aqueous ethanol Hot water (6590 C) Advantages/drawbacks Labor-intensive sample preparation procedure. Average recoveries > 70%. Big volumes of chlorinated organic solvents are used Extraction and clean-up in one step. Average recoveries > 90%. Reduced organic solvent consumption Use of an environmentally friendly solvent. Extraction and clean-up in one step. The use of crystobalite washed with EDTA prevents binding of TCs and AGs to metal impurities of the siliceous support. Need of automation of the extraction device to increase sample throughput Addition of modiers (e.g. methanol or formic acid) to water increases the extraction yield of the analytes. Average recoveries: 70102% Ref. [65]

SAs -LCs, AGs, TCs, Qs

Muscle Milk, muscle, cheese, milk

Alumina NS Sand (crystobalite)

[66] [6769,72,75]

SAs, MCs, Qs

Cheese, milk/yoghurt, eggs

Sand (crystobalite)

Qs

Eggs, muscle

MIP

H2 O/MeOH (90:10, v/v), 120 C. Aqueous formic acid (3050 mM), 7090 C ACN/TFA (99:1, v/v)

[70,71,74]

High selectivity in aqueous media without further clean-up. Retention of FQs on the MIP can be easily controlled by adjusting the pH of the solution. Higher recoveries (85104%) compared to other MSPD sorbents (C18 , silica, orisil, etc.)

[76]

Acronyms: AGs: aminoglycosides; triuoroacetic acid.

-LCs:

-lactams; MCs: macrolides; MIP: molecularly imprinted polymer; Qs: quinolones; SAs: sulphonamides; TCs: tetracyclines; TFA:

used, since higher percentages lead to protein denaturation and consequently, to shorter column lifetime. Moreover, a partial loss of the analytes can occur for those compounds that strongly bind to proteins. RAMs have found applications, mainly in bioanalysis and environmental analysis; however in food analysis only a few examples have been reported. Food matrices are much more complex, having a higher content of fat and proteins than biological uids or environmental samples. Since matrix composition determines column lifetime, this fact could explain the limited use of these sorbents in this particular area. Another limitation is the high price of RAM columns, as compared to common SPE sorbents. One of the few examples found in the literature about the application of RAMs to the determination of antibacterial residues in foodstuffs is the analysis of milk samples [78,79]. In the rst work, a column switching system consisting of a RAM-bovine serum albumin (BSA)phenyl column coupled to a C18 column has been applied to on-line sample clean-up and chromatographic determination of ve cephalosporins (CPs) in bovine milk [78]. Direct injection of untreated milk samples, allowed around 400 analysis with the same RAM column, using as loading mobile phase phosphate buffer pH 7.5/ACN (98:2, v/v), during 5 min for the exclusion of milk proteins. In another work, a switching column LC system, which combines an alkyl-diol silica (ADS)C8 RAM column and a C18 column, was evaluated for sample clean-up in the determination of TCs in milk samples [79]. Before loading into the RAM column, milk samples required a previous pretreatment with a solution of EDTAMcIlvaines buffer, to inhibit the complexation of TCs with metal ions and a centrifugation step, which removes proteins and fat. The clean-up with the RAM column removed the large peaks corresponding to milk proteins that appeared in the initial time window of the chromatograms. However, no information about the reusability of the RAM column is provided. 2.2.4. Turbulent ow chromatography (TFC) A relatively new technique used for sample preparation is the so-called turbulent ow chromatography (TFC) that has shown a great potential for on-line sample pretreatment, in terms of both, high sample throughput and high reproducibility linked to automation [23]. Basically, columns packed with large particle size sorbents (typically 60 m) allow on-line extraction using

