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Recent advances in LC-MS residue analysis of veterinary medicines in the terrestrial environment
` Barcelo M. Silvia D az-Cruz, Damia
Despite some European countries having banned the use of veterinary medicines for non-therapeutic purposes in food-animal agriculture, large amounts of pharmaceutical compounds, mostly antimicrobials, continue to be used for these purposes, as well as being used for the legitimate treatment of animal diseases. The high worldwide consumption of veterinary medicines has alerted scientists and the public in general, and efforts have been made to assess the concentrations levels of such residues (parent compounds, metabolites and degradation products) in the aquatic and terrestrial environments. As a consequence, reliable analytical methods have been developed for the identication and the determination of veterinary pharmaceutical residues in the environment. Probably because of the great complexity of solid environmental samples, only a few studies have been published on their determination in soils, sediments and sludge. Nevertheless, this information is needed to understand and to assess the impact of veterinary medicines on the environment. The important development of liquid chromatography coupled to mass spectrometry (LC-MS) in the environmental eld has made this technique a powerful tool for reliable and accurate determination of pharmaceutical residues in the terrestrial environment. We aim to review the latest studies, pointing out the progress achieved in the LC-MS analysis (screening, identication, conrmation and quantication) of veterinary pharmaceutical residues in solid environmental matrices (soil, sediment and sludge), with especial emphasis on sample pretreatment and instrumental detection. 2007 Elsevier Ltd. All rights reserved.
Keywords: Environmental analysis; LC-MS; Liquid chromatography-mass spectrometry; PLE; Pressurized liquid extraction; Sediment; Soil; Solidphase extraction; SPE; Veterinary medicine Abbreviations: APCI, Atmospheric pressure chemical ionization; API, Atmospheric pressure ionization; APPI, Atmospheric pressure photoionization; ASE, Accelerated solvent extraction; ESI, Electrospray ionization; EU, European Union; GC, Gas chromatography; GPC, Gel-permeation chromatography; HPLC, High-performance liquid chromatography; IP, Identification point; IT, Ion trap; LC, Liquid chromatography; LIT, Linear ion trap; MAE, Microwave-assisted extraction; MIP, Molecularly imprinted polymer; MISPE, Molecularly imprinted solid-phase extraction; MS, Mass spectrometry; MS2, Tandem mass spectrometry; PFE, Pressurized fluid extraction; PLE, Pressurized liquid extraction; Q, Quadrupole; QqQ, Triple quadrupole; SPE, Solid-phase extraction; SWE, Supercritical water extraction; TOF, Time of flight; UPLC, Ultra-performance liquid chromatography; USE, Ultrasonic extraction; UV, Ultraviolet; WWTP, Wastewater-treatment plant.

1. Introduction
az-Cruz*, M. Silvia D ` Barcelo Damia Department of Environmental Chemistry, IIQAB-CSIC, University of Barcelona, c/ Jordi Girona 18-26, Barcelona E-08034, Spain
*

Corresponding author. Tel.: +34 93 4006100; Fax: +34 93 2045904; E-mail: sdcqam@cid.csic.es

A huge diversity of pharmaceutical compounds is employed in food-animal agriculture worldwide for the purposes of treating or preventing infectious and noninfectious diseases, managing reproductive processes and promoting growth using sub-therapeutic levels (<0.2 g/kg) [1]. Compounds used belong to a variety of therapeutic classes, including antimicrobials, anti-inammatories, parasiticides,

anesthetics, sex hormones, antiseptics, bronchodilators and anti-fungals [2,3]. To date, however, data available mainly relate to antimicrobials (see Table 1), probably because these substances are sold in the greatest amounts, are highly toxic to environmental bacteria, as expected from their mode of action, and are the cause of general increasing concern as they can induce bacterial resistance, even at low concentration levels because of continuous (chronic) exposure [46]. 637

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Table 1. Antimicrobials most commonly used in food-animal agriculture Use Compounds Antimicrobial group b-lactams Sulfonamides Tetracyclines Macrolides Aminoglycosides

Table 2. Concentration range (ng/g) of selected antimicrobials in animal manure. Data from [78] Animal Tetracyclines Dairy Beef Hog Sheep Turkey nd52,000 nd600 nd25 nd10 nd310 Antimicrobial class Sulfonamides nd50 nd260 nd50 nd450 nd70 Macrolides nd5 nd850 nd6700 nd35 nd5

Treatment/prevention of infections Penicillin Sulfamethazine* Oxytetracycline, Chlortetracycline Erythromycin, Tylosin* Gentamycin Growth promotion** Penicillin Chlortetracycline Erythromycin, Tylosin* Bacitracin*, Virginiamycin*
* **

b-lactams Tetracyclines Macrolides Polypeptides

nd: not detected.

