Anal Bioanal Chem (2009) 395:9871008 DOI 10.

1007/s00216-009-2872-z

REVIEW

Analysis of antibiotics in fish samples

F. Caada-Caada & A. Muoz de la Pea & A. Espinosa-Mansilla

Received: 7 April 2009 / Revised: 21 May 2009 / Accepted: 26 May 2009 / Published online: 16 June 2009 # Springer-Verlag 2009

Abstract The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in highintensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish. Keywords Antibiotics . Fish . Analysis

Introduction Aquaculture is the production of marine or freshwater food fish, including finfish and shellfish, under controlled conditions (e.g. feed, medications, controlled breeding, and containment) that enhance production. The cultivation of marine species has been practiced through the ages. People have been farming fish for millennia and there is evidence of aquaculture in Egypt and China as early as
F. Caada-Caada (*) : A. Muoz de la Pea : A. Espinosa-Mansilla Department of Analytical Chemistry, University of Extremadura, Avda. Elvas, 06071 Badajoz, Spain e-mail: floricanada@gmail.com

2500 BC and 1100 BC, respectively [1]. The Romans also developed fish culture. Oyster culture prospered in ancient Rome and Gaul [2]. However, all of the early forms of aquaculture differed greatly from those practised today. Traditional aquaculture was characterized by minimal added inputs, small farm size, and low stock density. Throughout the last century there was a large increase in demand for seafood products. Initially, much of this demand was met by wild-caught fish, but as world fisheries continue to be over-exploited and depleted, aquaculture farms have undergone unprecedented growth, evolving as a significant contributor meeting demands for seafood. Most of the aquaculture systems in the world continue to intensify cultivation methods. These methods are characterized by high stock density and volume, heavy use of formulated feeds containing antibiotics, antifungals, and other pharmaceuticals, and the application of pesticides and disinfectants [3]. Antibiotics are a group of natural or synthetic compounds that destroy bacteria (bactericidal) or inhibit their growth (bacteriostatic). Antibiotics that are sufficiently nontoxic to the host are used as chemotherapeutic agents in the treatment of infectious diseases of humans, animals and plants. The accumulated scientific evidence is that some uses of antibiotics in food-producing animals can lead to antibiotic resistance in intestinal bacteria, and this resistance can then be transmitted to the general population, causing treatment-resistant illness. These uses of antibiotics can also create antibiotic resistance in non-pathogenic bacteria, the resistance genes of which can be transferred to disease-causing bacteria, resulting in antibiotic-resistant infections for humans. Dissemination of resistant microorganisms may occur in both hospitals and communities. It is recognized that another route of transmission of resistant microorganisms from animals to humans is through the food chain [4].

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F. Caada-Caada et al.

In fish farming (aquaculture, mariculture, etc.), the widespread use of antibiotics for treating bacterial diseases has been associated with the development of antibiotic resistance in Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, E. icttaluri, Vibrio anguillarum, V. salmonicida, Pasteurella piscida, and Yersinia ruckeri [4, 5], and controlled studies are needed to determine the effect of antimicrobial therapy on the ecology of aquaculture ponds, particularly at the micro-organism level. Another problem created by the excessive use of antibiotics in industrial aquaculture is the presence of residual antibiotics in commercialized fish and shellfish products [6]. Marine fish farms are surrounded by a wide range of marine ecosystems, where artisanal fishermen work, gathering shellfish and fishing wild fish for human consumption. Some of these wild species feed from leftover food pellets. The pellets are medicated with antibiotics and other drugs, which pass to the fish. Recently, Fortt et al. [7] confirmed that wild fish captured around salmon aquaculture areas ingest the pellets prepared for salmon. Residues of tetracycline and quinolones were found in wild fish captured around those aquaculture areas. In 1992, a general revision of chromatographic methods applied in food-animal tissues was reported [8]. In 1998, the current status of the application of liquid chromatographymass spectrometry (LCMS) in the analysis of antibiotics in animal food products for human consumption was reported [9] and MS data in the period 19871996 were specially summarized in the mentioned revision. Some reviews focussing on LCMS analysis of antibiotic and antibacterial agents in animal food [10, 11] and on the application of LCMSMS to the analysis of veterinary drug residues [12], have been recently published. In the reviews mentioned; most of the references are about milk, cattle, kidney muscle and pork tissues, and a limited number are about salmon and catfish samples. Since the 2000s, several reviews on the determination of antibiotic residues [1315] and/or growth-promoting agents in food-producing animals have been published [16, 17]. In this context, Samanidou et al. have published a review that summarises the analysis of antibiotic residues in fish [15], providing extensive information on HPLC methods. Recently, a review about analysis of antibiotics by capillary electrophoresis (CE) and capillary electrophoresis chromatography (CEC) in pharmaceuticals, biological, agrochemical, food, and environmental fields has been published [18]. In the literature, there are also monographic reviews on the analysis, in food matrices, of antibiotic residues belonging to the same family, for example quinolones [19, 20], sulfonamide residues [21], aminoglycoside [22], or aminoglycoside and macrolide residues [23]. In Table 1, reported reviews about antibiotic analysis in food samples are summarized.

Legislation Low-level doses of antibiotics in foodstuffs consumed for long periods may lead to an increase in resistant strains of bacteria. To protect human health, The European Union has established safe maximum residue limits (MRLs) for these drugs and other veterinary substances, for use as veterinary drugs in animal products entering in the human food chain. The use of veterinary drugs is regulated through EU Council Regulation 2377/90/EC [24], which describes the procedure for establishing MRLs for veterinary medicinal products in foodstuffs of animal origin. MRLs values for antibiotics in fish, as set by European Union, are summarized in Table 2. Council Directive 96/23/EC [25] contains guidelines for controlling veterinary drug residues in animals and their products with detailed procedures. Prohibition of the use of growth-promoting agents such as hormones and -agonists is laid down in Council Directive 96/22/EC [26]. The Codex Alimentarius Commission [27], created in 1963 by Food and Agriculture Organization (FAO) and World Health Organization (WHO), and other regulatory agencies around the world, for example the US Food and drug administration (FDA) [28], the Canadian Food Inspection Agency (CFIA) [29], the Australian Pesticides and Veterinary Medicines Authority (APVMA) [30], and the Health Department from Chile [31], have also set tolerance or maximum residue levels to ensure residues are not present in excess of the set tolerance levels and that no unapproved drugs are used. There are notably differences among MRLs or tolerances set by the different agencies. For instance, only the EU regulation permits the use of fluoroquinolones in fish (Table 2). The Codex Alimentarius Commission and Chile Health Department have established a MRL for flumequine in trout at 500 g kg1. On the other hand, the agencies have set different residue levels for the same drug. The MRL, for the sum of residues of tetracycline in fish, has been set at 100 g kg1 in the EU countries and Chile, at 200 g kg1 in Canada and Australia, and at 2,000 g kg1 in the US. Technical guidelines and performance criteria (detection level, selectivity, and specificity) for residue control in the framework of Directive 96/23/EC are described in Commission Decision 2002/657/EC [32], concerning the performance of analytical methods for the determination of organic residues and contaminants in living animals and animal products. According to these regulations, there is no obligation to use standardised methods in the residue control of food-producing animals. Indeed, it is necessary to provide for the progressive establishment of minimum required performance limits (MRPLs) of analytical methods, for substances whose use is not authorised or specifically prohibited in the Community. In March 2003,

Analysis of antibiotics in fish samples Table 1 Reviews about analysis of antibiotics in food samples Topic Veterinary antibiotics Veterinary antibiotics Antibiotics and antibacterial Legal restriction on veterinary drugs Veterinary drugs Basic drugs Antibacterials Antibiotics Antibiotics and growthpromoting agents Veterinary drugs and growth-promoting agents Antibiotics Quinolones Quinolones Sulfonamides Aminoglycosides Aminoglycosides and macrolides Technique Chromatography LCMS LCMS LCMS LCMSMS Multiresidue cation-exchange clean-up Several strategies Several strategies Several strategies Several strategies CE and CEC Pharmacokinetic studies using several strategies Several strategies Several strategies Several strategies Several strategies Sample Food-animal tissues Food-animal tissues Milk, cattle and kidney muscle, salmon, catfish Edible animal products Milk, cattle and kidney muscle, salmon, catfish Salmon, trout, farm animals Edible animal products Fish Edible animal products Edible animal products Food and feed samples Fish Food samples Edible animal products Milk and honey Fish and meat Ref. Robbitt [8] Niessen [9] Di corcia [10] Horie [11] Balizs [12] Stubbing [13]

989

Blasco [14] Samanidou [15] Stolker-Brinkman [16] Stolker-Zuidema [17] Castro-Puyana [18] Samuelsen [19] Andreu [20] Wang [21] Stead [22] McGlinchey [23]

the first MRPLs were published [33]. The MRPL was set at 0.3 g kg1 for chloramphenicol in meat, eggs, milk, urine, aquaculture products, and honey. For nitrofuran metabolites (furazolidone, furaltadone, nitrofurantoine, nitrofurazone) the MRPL was set at 1 g kg1 for poultry meat and aquaculture products. In comparison with other animal species, relatively few antibiotics are approved for use in aquaculture to date. In Fig. 1, the general structures of each family of antibiotics used in aquaculture are represented.