high solvent ow rates (typically 46 mL min1 ), without signicant back-pressure. Molecules with a low molecular weight diffuse faster than molecules with a high molecular weight. Therefore, the small analytes diffuse into the particle pores, whereas the high ow of the mobile phase quickly ushes the large sample compounds (e.g. proteins) into the waste, before they have the opportunity to diffuse into the particle pores. Once trapped the analytes on the turbulent ow column, a back-ushing elution desorbs the analytes and focuses them onto the analytical column for chromatographic separation. In the meantime during LCMS2 data acquisition, the turbulent ow column is reconditioned for the next sample injection. Thus, minimum sample pretreatment is required, saving considerably sample preparation time. Careful optimisation of the different on-line extraction steps is mandatory by choosing the appropriate mobile phase compositions, step times and solvent ow rates. However, a drawback associated with TFC is the high solvent consumption that implies the use of high working ow rates. As LC operators are coming under increasing pressure to reduce solvent consumption or to replace organic solvents (such as ACN) in their analytical methods, the applicability and environmental sustainability of this technique can be seriously compromised in the near future. The use of this technique in food analysis has been scarce so far. Two examples described in the literature deal with the determination of Qs in honey and animal tissues [80,81]. Sample preparation for honey just required a simple dilution with water, followed by ltration, before injection onto the TFC column (styrenedivinylbenzene copolymer 60 m, 60 ) [80]. More than 400 injections of honey extracts were made without deterioration of the TFC columns performance. However, the TFC clean-up was insufcient to avoid matrix effects that obliged to use standard additions calibration. The method has been successfully applied to the analysis of a set of 34 incurred honey samples, contaminated with CIPRO, noroxacin (NOR) and ENRO. In the case of animal tissues, samples were extracted with a mixture of ACN/H2 O (50:50, v/v), acidied with 0.01% formic acid and after centrifugation and ltration, an aliquot of the extract was directly injected onto the TFC system at 5 mL min1 [81]. Mean recovery rates for ENRO and CIPRO in different animal tissues were in the range 72105%. The run time was only 4 min, which enables a high sample throughput.

M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517

13

2.2.5. Molecularly imprinted polymers (MIPs) The use of MIPs as selective sorbents in SPE, so-called molecularly imprinted solid phase extraction (MISPE), is an emerging clean-up procedure for complex matrices. MISPE can compete with traditional SPE phases and immunosorbents, in terms of selectivity, stability and price. Selective extraction from complex samples, such as food, is assessed by a combination of the specic cavities within the MIP and the right choice of loading, washing and elution solvents that favour specic interactions within the cavities. The majority of the MISPE-based analytical methods are performed in off-line mode [82,83]. This is due in part to the nature of the organic elution solvents employed for analyte desorption from the MIP, that are usually not compatible with the common mobile phases employed in reversed-phase LC separations. But the main drawback of the MISPE procedures (when analyte itself is chosen as template molecule) is template leakage that can contaminate sample extracts, thus affecting the analytical results, or in the worst cases, giving false positive results. This can be avoided by the use of a structural analogue, similar to the target analyte in terms of size, shape and functional groups, which is usually referred as mimic-template. Moreover, a careful selection of the template molecule can hinder cross-reactivity to other structurally related compounds, allowing class-selective extraction for a whole group of compounds. Nevertheless, the number of MISPE applications in food analysis (12%) is still scarce, compared to other areas, such as environmental or bioanalysis [84]. A few examples of the application of MISPE in the determination of antibacterials residues in food samples have been reported in the literature [8587]. Furthermore, some companies, such as MIP Technologies AB (Lund, Sweden) [88] and Supelco [89], have recently launched new commercial MIP-based SPE cartridges, with the trademark name SupelMIPTM for different applications, including the determination of some antibacterials, such as CAP and Qs in prawns, milk, honey and bovine kidney samples. Recently, different authors have described the advantages of using the SupelMIPTM cartridges, compared to classical SPE, for selective determination of CAP residues in milk and honey by MISPE-LCMS2 [90,91]. Since CAP is a banned antibiotic in the EU since 1994, MIPs have been prepared through molecular imprinting with an analogue template, in order to prevent template bleeding. Excellent recoveries of CAP were obtained when applying MIP clean-up, due to an enhanced selectivity that allows detection at concentration levels 10 times lower than the MRPL. Sample pretreatment required just centrifugation of milk or dilution of honey with water, before loading onto the MIP cartridges, which results in high sample throughput (e.g. 18 milk samples can be processed within 3 h, instead of 8 h required by LLE). In another work, Daz-lvarez et al. have described a method for the determination of FQs in baby foods by MISPE-LC-UV [31]. Sample preparation consisted of ultrasonic extraction and clean-up using a CIPRO-imprinted polymer. The MIP showed high selectivity in particular, towards FQs and provided chromatograms free of matrix interferences; however poor recognition to other Qs limits the applicability of these cartridges. Further developments will likely be directed at improving online procedures coupling MIPs with LC determination. One example is the application of MIP-coated SPME bres [92,93]. Hu et al. have developed novel MIP-coated SPME bres for sample preparation in the determination of TCs in chicken muscle and milk by LC-FLD [94]. In this work, the MIP was copolymerized through chemical bonding with the surface of an activated silica capillary, by immersion of the bre in the polymerization mixture. The average thickness of the MIP coatings was of 20 m and the bres could be used for more than 100 extractions. The adsorption and desorption of the TCs were achieved in 30 min and 10 min,