Used in animals and not in humans. Only in cattle, swine and poultry production.

Parasiticides are another therapeutic group that causes concern since they have been proved to have strong effects on dung-pat fauna [7]. Studies on the inuence on the decomposition of dung organic matter of a selection of antimicrobials and parasiticides commonly used in the treatment of grazing cattle revealed signicant loss of organic matter in dung from heifers treated with parasiticides a-cypermethrin, fenbendazole, ivermectin and levamisole, and with antimicrobial spiramycin, with spiramycin and ivermectin causing the greatest adverse effects [8]. Given the strong link between steroid sex hormones (estrogens) and endocrine disruption in marine [9] and freshwater [10] sh, it is essential to assess their loads into the environment. The wastes produced in animal farms may be signicant sources of endogenous hormones, as well as hormones administered to treat the animals and to promote growth. According to a study by Raman et al. [11], typical concentrations of natural estrogens estrone (E1) and estradiol (E2) in dairy cow manure are 39 lg/kg and 18.4 lg/kg dry weight, respectively. A similar concentration was reported for E2 in broiler litter manure [12]. During manure storage, the concentration of E1 is expected to increase, since E2 undergoes oxidation generating E1, as observed in soil [13]. Two other steroids, trembolone and melengestrol, present in cattle dung are known to be detectable in manure-amended soil up to several months after fertilization [14]. A way to take even more prot from food-animal agriculture and at the same time to take apart the subproducts generated is to use manure as fertilizer on the nearest arable lands. This practice lowers the cost of crop production, improves the soil/water inltration and reduces potential soil erosion. When a medicinal product is administered to animals, residual concentrations of the parent compound along with metabolism products are 638
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present in their urine and excrement (see Table 2) to an extent that varies, depending mainly on the drug, the type of animal, and its age and body weight [15]. Quite a number of studies have revealed that veterinary pharmaceutical residues can persist in manure from days (tylosin) to months (ivermectin) [16]; as a consequence, manure spreading results in a source of contamination for the environment. Another important source of veterinary drugs in the environment is the sh-farming industry. Aquaculture treatments are employed to treat both eggs in hatcheries and free-living stock within pond or tank-based systems. A large proportion (up to 70%) of the pharmaceuticals administered, generally as food additives, is not ingested by the sh, due to overfeeding, loss of appetite by diseased sh and poor adsorption of the drugs [17]. They are, therefore, directly introduced into the aquatic environment since they are discharged into rivers through settlement ponds, and nally sorbed onto sediments and probably accumulate over a long period of time [18]. Oxytetracycline (OTC) is one of the most common antimicrobials used in aquaculture, and, according to a study by Lalumera et al. [19], the concentration of OTC in sediments within farms and the area close to discharge points of an active aquaculture area in Italy was as high as 246 ng/g dry weight. Once these residues reach the environment, they can be adsorbed, leached, degraded, and bioaccumulated, and their metabolites can revert back to the parent compound, as appeared to be the case for sulfamethazine and chloramphenicol, whose main metabolites in manure, N4-acetyl-sulfamethazine and chloramphenicol glucuronide, respectively, are decomposed by bacteria once excreted, thus reactivating the parent drugs [20]. The lack of information on production, sales, uses and dosages of veterinary medicines [21] is common in almost all countries, USA, and some European countries (e.g., Sweden and Denmark) being those where data are more abundant, although incomplete. Despite that, in Europe, the general trend has been to ban the use of antimicrobials as growth promoters; in 1986, Sweden was the rst to ban such use, and other countries (e.g.,

