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Analytica Chimica Acta 529 (2005) 249256

Determination of chloramphenicol residues in rainbow troutsby gas chromatographymass spectometry and liquid

chromatographytandem mass spectometry

Lucia Santos a, Jorge Barbosa b, M. Conceic ao Castilho c, Fernando Ramos c , ,Carlos A. Fontes Ribeiro a, M. Irene Noronha da Silveira c

a Faculty of Medicine, University of Coimbra, Portugalb LNIV, National Laboratory of Veterinary Research, Portugal

c Faculty of Pharmacy, University of Coimbra, Rua do Norte, 3000, 295 Coimbra, Portugal

Received 11 May 2004; received in revised form 7 July 2004; accepted 7 July 2004Available online 11 September 2004

Abstract

A methodology for the identication and quantication of chloramphenicol (CAP) residues was developed and validated. The methodis based on gas chromatographymass spectrometry (GCMS) for screening, in electron impact (EI) mode, after a solid phase extraction(SPE) step using C18 columns, and data acquired in selective ion monitoring (SIM) mode with the following ions: m / z 225, m/z 208 and m / z242. Conrmatory method consists on liquid chromatographytandem mass spectrometry (LCMS/MS), in ionspray-negative mode, after thesame extraction and clean-up procedure. The m / z 321 was selected as a parent ion and m / z 152 and m / z 194 as daughter ions. The data wereacquired in the negative multiple reaction monitoring (NMRM) mode, by monitoring the transitions 321 > 152 and 321 > 194. The methodgave a decision limit (CC ) and a detection capability (CC ) of 0.267 and 0.454 g kg 1, respectively.

The described methodology was applied on 40 samples of rainbow trout, collected in supermarkets of the Centre Region of Portugal, inorder to evaluate the presence of CAP residues.

Positive results were conrmed in 9 of the 15 suspected samples, which correspond to 22.5% of the whole samples collected. 2004 Elsevier B.V. All rights reserved.

Keywords: Chloramphenicol; Rainbow trout; GCMS; LCMS/MS

1. Introduction

Chloramphenicol (CAP), Fig. 1, is a broad spectrum an-tibiotic, effective against a wide range of microorganisms,

that has widely been used since the 1950s to treat food-producing animals, mainly because of its ready availabil-ity and low cost. However, CAP has been rapidly associatedto serious toxic effects, especially bone marrow depression,particularly severe when it assumes the form of the dose-independent, and fatal, aplastic anaemia [1,2] .

Corresponding author. Tel.: +351 239 859994; fax: +351 239 827126. E-mail address: [email protected] (F. Ramos).

The well-known risk of irreversible bone marrow disor-ders and carcinogenic properties of CAP [3], and the absenceof safe residue levels, has determined European Union (EU)prohibitionfor veterinaryuse, in 1994 [4,5] , andno maximum

residue limit (MRL) has been established for this antibiotic.Despite this legal ban, CAP has been found in severalanimal-derivedfoods, mainly aquacultureproducts,and otherfoodstuffs like honey, especially originated from Asiaticcountries.

Along with continued improvement of analytical methodsensitivity, the European Commission considers that it isnecessary to provide for the progressive establishment of minimum required performance limits (MRPL) of analyti-cal methods for substances for which no permitted limit has

0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2004.07.017

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250 L. Santos et al. / Analytica Chimica Acta 529 (2005) 249256

Fig. 1. Structural formula of chloramphenicol.

been established, and in particular for those substances, likeCAP, whose use is not authorised or specically prohibitedin the Community. So, Commission Decision 2003/181/EC[6] established a MRPL for CAP of 0.3 g kg 1 .

For conrmatory purposes at trace levels, CommissionDecision 2002/657/EC [7] requires that conrmatory meth-ods provide information on the chemical structure of the an-alyte and, so, mass spectrometry is the generally acceptedtechnique.

There are several recently published analytical methodsfor determination of CAP in various food matrixes, such asequine, porcine and bovine muscle [8], shrimp [9], chicken,turkey, pork, beef and sh in dry powdered form [10] andporcine tissues [11] , butnoneofthemreportingtoshmuscle.

In this study, an analytical method for screening andconrmation of CAP residues in rainbow trout musclewas developed. Gas chromatographymass spectrometry(GCMS) was carried out to screen rainbow trout musclesamples,and liquid chromatographytandem massspectrom-etry (LCMS/MS) was applied to conrm suspect samples.