respectively. Nevertheless, the dry extracts obtained after sample pretreatment have to be dissolved in toluene to favour specic interactions between the TCs and the imprinted polymers. 2.3. Multi-class antibacterials determination Laboratories involved in food safety control, need to analyse a large number of residues and contaminants in different food commodities, so the development of multi-class analytical methods is nowadays a demanding research area [95]. In order to take advantage of the multi-residue capability of the LCMS2 techniques, sample preparation methods should be able to effectively extract a broad range of compounds from food samples and, this is in fact a difcult task. The different physico-chemical characteristics of the target analytes, as well as the presence of high concentrations of fat and proteins in the food matrices complicate extraction and clean-up and thus, development of multi-class analytical methods is still a challenge. Most sample preparation protocols for antibacterial residues determination in foodstuffs reviewed above deal with a few individual compounds of one or at most two classes of antibacterials; but multi-class methods are still relatively scarce. However, recent trends in sample preparation are moving in this direction and multiclass LCMS2 methods for antibacterials in different foodstuffs are now beginning to become available (see Table 4). For instance, Aguilera-Luiz et al. have recently described a simple and fast extraction procedure for the determination of several antibacterial classes (e.g. SAs, Qs, MCs and TCs) in milk by ultraperformance liquid chromatographytandem mass spectrometry (UPLCMS2 ) [96]. Extraction was performed by treatment with acidied ACN, in the presence of EDTA to increase recoveries of MCs and TCs. Then, water and proteins were removed by salting out with magnesium sulphate and sodium acetate and afterwards, centrifugation and ltration of the organic phase, lead to clean extracts that can directly be analysed. The main advantages of this procedure are the simplicity and the high sample throughput, being possible to simultaneously extract 18 antibacterials with good recovery values (73108%) in less than 10 min. Moreover, separation of the analytes by UPLC is achieved in less than 10 min, so this method could be potentially applied in routine laboratories. In another work, Turnipseed et al. have described a LCMS2 method for multi-class residues determination in milk and dairy products, based on a rather laborious sample preparation procedure, that combines extraction with ACN, further clean-up with Oasis HLB cartridges and ultraltration [97]. Acceptable recoveries (>70%) were obtained for SAs, MCs and Qs; however for TCs (5060%) and -LCs (<50%) were rather low. Despite of the extensive clean-up procedure, a large degree of matrix ion suppression was observed for many compounds, making necessary to include matrix-matched calibration standards for quantication purposes. Furthermore, Stolker et al. have developed a method based on UPLCTOF-MS for the determination of 100 veterinary drugs in milk, including the main antibacterial families (MCs, PCs, Qs, SAs, TCs, NMZs, polyether ionophores, IPhs and amphenicols, AMPs), among other compounds of interest [98]. Sample preparation consisted of protein precipitation with ACN, followed by clean-up with Strata X cartridges. At the MRL concentration level the results for repeatability (RSDs < 20% for 86% of the compounds), reproducibility (RSDs < 40% for 96% of the compounds) and accuracy (80120% for 88% of the compounds) were satisfactory. However, despite of the high resolution MS detector used, the identication criteria are not fullled according the EU guidelines. So, this method can be used as screening, but samples suspected to be positive have to be conrmed by an MS2 technique. A multi-class LCMS2 method to screen antibacterial residues in table eggs has been developed [99]. A simple extraction proce-

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M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517