Chromatographic methods High-performance liquid chromatography (HPLC) is a versatile technique widely applied to quantify low levels of several residues in food. These chromatographic methods must be accomplished with confirmatory methods for organic residues or contaminants, which can provide information on the chemical structure of the analyte. Antibiotics used in veterinary practice have been classified above and are described in this paper. As was indicated in the Introduction, a review focussing on LCMS analysis of antibiotic and antibacterial agents in animal food [10] and, a second one, on the application of LCMSMS in the analysis of veterinary drug residues [12], have been recently published. In these reviews, most of the references are about milk, cattle and kidney muscle,

pork tissues, and a limited number are about salmon and catfish samples, but from 2000, the accelerated growth of aquaculture has resulted in a serious problem of contamination in fish food, and this has been extensively treated in the analytical literature. A liquid chromatographytandem mass spectrophotometry (LCMSMS) screening method has been developed targeting 23 pharmaceuticals and two metabolites, with different physicochemical properties, in fish tissues [34]. Detections limits were <6 ng g1 for most analytes. The method was subsequently used to screen for target analysis in fish from an effluent-dominant stream. Simultaneous determination of selected veterinary antibiotics in gilthead seabream, using liquid chromatographymass spectrometry, has recently been published [35]. Different classes of antibiotics, for example quinolones, tetracyclines, sulfonamides, and trimethoprim, were analysed in fish muscle and skin of medicated gilthead seabream.

Aminoglycosides Aminoglycosides are a large group of antibiotics that are particularly active against aerobic, Gram-negative bacteria, and act synergistically against some Gram-positive organisms. Aminoglycosides enhance bactericidal activity of betalactam drugs, making a useful combination for the treatment of serious infections. The aminoglycosides are used in

990 Table 2 MRL values for antibiotics in fish according to European Union legislation [24] Pharmacologically active substance Marker residue

F. Caada-Caada et al. MRL (g kg1)a 100b

Sulfonamides All substances belonging to the sulfonamide group Diaminopyrimidine derivatives Trimethoprim Penicillins Amoxicillin Ampicillin Benzylpenicillin Cloxacillin Dicloxacillin Oxacillin Quinolones Oxolinic acid Danofloxacin Difloxacin Enrofloxacin Flumequine

Parent drug

Parent drug

50

Amoxicillin Ampicillin Benzylpenicillin Cloxacillin Dicloxacillin Oxacillin Oxolinic acid Danofloxacin Difloxacin Sum of enrofloxacin and ciprofloxacin Flumequine

50 50 50 300 300 300 100 100 300 100 600 150 (salmonidae) 30 (salmonidae) 200 50 100 1000 50 100 100 100 100 300 500 500 150

Sarafloxacin Macrolides Erythromycin Tilmicosin Tylosin Florfenicol and related compounds Florfenicol Thiamphenicol Tetracyclines Chlortetracycline Oxytetracycline Tetracycline Lincosamides Lincomycin Aminoglycosides Spectinomycin Neomycin (including framycetin) Paramomycin Polymyxins Colistin Nitrofurans: No maximum levels can be fixed

Sarafloxacin Erythromycin A Tilmicosin Tylosin A Florfenicol Thiamphenicol Sum of parent drug and its 4-epimer Sum of parent drug and its 4-epimer Sum of parent drug and its 4-epimer Incomycin Spectinomycin Neomycin B Paramomycin Colistin

For fin fish these MRLs relate to muscle and skin in natural proportions

b Combined total residues for all substances within the sulfonamide group should not exceed 100 g kg1

veterinary medicine and animal husbandry, particularly for treatment of bacterial infections or for prophylaxis [36, 37]. Chemical structures of the most common aminoglycosides employed in aquaculture are shown in Fig. 1. A comprehensive review [22] of quantitative methods for the determination of aminoglycosides was published in 2000. Several methodologies were described and focussing

in the treatment of the samples. The use of solid-phase extraction (SPE) columns and cartridges has greatly increased the convenience and performance of sample preparation for HPLC analysis. Aminoglycosides are retained most successfully on weak cation exchangers or by ion-pairing on reversed phase media. Ion-exchange on carboxylic acid-bonded weak cation-exchange (CBA)

Analysis of antibiotics in fish samples Fig. 1 Chemical structures of the antibiotics used in aquaculture
H2N
NH2

991

O
O

S O

NHR

H2N

N O

Sulphonamides
R HN S O O OH O
5 4

Trimethoprim
O O

6 7 8 1

3 2

OH

N H

Penicillins

Quinolones

O F
5 6 7 8 1

4 3 2

OH

N HN

N H

Fluoroquinolones Macrolides: Erythromycin

is applied in SPE of neomycin and gentamycin from tissues. A method using liquid chromatography with tandem mass spectrometry was developed for the determination of 11 commonly used aminoglycosides in fish and other matrices [38]. Because the aminoglycosides in tissue are bound to proteins, harsh extraction conditions to liberate them from the matrix are required. These antibiotics are stable in the presence of acids, and protein precipitating agents, for example trichloroacetic acid, can be used for this purpose. The polar and cationic nature of aminoglycosides suggests the use of cation-exchange SPE instead of reversed-phase SPE. Aminoglycosides are readily adsorbed on a number of materials, and interactions with glass are strong. As a consequence, it is necessary to use plastic vials for the LC autosamplers. These compounds are weakly retained on a standard C18 column and the use of paired-ion chromatography is recommended. Heptafluorobutyric acid

has less effect on MS response than other volatile ionpairing agents. Spiked fish tissues at 20, 100, 500, and 1,000 g kg1 levels were analysed. Poor recoveries were obtained for several derivatives, for example neomycin. A recent review [23] focussing on extraction and cleanup methods, for example deproteination and solid-phase extraction, was reported for two antibiotic families, aminoglycosides and macrolides, in several food samples. The authors indicate the absence of UV chromophore or fluorophore groups for the aminoglycosides, which makes it necessary to use derivatising agents for detection by fluorescence. However, the derivatization steps render the analytical process more time-consuming and may even introduce impurities. Although light-scattering detection (ELSD) enables universal detection and it has been applied to several food samples, application in fish food analysis has not yet been reported. Mass spectrometry is currently the detection method of choice for aminoglycosides.