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Australia and Japan) have adopted the same measure [22]. Currently, in the European Union (EU), there are only four chemicals allowed as feed additives for the purpose of growth promotion (i.e. salinomycin, monensin, avilamycin, and avomycin), although it was intended to ban them by 2006 [23]. In the USA and Canada, other compounds are still licensed as growth promoters for farm animals, such as the synthetic esteroids trembolone acetate and melengestrol acetate [24]. In Europe, any new human and veterinary medicinal product to be authorized for sale has to go through an authorization procedure [25,26]. Assessment of their potential environmental risk became a goal of the authorization of veterinary drugs at the beginning of the 1990s [27]; currently, new guidance documents are being discussed in international bodies on which EU countries, Japan and USA are active, with Australia, New Zealand and Canada being observers [28,29]. In the USA, the Food and Drug Administration (FDA) has required environmental assessment reports for veterinary pharmaceuticals since 1980 [30]. Despite the lack of specic regulation regarding pharmaceutical residues in the environment, general guidelines for reliable compound determination based on a system of identication points (IPs) was described in the Commission Decision 2002/657/EC concerning conrmatory analysis of veterinary pharmaceutical residues in food-animal tissues [31]. According to this accepted system, different numbers of IPs are earned depending on the detection technique and analyzers employed. The main advantage of this IP-based method is the certainty that identications based on these criteria are done in a well-established, internationally accepted way [32]. Several methods have recently been developed for the analysis of quite a number of pharmaceutical residues in the environment; however, most of them applied to aqueous samples. These methods can be found in recent reviews [3335]. In order to provide more information concerning occurrence, fate and effects of veterinary pharmaceuticals in the terrestrial environment, a number of theoretical models can identify where and when measurable concentrations will occur, but it is necessary to calibrate and to validate the models using real data [36]. The low environmental concentrations (usually ng/g) and the complexity of environmental solid matrices make heavy demands on the analytical work, especially in preconcentration and purication processes. Another analytical challenge is the determination of drug metabolites or degradation products, since they should be included in monitoring programs because their behavior can substantially differ from that of the parent compound and selected environmental compartments

may be more susceptible to adverse effects caused by the metabolites or degradation products than might be expected if only the parent was considered [21]. New analytical methods therefore have to be developed and validated to determine low concentrations of drug residues and their transformation products in complex solid environmental matrices. In a recent interesting review [22], Sarmah et al. gave the latest general information regarding usage, production, occurrence, fate and effects of veterinary antimicrobials in the environment, including sediments and soils. In a previous survey [37], we focused on the environmental behavior and the analysis of both human and veterinary pharmaceuticals. At that time, data were hardly available, especially for veterinary drugs. The present review is intended to ll the gap in knowledge that still exists on the problems and the limitations of LC-MS-based analytical methodologies for reliable determination of veterinary pharmaceutical residues in solid environmental samples.

2. Sampling and sample preparation 2.1. Sampling Sample collection is the rst step in the analysis of every natural sample. Despite this initial part of the analytical processing being of critical importance, few authors report details of it [6,11,38]. The selection of the sampling device depends on the aim of the study, whether basic, at a supercial level or indepth. In collecting solid environmental samples, it is important to consider the inhomogeneous character of the matrix. Van Veen samplers are preferred when a large number of samples have to be collected, and corers are used when detailed information on the spatial distribution of the analytes is required. Cores can be sectioned with a chop saw tted with a diamond-tipped blade. Since the results can depend on the diameter of the corer, for sub-sampling, it is usually recommended to divide the sediment section and collect the inner part (which has not been in contact with the sampler walls). For sampling devices, stainless steel provides the best results. Collected samples are transported under cooled conditions in a portable refrigerator to the laboratory and then can be both directly stored in the dark (at about 4C or at 20C) or lyophilized, homogenized, sieved and nally stored until analysis. During storage, bacterial activity should be arrested in order to preserve the integrity of the sample. The use of chemical preservatives to halt bacterial activity is uncommon since the chemical composition of the sample is altered. As alternative, samples can be autoclaved to inactivate microorganisms and any enzymatic activity [39]. The material of the storage container can also affect samples. Glass is well adapted for several types of sediments and soils, dry or
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wet, and, in general, brown glass is preferred to prevent effects from exposure to light. 2.2. Sample preparation In order to detect, identify and quantify pharmaceutical residues released into the environment and accumulated in river and marine sediments, and in agricultural soils amended with animal manure, analytes need to be isolated from the sample matrix and concentrated to some extent, because of the extremely low level of concentration at which they occur. 2.2.1. Extraction Trends in analyte isolation include:  less solvent consumption;  improved extraction throughput (in some instances linked to automation);  higher recoveries; and,  better reproducibility. As a consequence, classical Soxhlet and ultrasonic extraction (USE) [40] techniques are being steadily replaced by recently developed techniques such as pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE) [4143] that fulll current demands for environmental analysis. Because there is often strong interaction between the drug residues and soils or sediments, the compounds are difcult to extract. The isolation of analytes from environmental solid samples therefore encompasses optimization of the physical properties of the extracting solvents, especially temperature and pressure, in order to enhance their capacity to extract analytes from a variety of solid matrices by dropping the surface tension and increasing analyte solubility and diffusion. Improving selectivity does not seem to be a current challenge in extraction, probably because it can be achieved later by efcient separation methods and/or highly selective detection techniques. 2.2.1.1. Pressurized solvent techniques. PLE, supercritical uid extraction (SFE), and supercritical water extraction (SWE) are based on similar principles. PLE