Table 4 Comparison of sample preparation methods for multi-class antibacterials determination in food samples. Drug classes SAs, Qs, MCs, TCs Matrix Milk Extraction/clean-up QuEChERS extraction with acidied ACNEDTA/magnesium sulphate and sodium acetate ACN/0.1% formic acid (v/v) followed by clean-up with Oasis HLB cartridges and ultraltration Deproteinization with ACN. Dilution of extracts with H2 O and clean-up with Strata X cartridges Extraction with sodium succinate buffer, centrifugation and clean-up with Oasis HLB cartridges Dissolution in H2 O, centrifugation and ltration. An aliquot was diluted with H2 O for AGs determination. The remaining portion was cleaned-up with Strata X cartridges Recoveries 73108% Advantages/drawbacks Simple and cost-effective sample preparation procedure. High throughput (extraction of 15 samples in less than 1 h) Poor accuracy for TCs and -LCs. Despite of the extensive clean-up a remarkable matrix ion suppression effect is observed Very powerful for multi-residue screening and quantication of 101 veterinary drugs, below or above the MRLs Poor accuracy and reproducibility (RSDs 1030%) for -LCs, SAs and TCs. Useful only for screening purposes Time-consuming and low throughput method: AGs have to be analysed in a separate set of samples and under different chromatographic conditions. CAP has to be analysed in a second chromatographic run, in negative ionization mode Rather laborious sample preparation that limits its applicability in routine analysis The injection order of the extracts (stacking injection mode) is very critical to avoid degradation of -LCs Low recoveries (<70%) for some Qs and poor precision (RSDs > 20%) for AMPs and SAs. Useful for screening but not reliable for quantitative determinations Rather extensive sample preparation procedure. Lower PCs recoveries were observed in kidney and liver samples probably due to the high enzymatic activity of these matrices High throughput (60 samples/day). Rather low recoveries (<68%) of TCs, Qs and MCs in kidney samples. Matrix effects results in poor precision for TCs and MCs High throughput (3040 samples/day). The method has been implemented for routine determinations, as part of the Residues Control Programme in an Ofcial Laboratory Easily adaptable to other food matrices and antimicrobial classes (e.g. MCs and LCs). Time-consuming sample preparation method Ref. [96]

SAs, Qs, MCs, TCs, -LCs SAs, Qs, MCs, TCs, -LCs, AMPs SAs, Qs, TCs, -LCs

Milk

Milk

SAs, MCs, Qs: >70%; TCs: 5060% and -LCs: <50% 80120% (for 88% analytes) 4580%, -LCs (<25%)

[97]

[98]

Eggs

[99]

SAs, Qs, MCs, TCs, AGs, LCs, CAP

Honey

65104%, ERY A (30%)

[100]

SAs, MCs, TCs, -LCs, Honey AGs, AMPs

SAs, Qs, MCs, AMPs, LCs, IPhs

Muscle

Four sequential LL extraction steps: (1) ACN; (2) 10% (v/v) TCA + ACN; (3) NFPA + ACN; (4) hydrolysis + ACN The four extracts were individually re-suspended in MeOH/H2 O (20/80, v/v) Extraction with ACN/MeOH (95:5, v/v) and defatting with n-hexane, followed by evaporation and dissolution in MeOH Bipolarity extraction with ACNaqueous McIlvaine buffer/ammonium sulphate, followed by clean-up with Oasis HLB cartridges Extraction with 70% MeOH and dilution with H2 O

TCs and AMPs: 68118%; AGs, MCs, SAs and -LCs: 26104% 70110%

[101]

[102]

SAs, Qs, MCs, TCs, LCs, -LCs

Muscle, kidney, liver

48148%

[103]

SAs, Qs, MCs, TCs, -LCs

Muscle, kidney

47121% (muscle), 26107% (kidney)

[104,105]

SAs, Qs, MCs, TCs, PCs

Muscle

Extraction with MeOH/H2 O (70:30, v/v) containing EDTA. Dilution with H2 O and ltration QuEChERS extraction with 1% (v/v) acetic acid/ACN and sodium sulphate, followed by d-SPE with an NH2 sorbent. NMZs require additional clean-up with strong cation exchange cartridges Extraction with H2 O/ACN or acetone/1% (v/v) formic acid

60.596.5%

[106]

SAs, Qs, NMZs, IPhs

Muscle

5589%

[107]

SAs, Qs, LCs, MCs, NMZs, IPhs, TCs

Honey, milk, eggs, meat

70120% (>80% of analytes)

Simple, cheap and straightforward method for [108] the extraction of 172 multi-class compounds (among pesticides, toxins and veterinary drugs), from different types of food matrices

Acronyms: AGs: aminoglycosides; AMPs: amphenicols; -LCs: -lactams; CAP: chloramphenicol; d-SPE: dispersive-SPE; IPhs: ionophores; LCs: lincosamides; MCs: macrolides; NFPA: nonauoropentanoic acid; NMZs: nitroimidazoles; PCs: penicillins; Qs: quinolones; SAs: sulphonamides; TCs: tetracyclines.