992 Fig. 1 (continued)

F. Caada-Caada et al.

Macrolides: Tilmicosin

Macrolides: Tylosin
OH
R1 R2 R3 R4 H OH N

R O O NH S
OH O OH OH O O NH2

O Cl Cl

R= F; Florfenicol R= OH; Thiamphenicol

R1=R4=H; R2=OH; R3=CH3; Tetracycline R1=H; R2=R4=OH; R3=CH3; Oxytetracycline R1=Cl; R2=OH; R3=CH3; R4= H; Chlortetracycline

Amphenicols Florfenicol is a broad-spectrum, primarily bacteriostatic, antibiotic with a range of activity similar to that of chloramphenicol. However, florfenicol does not carry the risk of inducing human aplastic anaemia that is associated with chloramphenicol. Chloramphenicol was the first antibiotic to be manufactured synthetically on a large scale. Chloramphenicol is associated with serious toxic effects in humans in the form of bone marrow depression, particularly severe in the form of fatal aplastic anaemia. Because of this, chloramphenicol has been banned for use in food-producing animals in many countries, including

the EU. Thiamphenicol and florfenicol were permitted as substitutes [39]. Although gas chromatography (GC) was, in the past, the analytical tool most often used to determine choramphenicol, florfenicol, and thiamphenicol in fish and shrimp samples, LCMSMS, without derivatization, is currently usually the tool of choice to determine antibiotic residues. From shrimp, the three amphenicols can be extracted with a basic ethyl acetateacetonitrile mixture, concentrated, and reconstituted with hexane. A C18 solid-phase extraction system was applied, and elution was with methanol. Sylon BFT was used as derivatizing agent and the analytes were determined by GC with electron capture detection. The

Analysis of antibiotics in fish samples Fig. 1 (continued)

O HO OH H H OH

993

ON2

CH

NR1

HN O OH SCH3

Nitrofurans

Lincosamides: Lincomycin
NH HO H O OH O

HN H OH

O H

Aminoglygosides: Spectinomycin
NH2 OH HO NH2 HO O O OH HO O H2N NH2 NH2 OH HO O O O NH2

Aminoglycosides: Neomycin

quantification limits were about 5 ng g1 for each analyte [40]. UV detection was proposed to determine florfenicol in plasma of catfish, lake trout, rainbow trout, and lake sturgeon, using an LC method [41] (quantitation limit <30 ng mL1). Chloramphenicol in seafood, fish, and shrimps was analysed by LCelectrospray ionization tandem mass spectrometry [42]. In 2003, an interesting multilaboratory validation method for chloramphenicol in shrimp and crabmeat, using LCMS was reported [43]. The

method was validated in three laboratories and satisfies the FDA and CVM (Center for Veterinary Medicine) US criteria for confirmation. Chloramphenicol in fish was analysed with APPI mass spectrometry [44], microcell electron capture detector mass spectrometry [45], SPELCESI MSMS [46], and GCMSNCI-SIM [47]. Florfenicol amine and florfenicol in fish can be quantified by LCUV [4850] and use of a GC microcell electron capture detector has also been proposed [51].

994 Fig. 1 (continued)

HO OH OH

F. Caada-Caada et al.

O H2N O NH2

O OH

NH2

HO OH

O H2N

NH2

HO OH

Aminoglycosides: Paronomycin
O HN O H2N O HN NH O H2N HO O O HN NH O NH2 N HN H N NH2 OH O O

NH H2N O NH

Polymyxins: Colistin

Fluoroquinolones and quinolones Quinolones and fluoroquinolones are an important family of synthetic antibacterials used in both human and veterinary medicine. In the veterinary field, they are used for the prophylaxis and for treatment of veterinary diseases in most types of farmed animals; they are also used in aquaculture [52]. The introduction of the fluorinated quinolones had important therapeutic advantages, because this antibiotic group has higher antibacterial activity than the parent compounds [53]. Their extensive administration to fish destined for human consumption has become a serious problem because their residues can persist in edible animal tissues. Quinolones may be directly toxic or be the source of resistant human pathogens representing a possible risk to human health [54]. Recently, Samuelsen et al., reported the pharmacokinetics of several quinolones in freshwater species and in sea water

species [19], for example oxolinic acid, flumequine, enrofloxacin, and sarafloxacin in channel catfish, eel, rainbow trout, Atlantic halibut, Atlantic cod, Atlantic salmon, and gilthead sea bream, among others. Maximum concentration and time to maximum concentration in plasma and tissues were established in each type of fish. HPLC is the most widely used analytical method applied to these compounds, and UV or fluorescence detection are usually employed. The high fluorescence quantum yield of fluoroquinolones enable use of highly sensitive analytical methods. Different methods to analyse several quinolonic and cinolonic antibiotics in trout, using HPLC fluorescence detection, have been compared [55]. The Initial step of extraction of these antibiotics from trout tissues is with sodium hydroxide solution; subsequently, a cleaning stage based on liquidliquid partition with chloroform is performed. Oxolinic acid and flumequine in salmon muscle and skin [56] and freeze-dried salmon

Analysis of antibiotics in fish samples Table 3 Chromatographic methods for determination of antibiotics in fish (from 2000) Analyte Aminoglycosides Spectinomycin Streptomycin Dehydrostreptomycin Amikacin Kanamycin B Paromomycin Apramycin Tobramycin Sisomycin Neomycin B Gentamycin C1 Amphenicols Chloramphenicol Florfenicol Florfenicol amine Thiamphenicol Sample Pretreatment Characteristics LOQ/LOD Ref.

995

Fish Pork muscle Veal liver

Low-pH extraction with trichloroacetic acid. Anionexchange step to remove the acid from the centrifuged extract. Retention in a weak cation-exchange solid-phase (SPE) cartridge

LC: C18 column using heptafluorobutyric acid as volatile ion-pairing agent

LOD (g kg1): 1540

Kaufmann [38]

MS Shrimp tissue Shrimp samples are extracted with basic ethyl acetate and an acetonitrilebasic ethyl acetate mixture, centrifuged, and evaporated. Sample clean-up by propylsulfonic acid and C18 SPE system. Analyte isolated from the plasma by C18 solid-phase extraction GC Derivatizing agent: Sylon BFT LOD (ng g1): 0.7 (Chloram.); 1.4 (Flor.); 2.4 (Flor. amine); 1.3 (Thiamp.) LOQ (ng mL1) 19-29 Pfenning [40]

Florfenicol

Channel catfish Lake sturgeon Lake trout Rainbow trout Striped bass hybrid Channel catfish

RP-LC

Vue [41]

UV detection

Channel catfish

Fish Shrimp Chloramphenicol Shrimp Fish Meat

Shrimp Crabmeat

Fortifies samples were extracted with 6 mol L1 HCl in shaking water bath set at 100 C for 3 h. After extraction with ethyl acetate, NaOH 30% solution was added to the hydrolysate to adjust the pH to 12.5. SPE extraction was used. Skinless catfish were homogenized, then frozen (40 C) for 24 h. 2 g catfish muscle was hydrolysed with 6 mol L1 HCl at 100 C (3 h) and extracted with ethyl acetate followed by basification. Extraction with acetonitrile and ethyl acetate (twice), defatted with hexane and clean-up by solid-phase extraction using an Oasis MCX cartridge. Sample in dry, powered form is liquidliquid extracted with ethyl acetatediethyl ether; d5chloramphenicol was used as internal standard. Clean step in a silica gel solid-phase extraction cartridge. Extracted with ethyl acetate, concentrated and evaporated.

LC (C8 column) with UV detection

LOD (g kg1): Wrzesinski 0.044 [48] LOQ (g kg1): 0.075

LC (with UV detection)

LOD (ng g1): 44 Wrezsinski [50]

GCECD

LC Electrospray ionization tandem mass spectrometry in negative-ion mode

LOD (ng g1): 0.5 LOQ (ng g1): 1.5 Decision limit: 0.01g kg1 Detection capability: 0.02g kg1

Zhang [51]

Mottier [42]

RP-LC andem triple quadrupole mass spectrometry (multilaboratory validation)

0.3g kg1

Hammack [43]

996 Table 3 (continued) Analyte Sample Yellowtail Flatfish Pretreatment Characteristics

F. Caada-Caada et al.

LOQ/LOD LOD (ng g1): 0.1 and 0.27

Ref. Takino [44]

Hairtail Yellow croaker Grass carp Silver carp Shrimp (some types) Shrimp

Extracted with acetonitrile and LC Atmospheric pressure evaporated to dryness followed by a clean-up step using a distribution photoionization (APPI) mass between acetonitrilehexane. spectrometry Extracted with ethyl acetate, defatted GC with hexane and derivatized with Microcell electronSylon BFT capture detection

LOD (ng g1): 0.04 LOQ (ng g1): 0.1

Ding [45]

Extraction with ethyl acetate, evaporated to dryness and clean-up using SPE (Oasis HLB) cartridge conditioned with methanol. Shrimp meat was extracted with ethyl acetatephosphate buffer (pH 6.88), centrifuged and the organic phase was reduced to dryness with a stream of nitrogen. The defatting extraction with hexane was then made and a second extraction with ethyl acetate was carried out. Tissue minced and homogenised was treated with citric acid + EDTA, Centrifuge (10 min) and then the supernatant was evaporated (under N2 stream).