allows rapid extraction (around 30 min. per sample) under high pressure and at temperature above the normal atmospheric boiling point) with excellent reproducibility, using quite a low volume of a wide variety of organic solvents. SFE and SWE are quite similar to PLE, although their use in the extraction of veterinary drug residues from environmental solid samples has not been reported. In optimizing a PLE procedure, all important parameters affecting extraction efciency (solvent, pressure, temperature, ush volume, and number of extraction cycles) should be considered. In addition to the pressure of the extracting solvent, temperature has a profound effect on the efciency of the PLE due to the importance of mass-transfer kinetics and solubility. A long exposure to solvents allows the matrix to swell, thus improving the penetration of the solvent into the sample interstices and the contact of the solvent with the analytes. That is why there is usually a pause between consecutive extraction cycles. More than one extraction cycle allows introduction of fresh solvent, maintaining favorable equilibrium between solvent and sample, hence improving partitioning into the liquid phase. Several applications of PLE to veterinary pharmaceutical residue analysis in the terrestrial environment have been described [6,23,4447]. In all cases, the extraction system was an ASE 200 from Dionex (Sunnyvale, CA, USA), accelerated solvent extraction (ASE) being the Dionex trade name for PLE. Table 3 lists most critical extraction parameters. Particular attention should be given to the PLE extraction of thermo-labile compounds (e.g., tetracyclines [6,44,47]) for which extraction has to be performed at room temperature. An attempt to enhance PLE extraction of tetracyclines was carried out by performing the extraction process in two extraction steps; in the rst, the temperature was set at 60C and in the second at 30C to prevent possible degradation from prolonged exposure at the higher temperature [47]. Another consideration when analyzing tetracyclines and other compounds known to form chelate complexes

Table 3. PLE methods applied in the extraction of veterinary medicines in solid environmental samples Compounds Macrolides, Ionophores, Tiamulin Sulfonamides, Penicillins Sulfonamides, Tetracyclines, Macrolides Antimicrobials (several therapeutic classes) Matrix 30 g soil 5 g sludge from inltration basins 10 g soil 10 g soil Solvent (v/v) 1% aqueous ammonia in MeOH Acetone/MeOH 50:50 MeOH/citric acid buffer (pH 4.7) 50:50 MeOH/water 80:20 T (C) 80 75 Room 100 P (bar) 140 150 150 140 Cycles 2 3 nr 3 Static time (min.) 10 5 3 10 Cell ushing (%) 70 60 nr 50 Ref. [23] [22] [5,21] [8]

MeOH, Methanol; nr, not reported.