dure consisting of treatment with succinate buffer and subsequent clean-up with Oasis HLB cartridges, allowed the detection of residues of SAs, TCs and Qs, in incurred eggs from dosed laying hens, at concentrations above 100 ng mL1 . However, the current methodology failed for quantitative analyses; because the recoveries of TCs and some SAs were rather low (4565%) and poor intra-lab reproducibility (1030%) was observed for all analytes. Since the administration of antibacterials to laying hens is forbidden and no tolerance levels have been established in eggs, then analytical methods should be able to detect low ng mL1 levels, in order to avoid false negative results. Honey is another matrix for which multi-class LCMS2 methods have been developed. Some authors have proposed a simple pretreatment which involves dilution of honey with water, centrifugation and ltration, followed by SPE [100]. In that way, 15 antibacterials of 7 different classes could be quantied at very low ng mL1 levels; only ERY A and monensin (MON), which had very low recoveries, could be detected and conrmed, but not quantied. Moreover, streptomycin (STR) determination needs to be performed on

a separate set of samples, due to the particular extraction and chromatographic conditions required for this antibiotic; whereas CAP had to be analysed in a second chromatographic run, because detection for this antibiotic is performed in ESI negative ion mode. Obviously, this fact leads to a relatively low sample throughput. In another work, a sample preparation method, consisting of four subsequent LL extraction steps has been applied to the extraction of 37 multi-class antibacterials from honey samples [101]. Instead of pulling the four honey extracts together (which leads to -LCs loss, due to the presence of residual amounts of acid in two of the extracts) or analysing them separately (which reduces considerably sample throughput), a single LCMS2 determination was performed using a stacking injection mode (taking 10 L of each of the four honey extracts). In this case, the injection order was very important to avoid losses of -LCs during LCMS2 acquisition. Up to 12 honey samples can be prepared within 5 h, using the above-mentioned sample preparation procedure. Animal tissues are among the food commodities that have attained more interest for the development of multi-class LCMS2

M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517

15

methods. One example is the determination of 130 veterinary drugs (including 61 antibacterials belonging to different families) in different meat samples (e.g. bovine, porcine and chicken muscle) by LL extraction with ACN:MeOH (95:5, v/v), followed by lipid removal with n-hexane [102]. Then, extracts are evaporated and the nal residue is dissolved in MeOH, before determination by LCMS2 . Acceptable recoveries (70115%) were obtained for most of the antibacterials, except for two Qs, NOR and CIPRO that showed recoveries below 70%. By contrast, other compounds (e.g. AMPs and SAs) that showed high enough recoveries, the RSD values exceeded 20%. Therefore, the methodology can be useful for screening but not conrmatory purposes, since the determination of some antibacterial families does not full the acceptable requirements of accuracy and precision. In the same way, Kauffmann et al. have recently described an UPLCTOF-MS method for the determination of about 100 veterinary drugs, including the most relevant antibacterial classes, in different meat matrices [103]. The applied extraction/SPE cleanup procedure which intends to recover the largest possible range of analytes, is rather extensive. It consisted of an extraction combining in parallel two solvents with different polarity (namely ACN and McIlvaine buffer), in the presence of high amounts of ammonium sulphate, which induces phase separation. In this manner, the presence of polar and non-polar liquid droplets in the emulsion improves the recovery of a wider polarity range of analytes, compared to the use of an organic solvent alone (which results in poor recoveries for polar PCs and TCs), or to a sequential extraction with an organic solvent and aqueous buffer (that induces foaming formation and high analyte losses). Moreover, further extract cleanup using Oasis HLB cartridges allowed the removal of major food components (proteins, fat and carbohydrates), avoiding signicant losses of most of the analytes, although very hydrophobic drugs (e.g. IPhs) were not quantitatively recovered. Granelli et al. have developed another method to screen 19 antibacterials in muscle and kidney samples [104,105]. In this case, a single extraction with 70% methanol and further dilution with water, before injection into the LCMS2 was the only sample treatment required. In this way, a high number of samples (60) can be analysed per day. However, the methods performance is clearly dependent on the type of matrix and compound class; thus recoveries of TCs, Qs and MCs from kidney samples were rather low (<68%) and signal suppression affecting mainly TCs and MCs, results in poorer precision for these two classes of antibacterials. In addition, the AGs which are highly polar antibiotics cannot be extracted in 70% methanol, and consequently could not be included in the present study. Another example is the work developed by Chico et al. [106], which consist of a simple and rapid method for the determination of 39 antibacterials (TCs, Qs, PCs, SAs and MCs) in different animal muscle tissues. The method combines extraction with MeOH/H2 O (70:30, v/v) in the presence of EDTA to properly extract TCs and determination by UPLCMS2 . No clean-up was applied in order to make the sample preparation as simple as possible and to achieve high sample throughput; but it was necessary to dilute the extracts with water to minimize matrix effects. The method has been implemented as a routine method in a Public Health Laboratory, instead of the ve plates test and LC methods previously used. Stubbings et al. [107] have recently reported a multiresidue/multi-class procedure to screen for the presence of SAs, Qs, IPhs and NMZs in animal tissues. The method uses a sample preparation procedure based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methodology. The optimal extraction was performed with ACN containing 1% (v/v) acetic acid with anhydrous sodium sulphate as drying agent, followed by d-SPE with a Bondesil-NH2 sorbent. Finally an aliquot of the extracts was evaporated and reconstituted in ACN/H2 O (90:10) before LCMS2