RP-LC ESI in negative-ion mode MSMS HPLC, GC, GCECD GCESI-SIM and ELISA methods.

LOD (ng mL1) : Gikas [46] 0.1 LOQ (g kg1):0.02 LOD (for GC MS, (g kg1): 1.0

Yellowfin tuna White shrimp Yellow eel Turtle and other food samples Fluoroquinolones/quinolones Oxolinic acid Gilthead seabream Flumequine Fish muscle, Oxytetracycline Skin Sulfadiazine Trimethoprim

Shen [47]

Atlantis dC18 150 2.00 mm. Gradient mode LCMS Electrospray ion source (ESI) in positive-ion mode

Nalidixic acid Oxolinic acid Pipemidic acid Piromidic acid Cinoxacin Flumequine

Trout muscle

Method A: Extraction in chloroform LC (C18 column) from tissue muscle fluorescence Method B: solidliquid extraction with detection sodium hydroxide and cleaning by liquidliquid partition in chloroform Extracted with NH3 and acetone, acidified, diluted and analysed. LC

LOQ<20g kg1 Romerofor all analytes Gonzlez LOD (g kg1): 7 [35] (oxolin. acid); 8 (flum.); 4 (oxitetr.); 6 (sulf.); 4 (trimet.) DurnMers [55]

Fish muscle and skin Freeze-dried salmon muscle

Oxolinic acid

Oxolinic acid Flumequine Sarafloxacin Ciprofloxacin Enrofloxacin

Salmon Trout muscle

Solidliquid extraction procedure: after LC (PuroSpher RPaddition of hydrochloric acid, 18E)fluorescence extraction detection used successively ethyl acetate, sodium hydroxidem and chloroform Extraction with acetonitrile basic LCfluorescence solution, centrifugation, evaporation detection and cleaning with hexane PLRP-S column

LOQ (g kg1): 30 (flum); 20 (oxolin. acid) LOD (ng g1): 3.2 LOQ (ng g1) 16.6 LOD (g kg1): 2 (sara.); 5 (oxolin. acid) 7 (flum.) No data

Hormazabal [56] Pouliquen [57]

Roudaut [58]

Catfish Shrimp

Extraction with acidified ethanol, solid-phase extraction and elution

LCfluorescence detection

Roybal [59]

Analysis of antibiotics in fish samples Table 3 (continued) Analyte Sarafloxacin Difloxacin Ciprofloxacin Enrofloxacin Sample Salmon Pretreatment with basic methanol. LOD (ng g1): 5 (for Sara., 10) Characteristics LOQ/LOD Ref.

997

Sarafloxacin Flemuquine Oxolinic acid Ciprofloxacin

Salmon muscle Clean-up by Discovery DS-18 cartridge LCfluorescence from homogenized samples in phosdetection Run 1: mobile phase phate buffer (pH 7.4). 18:82 acetonitrile buffer (pH 3.0) for cipro + enro + sara Run 2: mobile phase 34:66 acetonitrile buffer (pH 3.0) for oxo + flume

Ramos [60]

Enrofloxacin Sarafloxacin

Fish and other animal tissues

Extraction with acidic aqueous solution, LC (C18 narrow bore column) centrifugation, and filtration before fluorescence injection. detection Gradient mode

Difloxacin Norfloxacin Marbofloxacin Danofloxacin Flumequine Oxolinic acid Nalidixic acid Flumequine

LOD (g kg1): 411 LOQ (g kg1): 1336 CC (g kg1): 41154 CC (g kg1): 53207

Verdon [61]

Freshwater fish

Ciprofloxacin Enrofloxacin Sarafloxacin Danofloxacin Orbifloxacin Flumequine Oxolinic acid Piromidic acid Ciprofloxacin Enrofloxacin

Fish muscle Trout Abalone

Extraction with acetonitrile for 15 min (ultrasonic accessory), evaporated to dryness under N2 stream and defatted by liquidliquid partition with hexane. Extraction with acetonitrile and disruption using sonic probe. Evaporation to dryness under nitrogen stream. SPE clean-up using ENVI Chrom P cartridges.

LC (PLRP column) with fluorescence detection LC (C18 column) Gradient mode MSMS detection

LOD (g kg1):1 Kirbis [62]

LOD (g kg1): 13

Johnston [65]

Salmon

Sarafloxacin Norfloxacin Difloxacin Danofloxacin Enofloxacin Lomefloxacin Marbofloxacin Flumequine Oxolinic acid Nalidixic acid Pipemidic acid

Extraction with 75:25, 0.3% phosphoric acidacetonitrile, centrifugation and filtration. SPE clean-up using ENV + Isolute cartridge. Hexane was used to defat. The analytes were eluted with 25:75, 2% TFAacetonitrile.

LC (C18 column) gradient mode

Diode-array (DAD) fast-scanning fluorescence detection (FSFD)

LOD (g kg1): 0.28.9 and LOQ: 0.716.6 for fluorescent species LOD (g kg1): 8.730.0 and LOQ : 2932 for absorbing species

EspinosaMansilla [64]

998 Table 3 (continued) Analyte Piromidic acid Ciprofloxacin Enrofloxacin Sarafloxacin Difloxacin + six sulfonamides + amphenicols Norfloxacin Ciprofloxacin Sarafloxacin Danofloxacin Enrofloxacin Orbifloxacin Difloxacin Desethyleneciprofloxacin Oxolinic acid Flumequine Nalidixic acid Enrofloxacin Oxolinic acid Flumequine + erythromycin + dyes Norfloxacin Ofloxacin Danofloxacin Ciprofloxacin Enrofloxacin Sarafloxacin Difloxacin Oxolinic acid Flumequine Nalidixic acid Piromidic acid Ciprofloxacin Enrofloxacin Sarafloxacin Danofloxacin Oxolinic acid Nalidixic acid Flumequine Macrolides Analyte Erythromycin Oleandomycin Kitasamycin Josamycin Mirosamycin Spiramycin Shrimp Trout Salmon Sample Pretreatment Characteristics

F. Caada-Caada et al.

LOQ/LOD

Ref.

Salmon Shrimp

Sample was homogenized with acetic acidethanol and extracted with a propyl sulfonic acid solid-phase cartridge. The analytes were eluted with basic methanol and evaporated. Tissues were extracted with ammoniacal acetonitrile and the extract was defatted, evaporated, and the residue dissolved in basic phosphate.

LC (Inertsil phenyl ) Ion trap-MSn detection

Confirmatory method

Turnipseed [66]

Shrimp

LC (XDB-phenyl column) fluorescence detection and MS2 or MS3 confirmation.

LOQ (ng g1): 0.11.0

Schneider [67]

Shrimp

Salmon

Samples were extracted with ethyl acetate followed by solvent exchange into 0.2% formic acid and clean-up with hexane. Extraction method based on solid liquid extraction (SLE). Salmon was homogenized with acetonitrile, centrifuged and additional clean-up by addition of 0.2 g Bondesyl-NH2. The supernatant was evaporated to dryness and reconstituted with mobile phase. Tissues are cleaned, filleted, and homogenized with dry ice. Drugs are extracted using a mixture of ethanolacetic acid (1%), diluted in aqueous HCl and defatted by extraction with hexane. The analytes are isolated using cationexchange solid-phase extraction.

LC- fluorescence Electrospray-MS3 confirmation LCtime of flight (TOF) MS

LOD (ng g1): 2.33.0 LOQ (ng g1): 6.99.0 LOD (g kg1): 22.5 LOQ (g kg1): 7.510 CC (g kg1): 103218 CC (g kg1): 107234 LOD ( ng g1): 0.11.0

Karbiwnyk [68]

Hernando [69]

LCMSMS Triple-quadrupole mass spectrometer with electrospray

Dufresne [70]

Gilthead seabream

Extraction with NaOH (0.1 mol L1) and purification by solid-phase extraction (SPE) on Waters Oasis HLB cartridges

LCMSMS Electrospray ionization (ESI) Perfectil ODS analytical column in gradient mode

LOD (g kg1): 22.3 LOQ (g kg1): 68

Samanidou [71]

Sample Fish

Pretreatment Sample was homogenized at high speed with metaphosphoric acid with methanol as deproteinating extractant. The filtrate was evaporated at 40 C (under reduced pressure) and passed through an Oasis HLB cartridge.