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with metal ions is the composition of the extracting solvent, which has to isolate divalent and trivalent cations potentially present in the matrix. Often the complexation agent used is 0.2 M citric acid buffer, and the extraction solvent comprises a mixture of methanol and buffer (1:1, v:v) adjusted to pH 4.7 [6,44]. Other combinations have been assessed by using methanol, McIlvine buffer (citric acid and potassium phosphate) and EDTA; however, this extraction buffer precipitates after few hours, so it may cause blockage in the connections, tubes and valves of the PLE instrument when long extraction sequences are to be run. Nevertheless, when extractions are carried out by sonication or mechanical shaking, this extracting solvent combination is useful, as applied in the isolation of OTC, sulfachloropyridazine and tylosin from manure and soil [48]. 2.2.1.2. Microwave-assisted extraction. In MAE, modication of the physical properties of the extracting solvents is achieved by heating the liquids using microwave energy. As a consequence, only solvents with the ability to absorb microwave energy, which requires the presence of dipoles (i.e. polar solvents), can be used. Besides the nature of the solvent, temperature and irradiation time are the two most important parameters in MAE, because of they affect both extraction efciency and also analyte degradation. In a recent study [49], mechanical shaking and MAE were compared in terms of recovery and precision as applied to the extraction of two quinolone antimicrobials, namely umequine and oxolinic acid, from two marine and lake sediments, and one agricultural soil spiked at 50-ng/g, 100-ng/g and 250ng/g levels. In this work, both optimized extraction methods were carried out with a mixture of 1-mM phosphoric buffer at pH 2 and dichloromethane (50:50). The mechanical shaking lasted 15 h and the MAE 22 min at 90C. Data obtained revealed that:  mechanical shaking resulted in lower recoveries (5391%) than those from MAE (8294%), the larger differences being for oxolinic acid, which is the most retained antimicrobial; and,  MAE extraction efciency did not depend on the nature of the matrix. Relative standard deviations (RSDs) for repetitivity and reproducibility were similar with values in the range 37% and 59% for MAE and extraction by mechanical shaking, respectively. Since these extracts may contain interfering co-extracted components as a consequence of the intensive extraction process performed, and that would lead to an unreliable analysis, further purication is needed. 2.3. Purication Monitoring requirements are steadily covering substances beyond priority pollutants. There is a preference for multi-residue analytical methods, which

imply, in most instances, large sets of compounds that exhibit great differences in their physicochemical properties. Typically, extracts from solid samples are puried following schemes similar to those employed for pharmaceutical residues from aqueous samples, which are mainly based on solid-phase extraction (SPE) [50], the leading technology for extracting organic compounds, with a range of different adsorbing materials. Prior to SPE clean-up, the organic extracts are usually evaporated under a gentle N2 stream and then reconstituted in a methanol:water solution below 10% methanol (v/v) in order to prevent the continuous elution of analytes from the SPE-sorbing material. Polymeric Oasis HLB cartridges (lipophilic divinylbenzene + hydrophilic N-vinylpyrrolidone) (Waters, Milford, MA, USA) were mostly employed because of their more rugged extraction efciency, improved recovery for both polar and non-polar compounds and greater capacity than reversed-phase silica-based materials. Other sorptive phases used include Lichrolut EN, Lichrolut C18, Oasis MCX, and Widepore CBX. Finally, analyte concentration is typically carried out to allow determination at the low-ng/g level. Given that sample preparation is quite a large eld, we describe only really innovative achievements. When monitoring the illegal use of veterinary drugs in animal production (e.g., clenbuterol and aniline-like b2-agonists), a broad group of molecules share the same pharmacological effect. In addition, improved analytical selectivity is required for reliable analysis. These requirements have led to the development of new sorptive phases, with molecularly imprinted polymers (MIPs) outstanding for allowing a higher degree of selectivity to be achieved. MIPs have been used as selective sorbent with several analytical techniques (e.g., liquid chromatography (LC), capillary electrophoresis, capillary electrochromatography, immunoassay and molecularly-imprinted SPE (MISPE)). MISPE is analogous to immunoafnity extraction, which relies on the extremely highly selective interaction between antigen and antibody [51]. The main benet of the technique is that the selectivity of the MIPs can be determined by the selection of the template molecule employed in its preparation leading to exhaustive sample purication. In the past decade, there was a limited number of reports dealing with the application of MISPE to biosamples (biological tissues and uids) [52 54] and a few in environmental analysis [52,55,56]. However, recent developments in MIP technologies have allowed new studies [5759]. Although none has yet been applied in the purication of extracts from solid environmental samples, similar clean-up procedures to those for biological uids [59] or tissues [58] could be employed. For example, Widstrand et al. [59] evaluated MISPE as a purication technique in the determination of eight b-agonists in calves urine, using MIP4SPE b-agonist columns from MIP Technologies (Lund, Sweden). The
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columns were conditioned with methanol, water and sodium acetate at pH 6.7. For washing, water and acetonitrile containing 1% acetic acid were employed. Finally, elution of b-agonists from the MIP was carried out with methanol containing 1% acetic acid. Other solid-liquid based extraction techniques, such as gel-permeation chromatography (GPC), are rarely used to purify soil extracts; however, an example can be found in the work by Kreuzig et al. [60], where residues of sulfadiazine in manure fertilized soils were investigated. Sephadex LH-20 (Gilson, Du sseldorf, Germany) and a solution of 0.1-M acetic acid in methanol as elution solvent were employed. GPC has also been used for purifying acetone extracts from plant samples grown in agricultural elds containing diazinon, orfenicol, levamisole, trimethoprim, enrooxacin, OTC, sulfadiazine, amoxicillin, tylosin and phenylbutazone [40] in uptake studies from soils into plants. In order to enhance the purication process, a tandem SPE set-up is a good approach and may be a future trend, as the latest studies show. This approach is not much more time consuming compared to a single clean-up, although it has not so far been fully automated. Combinations of ion-exchange (weak and strong) and polymeric cartridges reduce the ionic humic acids co-extracted from the soil/sediment matrix. Good results were obtained by Jacobsen et al. [44] when combining a strong anion-exchange cartridge, SAX (Isolute, IST. Mid-Glamorgan, UK) with the polymeric Oasis HLB cartridge in the purication of a manure-fertilized-soil extract contaminated with tetracyclines, sulfonamides and macrolides. The SAX cartridge (on the top) retained anionic humic acid components present in the sample and prevented the blockage and the overload of the HLB sorbent. In order to allow the retention of polar analytes in the HLB sorbent, solution pH has to be optimized to maintain the polar analytes in neutral or cationic form. In this study, a pH of 4.7 proved to be suitable. The same tandem combination was also successfully applied by Kay et al. [38], Aga et al. [47] and Blackwell et al. [48] when analyzing several antimicrobials in manure and manure-fertilized soils.