determination. Determination of NMZs required additional cleanup after d-SPE with Bond Elut SCX cartridges. Validation has been performed on chicken muscle samples, showing that all analytes can be screened at their MRPL/MRL levels. Furthermore, recent work has demonstrated that the method is also applicable to MCs and lincosamide (LCs) antibiotics. Lastly, a very interesting work describes for the rst time a fast and straightforward generic procedure for the extraction of a wide variety of residues and contaminants (namely, pesticides, mycotoxins, plant toxins and veterinary drugs) from different types of food and feed matrices [108]. Among the veterinary drugs, several antibacterial classes were included (e.g. SAs, Qs, LCs, MCs, NMZs, IPhs, and TCs). The new procedure involves basically the extraction of samples with water/ACN or acetone/1% (v/v) formic acid and further determination by UPLCMS2 . As sample preparation does not involve any clean-up, thus to minimize matrix effects small volumes of extracts (typically 5 L) were injected. Despite the simplicity of the procedure and the inherent complexity of the extracts, adequate recoveries were obtained for the majority of the analyte/matrix combinations (typical values for antibacterials were in the range 70120%). Moreover the use of UPLC allows high speed analysis, since all analytes eluted within 9 min. Up to our knowledge, this is the rst analytical method described in the literature that really ts to the purpose of generic residue/contaminant determination in food.

3. Conclusions and future trends In this article we reviewed recent progress in sample preparation for the determination of antibacterial residues in foodstuffs. Table 5 summarizes the main extraction and clean-up techniques applied nowadays to this aim. Progress made over the past years in topics like mass spectrometers design, innovative chromatographic technologies, laboratory automation/miniaturization and more efcient materials for SPE drive the trend towards sensitive detection together with high sample throughput and less time devoted to sample clean-up. But still in many cases, sample preparation remains as the crucial step in food residue determination. Very often, analyte extraction efciency is the key aspect in sample preparation. The number of sample preparation steps should be kept as low as possible, as each step or transfer may result in losses of the target compounds. Therefore, a rapid and one-step sample preparation procedure can reduce costs and error sources. New sample extraction procedures have evolved to the use of much less solvent and smaller sample sizes. However, in such cases, care must be taken to ensure a representative portion from such naturally inhomogeneous food samples. Another aspect is related to the automation of the extraction and clean-up steps to allow on-line sample preparation and increase the robustness of the analytical methodology. A major advantage of on-line clean-up procedures is the speeding up of the analytical methodology. Moreover, many on-line sample preparation procedures allow the direct injection of the entire sample with a signicant enhancement in sensitivity. Nevertheless, optimisation of individual steps in off-line SPE methods is fairly exible, while in on-line systems a compromise must be often reached. In other cases, too much technical complexity or the high price of the instrumentation (e.g. PLE) has restricted the widespread use of on-line and automatic techniques. Selective sorbents like RAMs and MIPs are perhaps among the most innovative materials into the sample preparation eld, although they are far from being ready for routine food analysis. Nowadays, trends in food residue determination are clearly moving to the development of multi-analyte methods for the

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M.D. Marazuela, S. Bogialli / Analytica Chimica Acta 645 (2009) 517