Characteristics LCMS Electrospray ionization (ESI and selected ion monitoring (SIM)) TSLgel Super ODS

LOQ/LOD LOQ (g g1): 0.01

Ref. Horie [73]

Analysis of antibiotics in fish samples Table 3 (continued) Analyte Tilmicosin Tylosin Erythromycin Sample Pretreatment Characteristics column with gradient mode Trout Sample liquid extraction (acetonitrile) and defatting with hexane LCMSMS-ESI Reversed-phase gradient mode CC (g kg1): 220 CC (g kg1) LOQ/LOD Ref.

999

Lucchetti [74]

Erythromycin Josamycin Roxithromycin Spiramycin Tilmicosin Tylosin Troleandolycin Nitrofurans Furazolidone + 3-amino-2-oxazolidinone (AOZ)

Fish

Triple quadrupole 238 mass spectrometer with turbo ion spray in positive-ion mode Pressurized liquid extraction (PLE) with LC-MS LOQ (g kg1): 50 methanol at 80 C (pressure 1500 psi) Electrospray ionization (ESI)

Houda Berrada [75]

Fish

Solid-phase extraction (SPE)

Farm fish

Extraction with dichloromethane

Nitrofuran metabolites

Shrimp Fish

Extraction/derivatization step Solid-phase extraction clean-up with polymer phase cartridge Extraction/derivatization step Liquidliquid extraction clean-up

LC Hypersil ODS column Derivatizing agent: 2nitrobenzaldehyde UVvisible detection HPLCUV and LCESI- LOD (ng g1): MSMS 0.4 (furazolidone) and 0.05 for AOZ LCMSMS

Connely [76]

Hu [82]

Edder [78]

Nitrofuran metabolites

Shrimp

LOD (g kg1): 0.10.3 LOQ (g kg1): 0.10.5 Derivatization with 2-naphthaldehyde LCDAD LOD (g kg1): (NTA) ChromSpher 5 Pesticide 0.210.27 Liquidliquid extraction with ethyl (2504.6 mm, 5 m) LOQ (g kg1): 0.690.91 acetate, evaporation and redissolution column at 40 C of the residue in acetonitrilewater gradient mode LCAPCI-MSMS Ultrasphere OS UV detection, 214 nm LOD (g g1): 0.05

Chu [79]

Chumanee [83]

Penicillins Penicillins

Catfish Extraction with acetonitrile Salmon (and other food products of animal origin) Trout muscle Muscle

Samanidou [85]

Sulfonamides Sulfonamides (13) Sulfonamides (10)

Matrix solid-phase dispersion technique LCESI-MS with hot water Liquid extraction with ethyl acetate and HPLCDAD at 270 nm clean-up with cation-exchange solid- C8 column in gradient phase extraction mode Solid-phase extraction HPLC post-column derivatization fluorescence detection Several LCMS

LOQ (g kg1): 313

Bogialli [91] Pecorelli [92]

Sulfonamides (14)

Catfish Shrimp Salmon Salmon

Sulfonamides (review)

LOQ (ng g1): 5 Gehring (cat fish), 10 [93] (shrimp) and 20 (salmon) Wang [21]

1000 Table 3 (continued) Analyte Sample Pretreatment Characteristics TLC HPLC and others

F. Caada-Caada et al.

LOQ/LOD

Ref.

Sulfonamides (17) + ormetoprim Tetracyclines Oxytetracycline Tetracycline Oxytetracycline

Shrimp and other animal products Salmon

Liquid extraction with water acetonitrile, defatting with hexane, and extraction with chloroform EDTAMcIIvaine buffer and clean-up by solid-phase extraction with a polymeric sorbent EDTAMcIIvaine buffer and trichloroacetic precipitation of proteins EDTAMcIIvaine buffer, acid precipitation of proteins, SPE clean-up (Strata cartridge) Liquid extraction with acetonitrile citrate buffer containing Mg(II) Monolithic capillary as extraction medium, EDTAMcIIvaine buffer at pH 4 No protein precipitation.

LCMSMS C18 column

LOD (ng g1): 0.10.9

Potter [94]

Salmon

Fish muscle

HPLC post column derivatization with fluorescence detection HPLC with UV detection

LOQ (g kg1): 50 LOQ (g g1): 0.04

Pena [98]

Coyne [99]

Oxytetracycline

Salmon

Tetracyclines Fluoroquinolones Tetracycline Oxytetracycline Chlorotetracycline Doxyciline

Catfish Fish muscle

HPLC mobile phase containing Mg(II) Fluorescence detection HPLC LOQ (ng g1): Fluorescence detection 0.151.5 In-tube solid phase LOD (ng g1): microextraction coupled 22, 16, 30 and with HPLC 21

Rupp [100]

Schneider [101] Wen [102]

muscle [57] have also been determined by HPLC with fluorescence detection. Oxolinic acid and flumequine up to 600 g kg1 and sarafloxacin up to 120 g kg1 can be determined by HPLC and fluorescence detection; previous extraction from muscle was by use of basic acetonitrile solution [58]. Four fluoroquinolones were determined in catfish, shrimp, and salmon by HPLC with fluorescence detection, using isocratic elution with acetonitrileacetic acid (2%) and a PLRP-S polymer column. Extraction from fish uses acidified ethanol and retention on a cation-exchange solid-phase column [59]. Many papers describe use of HPLC with fluorescence detection for analysis of these antibiotics in different fish samples [60 63]. In salmon samples, fourteen fluoroquinolones and quinolones can be determined by use of an RP-C18 column and gradient HPLC with fluorescence detection [64]. In order to unambiguously identify animal drug residues in a matrix, structural information can be obtained from mass spectral analysis coupled with a chromatographic system. Hence, HPLC coupled with MS detection is being widely applied. Eight quinolones and fluoroquinolones have been determined in trout, prawns and abalone, using HPLC with electrospray ionisation tandem MS. Limits of detection were 1 3 g kg1 depending on the analyte and matrix [65]. Ion-trap liquid chromatographymass spectrometry (LCMSn) has been shown to be a valuable tool for confirmation of animal drug residues in aquaculture products, for example salmon tissues [66]. A multiresidue method for analysis of fluoroqui-

nolones in shrimp samples has been developed, by conjunction of fluorescence detection (LCFL) for quantification and multiple stage mass spectrometry (MSn) for confirmation studies [67], and LCFL and MSn has also been applied to determination of quinolones in shrimp samples [68]. Liquid chromatography with time-of-flight mass spectrometry (LC TOF-MS) has been used to quantify and confirm enrofloxacin, oxolinic acid, flumequine, erythromycin, and other drugs in edible portions of salmon [69]. Recently, multiresidue determination of seven quinolone antibiotics in gilthead seabream [70] and quinolones and fluoroquinolones in fish and shrimp [71], using LCtandem mass spectrometry, have been reported. LCtandem mass spectrometry seems to be the current analytical method of choice for routine quality control of quinolones/fluoroquinolones in fish.

Macrolides Macrolides are the most effective medicine against diseases produced by mycoplasmas and have been widely used in the rearing of food-producing animals, including fish, to prevent and treat diseases. The antimicrobial spectrum of macrolides is slightly wider than that of penicillins and, therefore, macrolides are a common substitute for patients with a penicillin allergy [72]. Determination of macrolide antibiotics is mainly carried out by microbiological assays. For high-performance liquid

Analysis of antibiotics in fish samples Table 4 Other methods used to determine antibiotics in fish Analyte Sample Pretreatments Characteristics LOQ/LOD Ref.