3. Separation and detection Analytical methods are usually based on LC or gas chromatography (GC) since multi-residue analysis requires the application of separation techniques. Because most veterinary medicines, and even more their transformation products, are highly polar, water soluble and non-volatile or thermally labile, LC is the technique of choice for their determination. Given that the aqueous, puried and pre-concentrated extract of a solid environmental sample is quite similar to a natural water sample, detailed practical information 642
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regarding separation and detection (e.g., mobile phases and additives, and parent and product-fragment ions) can be found elsewhere (e.g., in the collection of reviews on water analysis and references cited therein (see Introduction)). Briey, LC of veterinary drugs in extracts obtained from solid environmental samples has usually been carried out with C18 LC columns. As mobile phases, mixtures of water-methanol and acetonitrile at different pH have normally been used. Modication of the mobile phase is usually performed in an attempt to improve the sensitivity when mass spectrometry (MS) detection is to be used, and has been accomplished with volatile substances (e.g., acetate, formate or formic acid) to avoid clogging at the interface and a build-up of deposits in the ion source. GC is less commonly used in veterinary drug analysis due to the mostly polar and water-soluble character of such substances, which require a tedious derivatization step. For example, N-trimethylsulfonium hydroxide in methanol was used by Boxall et al. [40] as the derivatizing agent in the analysis of anti-inammatory phenylbutazone in soil and plant samples, and the mixture N-methyl-N-(trimethylsilyl)-tri-uoroacetamide:trimethylsilylimidazole: dithioerytrol (100:2:2, v:v:v) by Burnison et al. [61] in the determination of natural and synthetic estrogens, and phytoestrogens in pig manure. There has been signicant progress in separation techniques through development and availability of new stationary phases for LC that have improved analyte separations, and column stability and durability. Moreover, there has been a trend to use microbore LC columns (about 1-mm. i.d.), which, compared with conventional columns (24-mm. i.d.), allow greater separation efciencies, sensitivities and reduced solvent consumption and analysis cost [62]. However, the most outstandingly novel approach to chromatographic separation is the development of ultraperformance liquid chromatography (UPLC), which has sensitivity 23 times greater than high-performance liquid chromatography (HPLC) by using columns packed with particles of <2lm, resulting in better chromatographic resolution, increased peak capacity and reduced run time [63]. Only one application of UPLC in environmental analysis has been reported to date; Petrovic et al. [64] developed a multi-residue method based on MS detection for screening and conrmation of human and veterinary pharmaceutical residues, including erythromycin, sulfamethoxazole and trimethoprim, in wastewaters with limits of detection (LODs) in the range 10500 ng/L. Fig. 1 shows the short analysis time and the optimal separation attained. Since the introduction of atmospheric pressure ionization (API) techniques, LC-MS has played an increasingly important role in environmental analysis. Electrospray ionization (ESI) and atmospheric pressure