Table 5 Summary of the main extraction and clean-up techniques used for sample preparation for antibacterials determination in food samples. Technique PLE Type of matrix Solid, semi-solid foodstuffs Mode Off-line Advantages/drawbacks Automated extraction and low sample manipulation. Reduced organic solvent consumption compared to Soxhlet or conventional LLE. Possibility of using H2 O as extractant (PHWE). Expensive lab-equipment is required Smaller sample sizes, reduced organic solvent and shorter extraction times compared to conventional extraction procedures. Samples with high water content (>30%) prevent diffusion of the extraction solvent resulting in low analyte recovery. This problem can be circumvented by lyophilization of samples prior to extraction Generic extraction strategies that allow multi-class determinations. Simple and cost-effective procedures that allow high sample throughput. Ideal for routine laboratories that have to analyse a large number of samples. Mostly appropriate for screening purposes. Matrix effects are often observed in complex matrices, due to the absence of clean-up Simplied and speeded up sample clean-up compared to conventional cartridge-based SPE (e.g. d-SPE allows tripling or quadrupling the number of samples prepared in a single day). Clean-up efciency can be modulated by the type of the dispersive media used (C18 , NH2 -sorbent, etc.) MSPD offers the possibility of performing extraction and clean-up in one step. Extraction efciency is enhanced since the entire sample is exposed to the extractant. Clean-up efciency can be modulated by the type of the dispersant (C18 , silica, alumina, MIPs, etc.). MSPD with water as extractant allows selective extraction of polar and medium polar compounds by adjusting water temperature. MSPD is amenable of automation using PLE extraction devices Integrates sample extraction, concentration and LC introduction in one single step. Does not require deproteinization or defatting steps prior to extraction. The polymer monolith capillary is stable within the entire pH range. Small sample volumes are required and no organic solvents are used. Matrix compounds can affect MS signal and/or extraction efciency Allow direct injection of untreated or minimally treated samples, thus simplifying the whole analytical procedure. High-throughput analysis and easy automation. No more than 20% of organic solvent should be used, since higher percentages lead to protein denaturation and consequently, to shorter column lifetime. RAMs are expensive compared to classical SPE sorbents. Extraction and clean-up in one step. Minimal sample pretreatment is required. High throughput and easy automation. High solvent consumption due to the high working ow rates (typically 24 mL min1 ) Selective concentration of a single analyte or a class of substances from complex matrices. Can be prepared in different formats (bulk, micro/nanoparticles, membranes, etc.) and used in SPE, SPME and MSPD applications. Template bleeding can led to contamination of extracts. This problem can be circumvented by using a dummy template. A careful choice of the template molecule can open the way to sample screening for whole analyte classes

MAE

Solid foodstuffs

Off-line

Dilute-and-shoot, QuEChERS

Solid, semi-solid, liquid foodstuffs

Off-line

d-SPE

Solid, semi-solid, liquid foodstuffs

Off-line

MSPD

Solid, semi-solid, liquid foodstuffs

Off-line

PMME

Solid, semi-solid, liquid foodstuffs

On-line

RAMs

Semi-solid and liquid foodstuffs

On-line

TFC

Solid, semi-solid, liquid foodstuffs

On-line

MIPs

Solid, semi-solid, liquid foodstuffs

On/off-line

Acronyms: d-SPE: dispersive-SPE; MAE: microwave-assisted extraction; MIPs: molecularly imprinted polymers; MSPD: matrix solid phase dispersion; PMME: polymer monolith microextraction; PLE: pressurized liquid extraction; QuEChERS: Quick, Easy, Cheap, Effective, Rugged and Safe; RAMs: restricted access materials; SPE: solid phase extraction; SPME: solid phase microextraction; TFC: turbulent ow chromatography.

determination of a broad range of compounds in a single analysis. Multi-class methods are favoured by regulatory agencies as part of their mandate to monitor the food supply for a variety of regulated veterinary drugs and other chemical contaminants. Another interesting research area that is just beginning is the determination of metabolites and degradation products of food contaminants, especially those that are considered genotoxic or carcinogenic. In both cases, generic solvent extraction procedures, so called extraction/dilute-and-shoot type methods, are preferred to simultaneously extract a wide variety of residues and contaminants from various food matrices. Thus, in the near future the development and application of generic extraction approaches will undoubtedly help in routine monitoring tasks since it implies a drastic reduction of both effort and time. References
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