1001

Capillary eletrophoresis Oxolinic acid Feed and cultured Solid-phase extraction (SPE) fish samples procedure Electrolyte: buffer solution (10 mmol L1 phosphate, pH 9.0) and methanol (9:1) Danofloxacin Fish samples SPE procedure taken from local Enrofloxacin Electrolyte: markets Flumequine 60 mmol L1 (NH4)2CO3 at pH Ofloxacin 9.2 Pipemidic Sulfamethazine Fish samples Solvent extraction/deproteination taken from Sulfathiazole Dispersive SPE clean-up markets, Sulfadiazine Electrolyte: slaughterhouses, Sulfachloropyridazine 60 mmol L1 ammonium and fish farms Amoxicillin acetate (pH 8.0) and 10% methanol Ampicillin Oxacillin Penicillin V Clanofloxacin Enrofloxacin Ofloxacin Flumequine Norfloxacin Fish muscle Mobile phase: samples Fleroxacin 27% ACN, 5 mmol L1 Na2HPO4 buffer (pH 4.0), 11 Ciprofloxacin mmol L1 SDS and 0.01% Lomefloxacin TEA Enoxacin Ofloxacin Gatifloxacin Enrofloxacin Eel liver samples Extraction with dichloromethane Ciprofloxacin Mobile phase: 20 mmol L1 phosphate buffer solution pH 8.00 Oxytetracycline Raw and cooked Sep-Pack C18 SPE procedure Mobile phase: channel cat fish 0.2 mol L1 phosphate buffer of pH 2 ELISA Sarafloxacin Fish and Shrimp 1:1 mixture of methanol and samples phosphate buffer pH 7.4 Norfloxacin Difloxacin Ciprofloxacin Pefloxacin Ofloxacin Cinoxacin Danofloxacin Enrofloxacin Marbofloxacin Lomefloxacin Enoxacin Flumequine

CE with UV detection at 254 nm

LOD: 0.08 g mL1 Saad Linear range: 0.540 [103] g mL1

CE with MS detection using LOD 20 ng g1 single-quadrupole and multiple-stage MS using an ion-trap (IT) quadrupole CEion trap (IT) MSMS LOD below 18 ng kg1 LOQ below 50 ng kg1

JuanGarca [104]

JuanGarca [105]

Pressurized-CEC UV detection at 287 nm

CZE with end-column amperometric detection

LOD (mg L1): 0.2 (nor.) 0.4 (fle.) 0.4 (cipro.) 0.5 (lome.) 0.8 (oflo.) 1.0 (gati.) LOD (mg kg1): 13.68 (enro.) 14.35 (cipro.)

Lu [106]

Wang [107]

CE with UV detection at 265 nm

Huang [108]

Direct screening ELISA assay

Assay detection Capabilities (CC): <10 g kg1 for most of them except: Sarafloxacin <4 Oxolinic acid <25 Flumequine <100 Cinoxacin <200

Hiet [109]

1002 Table 4 (continued) Analyte Sample Pretreatments Characteristics

F. Caada-Caada et al.

LOQ/LOD

Ref.

Oxolinic acid Nalidixic acid Sulfisozole Fish muscle samples Sulfathiazole Sulfameter Sulfamethoxypyridazine Sulfapyridine Sulfamethizole Sulfachloropyridazine Chloramphenicol Shrimp, eel, yellow-fin tuna and crab meat samples 3-Amino-2Shrimp samples oxazolidinone (furazolidone metabolite) 3-Amino-2oxazolidinone (furazolidone metabolite) Bioluminescence Tetracycline Oxytetracycline Chemiluminescence Tetracycline Tetracycline Fish muscle samples

1:5 dilution of fish extract with 1% bovine serum albumin (BSA)PBS

Direct screening ELISA assay

LOD <100 g kg1

Zhang [110]

Extraction with phosphate buffer Direct screening ELISA solution (PBS, pH 6.88)ethyl assay acetate, defatting with hexane Protease addition, hydrolysis, hydrochloric acid, derivatization in acid with o-nitrobenzaldehyde Hydrolysis, hydrochloric acid, derivatization with benzaldehyde in methanol solution, Sep-Pack MAX SPE procedure Bioluminescent Escherichia coli K12 strain sensor bacteria LuminolH2O2 Direct screening ELISA assay

LOD: 0.110 g kg1 LOD: 0.4 g kg1

Shen [47]

Cooper [111] Diblikova [112] Chang [113]

Indirect competitive screening (ic-ELISA) assay

LOD: 0.30.4 g kg1

Rainbow trout samples

Luminescence detection

LOD: 20 g kg1 (tetra.) 50 g kg1 (oxy.) LOD: 1.7108 g mL1 LOD: 1109 g mL1

Pellinen [114]

Fish and shrimp samples

Fish samples

Molecularly imprinted on-line solid-phase extraction Ce(IV) + rhodamine B Alkaline degradation derivatization on alkalineactivated solid silica gel G plates

Micro-flow injection system on a chip with chemiluminescence detection Flow-injection with chemiluminescence detection

Zhang [115]

Xiong [116]

Fluorescence Tetracycline Oxytetracycline Chlorotetracycline

Fish muscle samples

Solid-surface fluorescence LOD: 16 g kg1 with CCD camera detection LOQ: 53 g kg1

Sun [117]

chromatography (HPLC) of macrolide antibiotics, the most popular detector is a UV spectrophotometer. Some macrolides, for example tilmicosin (TLM) and spiramycin (SPM), have relatively strong UV absorption, however, erythromycin (EM) does not have a specific UV chromophore. Liquid chromatography mass spectrometry (LCMS) is the most promising technique for separation and determination in fish and other food samples. Eight macrolides at the 0.01 g g1 level in fish have been determined using LC ESI MS detection [73]. Erythromycin in trout can be determined by LCtandem mass spectrometry after previous liquidliquid extraction [74]. Decision limit (CC) and detection capability (CC) were calculated to be 220 and 238 g kg1, respectively. Seven macrolides in fish were

determined by using pressurised liquid extraction (PLE) and liquid chromatographymass spectrometry with electrospray ionization (LCESI MS) [75]. The results reveal PLE is a rapid quantitative technique for use with small initial sample sizes.

Nitrofurans Nitrofuran antimicrobials, for example furazolidone, had been widely used on food-producing animals, prior to their prohibition within the EU, because of their potential harmful effects on human health. Nitrofurans are included in Annex IV of the European Union Council Regulation

Analysis of antibiotics in fish samples

1003

[24] that lists banned substances. For nitrofuran metabolites the MRPL was set at 1 g kg1 in aquaculture products. Illegal use of furazolidone can be monitored most effectively by testing for bound residues containing the 3-amino-2-oxazolidinone (AOZ) moiety which, unlike the parent drug, is stable and can be detected for prolonged periods after cessation of treatment. The development of an extraction and clean-up procedure for this metabolite from liver, using solid-phase extraction, and HPLCUV determination of residues of furaltadone, furacilin, nitrofurantoin, and furazolidone in fish and shrimp by SPEHPLC, have been reported [76, 77]. Analysis of nitrofuran metabolite residues in shrimp have also been performed [78, 79]. The methods are based on an extraction/derivatization step, followed by SPE [78] or liquidliquid extraction clean-up [79]. The separations were carried out by liquid chromatography coupled with tandem mass spectroscopic detection. Other HPLCMS methods focussing on determination of metabolite residues of the banned nitrofurans, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone in shrimps has been reported [80]. Sample preparation involved hydrolysis and derivatization with nitrobenzaldehyde overnight at 37 C, extraction of the nitrophenyl derivatives, evaporation, and reconstitution of the sample before injection. An HPLCMSMS method for determination of nitrofuran metabolites, AOZ, 1-aminohydantoin hydrochloride (AHD), 3-amino-5-morphinomethyl-2-oxazolidinone (AMOZ), and semicarbazide (SEM) in fish and shrimps, has been reported [81]. Determination of residual furazolidone and its main metabolite (AOZ) in feed fish, using HPLCUV and LCMSMS, has been accomplished [82]. Recently, the synthesis of derivatives of metabolites from furazolidone, furaltadone, nitrofurazone, and nitrofurantoin, using a new derivatizing reagent, 2naphthaldehyde (NTA), has been described. The reaction product was analysed by liquid chromatography with diode-array detection (LCDAD), to determine nitrofuran metabolites in shrimp after two steps of liquidliquid extraction [83].

Several reviews including penicillins can be found in the literature covering the last two decades, as has been indicated in the introduction of this review. Recently, Samanidou et al. published an interesting review on HPLC analysis of penicillin residues in food products of animal origin [85]. Amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin, and bencilpenicillin are controlled by the EU, and maximum residue limits are fixed in edible animal tissues. However, specific methods to determine penicillins in fish samples by HPLC are very limited. Samanidou reports that penicillin G was determined in Chinook salmon by using acetonitrile (ACN)phosphate buffer as mobile phase, using a previous SPE procedure [86], and ampicillin in catfish [87] by HPLC with fluorescence detection. Later, in 2007, a general method to determine penicillin G in feed by HPLC, with solid-phase extraction, was reported [88].