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Figure 1. UPLC-Q-TOF total ion chromatogram of a 100-ng/ml standard solution of 23 pharmaceuticals, among them some veterinary medicines, such as erythromycin, used in both treatment and growth promotion (Reproduced with permission from [64]).

chemical ionization (APCI) are the most popular API techniques. Liquid-introduction APCI was developed because ESI was not sensitive with less polar chemicals. Despite APCI expanding the range of low-polarity and/or low-mass compounds that were amenable to LC-MS analysis, there are still compounds that do not ionize or are not sensitive with APCI. In 2000, atmospheric pressure photoionization (APPI) emerged as a new ionization approach [65] for expanding the range of applications of API interfaces to low-polarity and non-polar compounds that can hardly be detected by either ESI or APCI. Briey, APPI is a modication of the APCI source where the corona is replaced by a gas-discharge lamp, emitting radiation in the UV region that can selectively ionize the analytes in the presence of the LC mobile phase [66]. According to the rst application by Robb et al. [65], through the use of an APPI source (10-eV photons, vacuum, UV lamp) along with a dopant agent (toluene) to enhance sensitivity, since the dopant agent is rst ionized and then aids ionization of analytes in further reactions, it is possible to obtain analyte-signal intensities 8 times those attained using an APCI interface. Results indicated that APPI followed a similar behavior to APCI, so compounds amenable to being efciently ionized by APCI could also be successfully ionized by APPI, and through similar fragmentation pathways, despite the initial ions being different. To illustrate such ndings, Fig. 2 shows some applications, for which APPI was reported to be more suitable than APCI as ionization source. This new API interface has found only limited applications so far to natural samples detecting

chloramphenicol in sh meat [67] and cyclosporine A in rat plasma [68]. Perhaps this ionization technique will become a routine complement to ESI and APCI for the LC-MS analysis of a broader number of pharmaceutical residues. Traditional MS methods using single-quadrupole MS produce low fragmentation, so abundant stable pseudomolecular ions, [M+H]+ or [M+H], are obtained. However, this technique can prevent reliable analysis due to the co-fragmentation of matrix components other than those targeted. Examples of such limitation can be found in the conrmation analysis of trembolone in cattle manure, where matrix interferences prevented the reliable determination of its heptauorobutyril derivative by GC-MS, and the quantication of melengestrol acetate in soil by LC-MS because the sensitivity was not sufcient [14]. To face these shortcomings, by using LC-tandem MS (LC-MS2), it is possible to distinguish individual compounds having the same molecular mass (isobaric) by the different fragments obtained after an induced collision with an inert gas. So, whenever possible, it is preferable to use tandem-MS detection for better selectivity and sensitivity, in particular, in complex matrices, such as soils and sediments. The most important characteristic of a tandem mass spectrometer is the nature of the nal analyzer since it usually provides the analytical mass spectrum. Congurations combining two different principles of mass analyzers are designed as hybrid mass spectrometers. A number of combinations are possible, but, often, the rst mass spectrometer is a quadrupole, and the nal MS analyzer may be, e.g., ion trap (IT) or time-of-ight (TOF).
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Figure 2. Comparison of APPI and APCI sources for the ionization of a standard solution containing carbamazepine, acridine, naphthalene and diphenyl sulde. Mobile phases used were: (a) methanol/water; and, (b) acetonitrile/water (Reproduced with permission from [65]).