Sulfonamides The sulfonamide group includes a large number of compounds which have been used for a very long time, including sulfadiazine, sulfamethizole, sulfamethoxazole, sulfasalazine, sulfisoxazole, and various high-strength combinations of three sulfonamides. At present, sulfonamides and other drugs (chlortetracycline, penicillin, and several ionophores) are the most common contaminating antimicrobials in animal feed, generating potentially serious problems in human health, for example allergic or toxic reactions. In addition, some sulfonamides have been found to be potentially carcinogenic and this fact has become a cause for considerable debate in food safety [89, 90]. As summarized in Table 2, the EU has set an MRL for total sulfonamide concentration in fish at 100 g kg1. A liquid chromatographymass spectrometry assay for analysing sulfonamides in fish muscle has been reported with an estimated limit of quantification of 313 g kg1 in trout fillet [91]. This method is based on the matrix solidphase dispersion technique, with hot water as extractant, followed by liquid chromatographymass spectrometry (LCMS). Ten sulfonamides can be determined in muscle samples by application of a multiresidue method, according to European Union regulation 2002/657/EC, using HPLC and diode-array (DAD) detection. CC and CC are described for each sulfonamide [92]. Multiresidue determination of 14 sulfonamides in catfish, shrimp, and salmon, using a post-column fluorescence derivatization HPLC method, was recently reported [93]. The method was validated at 5, 10, and 20 ng g1 levels. A review, including HPLC, LCMS, GC, and other methods, has been published about the analysis of sulfonamides in edible animal products [21]. Data for sulfamonomethoxine and sulfadiazine in salmon and shrimps were obtained by use of

Penicillins -Lactam antibiotics consist of a broad class of antibiotics which include penicillins, cephalosporins, monobactams, carbapenems, and -lactamase inhibitors. They are probably the most widely used groups of antibiotics in veterinary medicine for treatment of bacterial infections of animals used in livestock farming [84]. According to EU Council Regulation 2377/90/EC [24], penicillins are the -Lactam antibiotics licensed for aquaculture. In Table 2, suitable penicillins and their MRLs are summarized.

1004

F. Caada-Caada et al.

HPLC. Seventeen sulfonamides and the potentiators, ormetoprim and trimethoprim, in salmon, were quantified by LCtandem mass spectrometry [94]. The limit of detection varied from 0.10.9 ng g1.

Tetracyclines Tetracyclines are broad-spectrum antibiotics widely used in human and veterinary medicine and feed additives to treat and prevent diseases. They are also used for promoting growth in the farming industry. Tetracycline, chlortetracycline, doxytetracycline, and oxytetracycline are the tetracyclines most widely used with food-producing animals. The intensive culture of aquatic organisms involves the risk of outbreaks of infectious diseases, because of uncontrolled application of several antibiotics agents, with special emphasis of tetracycline derivatives [95, 96]. Oxytetracycline an tetracycline are widely used in salmon treatment. The EU established its maximum residue limit as 100 g kg1 for muscle tissue, including salmonidae and finfish. Chromatographic analysis of tetracycline antibiotics in food was reviewed by Oka et al. [97], including some references to fish tissue analysis. Later, several papers about analysis of tetracylines, using chromatographic systems, were published. Oxytetracycline and tetracycline residues can be extracted from salmon with polymeric sorbent. A liquid chromatographic post-column derivatization method, with fluorescence detection, was optimized and validated for salmon fortified at 50, 100, and 200 g kg1 levels [98]. Oxytetracycline in fish muscle was determined by a photometric HPLC method, with liquidliquid extraction and precipitation of proteins in previous steps [99]. The ability of the fluoroquinolones to form fluorescent metal complexes has been used to develop a fluorimetric HPLC method, by detection of the oxytretracycline-Mg derivative [100]. The proposed method was used to analyse farmed Atlantic salmon muscle tissues. In catfish muscle, tetracyclines and fluoroquinolones can be analysed using a fluorescent HPLC method, after extraction with ACN and citrate buffer containing magnesium chloride [101]. In-tube solid-phase microextraction, coupled with HPLCDAD, has been reported for simultaneous analysis of four tetracyclines in fish [102]. The analytes were extracted with EDTA MacIlvaine buffer then injected directly for analysis. Most published methods used sample pre-treatment for determination of antibiotic residues in fish samples. Liquidliquid extraction (LLE) and/or liquidsolid extraction (LSE), alone or in combination with clean-up by solidphase extraction (SPE), are the procedures most often used. Dufresne et al. [70] described liquidsolid extraction of eight fluoroquinolones from fish tissue with a mixture of ethanol and 1% acetic acid, diluted in aqueous HCl, after

defatting by extraction with hexane. The compounds were further isolated by use of cation-exchange solid-phase extraction. After evaporation and reconstitution steps the samples were analysed by LCMSMS. Recoveries were in the range 5796% depending on analyte and commodity. Verdon et al. [61] employed a simpler procedure to extract ten fluoroquinolones from animal tissues. It involved extraction of the residues from animal tissues with acidic aqueous solution (5% trichloroacetic acid), centrifugation, and filtration. The filtered extract was analysed by LC with fluorescence detection. In this case, recoveries, which ranged from 2168%, were poorer than those in the previous extraction procedure. Schneider et al. [101] proposed a multiresidue method for simultaneous determination of five fluoroquinolones and three tetracyclines in catfish muscle. The method involved solid liquid extraction of the analytes with a 1:1 (v/v) mixture of acetonitrile and citrate buffer containing magnesium. The supernatant was centrifuged and evaporated, at 40 C under a stream of nitrogen. The residues were then resuspended and filtered for analysis by LC with fluorescence detection. The presence of magnesium ions both improved the recovery of fluoroquinolones and promoted the fluorescence of the tetracyclines. The recoveries ranged from 6092%. The application of liquidsolid extraction combined with of propylsulfonic acid (PRS) and C18 SPE columns in tandem was proposed by Pfenning et al. [40] for simultaneous determination of residues of amphenicol drugs in shrimp. The polar component, florfenicol amine, is retained on the PRS SPE column whereas the nonpolar components chloramphenicol, florfenicol, and thiamphenicol were retained on the C18 SPE column. The C18 SPE column was eluted with methanol and the PRS SPE column was eluted with basic methanol plus counter ion. The combined eluates were evaporated, reconstituted in acetonitrile, and derivatized with Sylon BFT. The analytes were determined by GC with electron-capture detection. The recoveries ranged from 84 to 101%. In Table 3 are summarized chromatographic methods to determine antibiotics in fish and procedures for antibiotics extraction.

Other methods Capillary electrophoresis The lack of volatility and/or thermal instability of many antibiotics does not permit their analysis using GC; HPLC is the technique of choice for their analysis. However, capillary electrophoresis (CE) techniques, together with other working modes, for example micro emulsion electrokinetic capillary chromatography (MEEK), capillary electrochromatography (CEC), cyclodextrin-modified elec-