This kind of hybridization is now driving new instrument development because it provides additional or superior performance characteristics [69]. IT analyzers can perform multiple stages of fragmentation in time (MSn) and trap the ions resolving isobaric fragments. As a consequence, their extensive application in the environmental eld will help to infer pathways to identify unknowns (metabolites and degradation products). TOF is an attractive alternative analyzer to identify unknown residues because of its accurate mass measurement of fragments at high sensitivity. Although environmental applications of hybrid analyzers are still scarce, several authors reported the use of LC-Q-TOF in the analysis of pharmaceutical residues (screening, conrmation of identity and determination) in natural waters [7072], drinking waters [71] and wastewaters [64]. To date, however, there has been only one simple laboratory experimental study concerning solid samples on the degradation process of trimethoprim in nitrifying activated sludge [73]. According to the literature surveyed concerning terrestrial environmental analysis, veterinary medicine residues have usually been ionized in positive-ioniza644
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tion mode, mainly using ESI; there were exceptions, in the studies by Schlusener et al. [46] and Lofer et al. [39], where APCI was selected. In the determination of parasiticides abamenctin and ivermectin, negativeionization mode was used [39]. MS analyses were conducted using tandem-MS analyzers (with multiple reaction monitoring (MRM)), and MS detection [47,61] for improved sensitivity (with selected-ion monitoring (SIM)). IT-tandem-MS spectrometers have rarely been used [74,75], triple quadrupole (QqQ) analyzers being the most frequently employed [6,19,23,40,44,45].

4. Summary and future outlook In a near future, there will be a demand for suitable, reliable, and very selective analytical methods that can identify and conrm the presence of veterinary drugs at ultra-trace level when their use as growth promoters becomes illegal. Future work should also cover veterinary medicines not yet considered a threat despite their wide use, as that will help to clarify their occurrence,

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fate and environmental risk. Since there is an absence of studies regarding solid environmental samples, data is urgently needed on occurrence, fate and transport of veterinary pharmaceutical residues for an integrated environmental risk assessment of the soil/sediment/ water system. PLE is clearly the method of choice for solid-sample extraction, with SPE as the next step in sample preparation as a method of purication. A signicant increase in selectivity can be achieved using selective adsorbing materials. Nowadays, the approach using MIPs appears promising because of their high selectivity and their ease and cost-effective preparation, not only as adsorbing materials but also as stationary phases in chromatographic separations. Nevertheless, further research is required to isolate quantitatively a collection of compounds belonging to the same therapeutic class by MISPE. LC-tandem MS is certainly a powerful tool for analyzing a large number of veterinary-medicine residues in the environment, especially for complex samples (e.g., soil, sediments and sludge), leading to more comprehensive assessment studies. Usually, high sensitivity and selectivity is obtained using API techniques in combination with MS2 analyzers. Although some authors have used LC-MS in SIM mode with single-MS analyzers, the majority of papers reviewed have applied LC-MS2 with QqQ or IT to determine veterinary-medicine residues in the terrestrial environment. QqQ instruments operated in MRM mode are preferred when sensitive as well as selective information is required. However, when structural information is needed (e.g., in screening and identifying unknowns, such as metabolites and degradation products), hybrid MS2 instruments are useful. The trends in mass-analyzer development therefore focus on hybridization with conventional mass analyzers to allow additional instrument functionality in MS2 detection, as required in the analysis of complex samples. New generations of TOF-based and Q-TOF-based analyzers have emerged recently with improved sensitivity and expanded effective linear dynamic range, and, in due time, they will probably allow performances comparable to those of QqQ detectors in addition to the valuable structural information provided. The combination of highly-selective QqQ scans with the highly-sensitive IT product scans in the hybrid Q-LIT analyzer [76] will allow reliable identication and unequivocal conrmation of analytes, even in very complex samples. Other powerful hybrid mass spectrometers, to be explored in environmental analysis of pharmaceutical residues (e.g., IT-TOF instruments [77]) will help in screening and identifying transformation products of veterinary medicines promoted by the extremely high sensitivity of IT and the excellent resolution of TOF.

Acknowledgements This work has been funded by the EU Framework VI Programme (Project NOMIRACLE, Contract No: 003956-2) and Coordination Action RISKBASE (Contract No: 036938 GOCE) and by the Spanish Ministry of Education and Science (Project CTM2006-26225-E). M.S. D az-Cruz n y Cajal contract from the acknowledges her Ramo Spanish Ministry of Education and Science. References
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