Analysis of antibiotics in fish samples

1005

trokinetic capillary electrophoresis (CD-EKC) or nonaqueous capillary electrophoresis (NACE), are alternatives to HPLC methods for analysis of antibiotics. Capillary electrophoresis has inherent characteristics as analytical tool, for example speed of analysis, high efficiency, separation selectivity, small sample size capability, and low reagent consumption. However, very limited work has been reported on the application of CE to the analysis of antibiotics in fish samples. Castro-Puyana et al. [18] published a review on recent developments in the determination of antibiotics by CE and CEC. The most employed CE separation modes were capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), although MEEK was also employed. CE has also been coupled to MS for use as a specific and confirmatory detection technique for the analysis of antibiotics. Oxolinic acid has been determined in fish muscles by CE [103]. The muscle sample was spiked with nalidixic acid, used as internal standard, then homogenized with a blender using 10 mmol L1 phosphate buffer at pH 7.0. The slurry was sonicated, centrifuged, and the supernatant was filtered and re-extracted twice with a 10 mmol L1 phosphate buffer. The combined extracts were filtered and the supernatant was cleaned-up using SPE cartridges. Danofloxacin (DAN), enrofloxacin (ENRO), flumequine (FLU), ofloxacin (OFL), and pipemidic (PIP), have been determined in fish tissues by CE with mass spectrometric (MS) detection [104]. The sample was mixed with 50 mmol L1 NaH2PO4 (pH 7.0), extracted twice with dichloromethane. After centrifugation, the organic layers were extracted with 0.5 mol L1 NaOH. The aqueous phase was separated from the dichloromethane by centrifuging, adjusted to pH 7.0 with 200 mmol L1 H3PO4, and defatted by extraction with hexane. The aqueous sample extract was passed through a C18 SPE column, previously conditioned with methanol and distilled water. Elution was performed with 2 mL 4% TFA in waterACN (25:75v/v), followed by 1 mL ACN. A specific CEMS method has been developed for simultaneous determination of 12 antibacterial residues (four sulfonamides: sulfamethazine, sulfathiazole, sulfadiazine, and sulfachlorpyridazine; four -lactams: amoxicillin, ampicillin, oxacillin, and penicillin V; and four quinolones: danofloxacin, enrofloxacin, ofloxacin, and flumequine), in fish and live stock [105]. The LODs and LOQs (below 18 and 50 ng kg1, respectively) were in all cases lower than the maximum residue limits for these compounds in fish. The method was applied to fish samples obtained from markets, slaughterhouses, and fish farms. Seven fluoroquinolones have also been determined in spiked fish samples by use of a CEC method [106]. The method was simple (isocratic elution), rapid, accurate, and

precise for simultaneous separation and determination of the selected fluoroquinolones. Amperometric detection enabled the determination of enrofloxacin and its metabolite ciprofloxacin in spiked fish (eel liver) samples by CZE. In this work, a simple pretreatment method using dichloromethane for extraction was developed and proved effective in obtaining good recoveries and short analysis time. Good reproducibility and low LODs were obtained, which allowed the analysis of contaminated animal tissue samples [107]. Catfish fed with the antibiotic oxytetracycline (OTC) for 10 days were cooked by four different procedures. Portions (5 g) of minced fish were mixed with 2 mL 50% TCA, 30 mL 1 mol L1 HCl, and 500 mg EDTA disodium, the mixture was centrifuged, and the supernatant was filtered. The pellet was re-extracted with 1 mL 50% TCA and 15 mL 1 mol L1 HCl, and the filtrates were combined and applied to Sep-Pak C18 SPE cartridges. The CE migration time was 10.9 min and mean OTC recovery from spiked catfish was 92.9% for added levels of 0.125 g g1 OTC [108]. ELISA methods In order to detect residues in food and tissues, bioassay techniques are widely used as screening methods. These methods generally do not distinguish between members of a class of antibiotics, but provide a semi-quantitative estimate of total residues detected. Nevertheless, they continue to be used because of their simplicity and low cost. Immunoassays are rapid and convenient, because they do not require sample preconcentration and clean-up steps. However, ELISA methods often have a high potential for nonspecific binding between nontarget analytes and antibodies and are consequently prone to matrix effects. Chemical compounds, for example protein, fat, solvents, and others, present in animal product samples or sample extracts might affect the binding of antibody and analytes, and they also might affect other aspects of the assay. A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed to detect a broad range of quinolones in various matrices, including fish and shrimp samples. In the optimized generic test, antisarafloxacin antibodies in combination with norfloxacin conjugate resulted in 50% binding inhibition at 0.21 ng mL1 for sarafloxacin in buffer. The method was able to detect 15 quinolone residues at levels lower than the established MRLs [109]. Direct competitive enzyme-linked immunosorbent assays (ELISA) have been developed to detect a broad range of sulfonamides in various matrices [110]. Screening for this class of antibiotics in fish was accomplished by simple, rapid extraction methods carried out with only

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phosphate-buffered saline (PBS). Matrix effects could be avoided by 1:5 dilution of the fish extract. Studies on screening, determination, and confirmation of chloramphenicol in several matrices, including seafood, by enzyme-linked immunosorbent assay (ELISA), highperformance liquid chromatography with UV detection (HPLCUV), and gas chromatography in combination with electron-capture detection (GCECD) and mass spectrometric detection (GCMS) have been carried out [47]. Methods have been developed for both qualitative and quantitative detection of chloramphenicol. ELISA was carried out for screening; HPLC, GC, and GCMS were applied to determine and confirm chloramphenicol residue concentrations in suspect samples. A sensitive and specific monoclonal ELISA for determination of tissue bound furazolidone metabolite 3-amino-2oxaxolidinone (AOZ) has been described [111, 112]. The procedure enables the detection of AOZ in matrix supernatant after homogenization, protease treatment, acid hydrolysis, and derivatisation with o-nitrobenzaldehyde of AOZ released from the tissue. The p-nitrophenyl 3-amino-2oxazolidinone (NPAOZ) formed was determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.055.0 g L1. A polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for determination of animal edible tissue-bound furazolidone metabolite(AOZ) [113]. Bioluminescence methods Bioluminescent Escherichia coli K-12 strain for specific detection of the tetracycline family of antimicrobial agents has been optimized to work with fish samples [114]. The biosensing strain contains a plasmid incorporating the bacterial luciferase operon of Photorhabdus luminescens, under the control of the tetracycline responsive element from transposon Tn10. The procedure for extraction of oxytetracycline from rainbow trout tissue was optimized. There was neither need for centrifugation of homogenized tissue nor use of organic solvents. The assay was able to detect oxytetracycline residues below the European Union maximum residue limits, and the results correlated well with those obtained by conventional HPLC. Chemiluminescence methods A micro-flow injection system on a chip has been prepared for determination of tetracycline in fish and shrimp samples. The method is based on luminolH2O2 chemiluminescence [115]. Spiral flow cell channels were fabricated on poly(methyl methacrylate) (PMMA) by use of a laser. The sampling time was controlled precisely by a timer. The linear range was 2.0107 to 1.0105 g mL1 with a detection limit of 1.7

108 g mL1. The method is simple, rapid, and highly sensitive and reagent consumption is very low. A molecularly imprinted polymer solid-phase extraction (MISE) method combined with flow-injection chemiluminescence (FI-CL), for determination of residual tetracycline (TC) in fish samples, has been presented [116]. The molecularly imprinted polymer (MIP) of TC was synthesized and particles of this MIP were packed into a polytetrafluoroethylene (PTFE) tube, which was connected to the sampling loop of an eight-way injection valve and served as the MISE column for on-line selective adsorption of TC. The eluent CH3CNHNO3 (4:1, v/v), was used for extracting the adsorbed TC, which could be detected by its good enhancing effect on the CL reaction between Ce(IV) and rhodamine B. The CL intensity is a linear function of TC concentration in the range 4109 to 4107 g mL1. The proposed MISPE-CL method has been successfully applied to the determination of TC in fish samples. Fluorescence methods A method for screening of tetracyclines (TCs), including tetracycline (TC), oxytetracycline (OTC), and chlorotetracycline (CTC), in different fish muscle matrices has been proposed [117]. This method was based on in-situ fluorescent derivation of TCs, to convert the weakly fluorescing TCs into highly fluorescent species, on alkaline-activated solid silica gel G plates (SGGPS). By coupling solid-surface fluorescence (SSF) with a charge-coupled device (CCD) camera imaging, a CCD camera-based SSF (CCD-SSF) method has been developed. The trueness of the method was validated by HPLC, and no significant difference between CCDSSF and HPLC was found at the 95% confidence level. The newly developed CCD-SSF method has been applied to the screening of TC residues in fish muscle samples. The method has been demonstrated to have advantages such as its simplicity, high throughput, low cost, use of fewer pollutants, and reasonable sensitivity. In Table 4 are summarized the above reported methods and sample pretreatments.

Conclusions Many methods have been developed for analysis of a wide variety of antibiotics in fish. The antibiotics most often analysed are the fluoroquinolones, amphenicols, tetracyclins, penicillins, and aminoglycosides. LCMSMS is currently regarded the tool of choice for analysis of antibiotic residues in animal-derived food. Use of LCMSMS also gives much needed structural information. By contrast, rapid, inexpensive screening methods (e.g., immunoassays and biosensors) are also required, and are being developed.

Analysis of antibiotics in fish samples Acknowledgements Financial support was provided by DGI-MCI of Spain (Projects CTQ 2008-00468/BQU and CTQ 2008-06657C02-01/BQU).

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