GCMS, in negative chemical ionisation (NCI) mode isthe more recommended and used technique for CAP deter-mination,because of itsbetter sensitivity [12-15] , when com-pared with GCMS in electron impact (EI) mode [16]. How-ever, GCMSinEImode wastheonly GCtechniqueavailablein our laboratory and, therefore, the one that was used in thepresent study.

Developed methodology was applied on 40 rainbow troutsamples, of aquaculture origin, collected in supermarkets of the Centre Region of Portugal, in order to evaluate the pres-ence of CAP residues.

2. Experimental

2.1. Materials and reagents

Chloramphenicol (CAP) was obtained from SigmaAldrich (Steinheim,Germany). CAP-d5, obtainedfromCam-bridge Isotope Laboratories (Andover, USA), was used asinternal standard. GC derivatisation reagent [HMDS (hex-amethyldisilazane):TMCS (trimethylchlorosilane):Pyridine;2:1:10] was obtained from Macherey-Nagel (D uren, Ger-many). All other reagents were provided by Merck (Darm-stadt, Germany), except for acetonitrile, ethyl acetate andphosphoric acid 85%, obtained from Carlo Erba (Milan,Italy).

Puried water was obtained with a Milli-Q apparatus(Millipore, Bedford, MA, USA). The following deviceswere used: a Mettler Toledo AG285 balance (Greifensee,Switzerland), a Selecta Meditronic centrifuge (Barcelona,Spain), anEdmundB uhler7400 horizontalshaker (T ubingen,Germany), a Stomacher Homogenizer (Lameris, Utrecht,

Netherlands), a CD7400 WPA pHmeter (Cambridge, UK),a vortex mixer (Retsch, Haan, Germany), a Stuart Scienticvacuum evaporator (purchased from Reagente 5, Porto, Por-tugal), Gilson micropipettes (Villiers-le-Bel, France) of 100,50 and 20 l, and membrane lters Schleicher & Schuell,0.2 m, 50 mm (Dassel, Germany). Solid-phase extraction(SPE) columns (Chromabond C18 500mg/3ml) were pur-chased from Macherey-Nagel.

A Hewlett Packard (HP) equipment (Soquimica, Lis-bon) comprising an HP5890 series II GC chromatograph,an HP6890 autosampler, an HP5972 MSD detector, an HPVectra VL2 4/50 computer and an HP Deskejet 520 printerwas used. A Permabond OV125m 0.25mm i.d., 0.25 mlm thicknesses (Macherey-Nagel) was used as CG column.

The LCMS/MS system consisted of an Agilent 1100 LCequipment (Palo Alto, USA), an MS/MS detector, from Ap-plied Biosystems Model Sciex API 2000 (Foster City, USA),with TurboIon Spray ionisation source, an Eclipse XDB-C18, 2.1 mm 150mm, 5 m Agilent LC column and aZorbax Eclipse XDB-C8, 2.1mm 12.5 mm, 5 m Agilentpre-column.

Helium N55 and nitrogen N45 were supplied by Sogafer(Coimbra, Portugal).

2.2. Standard solutions

CAP stock solution (1 mg ml 1) was prepared by weigh-ing accurately 50.0mg of CAP into a 50.0 ml volumetricask, diluted with methanol, and stored at 4 C, protectedfrom light. An intermediate solution was prepared in order toachieve a standard solution containing 50 g ml 1 , by dilut-ing the previous stock solution with methanol.

Taking this solution, suitable CAP working standard so-lutions were prepared, by diluting with the referred solvent.

CAP-d5 standard solution was prepared by diluting theinitial 1mg ml 1 solution in a proper volume of methanol, inorder to obtain a 50 g ml 1 solution.

Table 1Validation data ( n = 9)a

Criteria Data

CC ( g kg 1) 0.267CC ( g kg 1) 0.454RecoveryFortication level (% S.D.)

1.0 g kg 1 117.15 0.121.5 g kg 1 107.72 0.062.0 g kg 1 92.80 0.03

Within-repeatability (% S.D.) 5.19 0.21Within-reproducibility (% S.D.) 9.29 0.12

a nnumber of samples.

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L. Santos et al. / Analytica Chimica Acta 529 (2005) 249256 251

Fig. 2. Mass spectrum of trimethylsilylated CAP. GCMS, EI mode.

Fig. 3. GCMS chromatogram, EI mode, SIM scan, of a suspect sample (extracted ions: m / z 225 (top) and m / z 230 (bottom)).

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2.3. Phosphate buffer

Phosphate buffer 0.1 M, pH 6.0, was prepared by mixingthe two following solutions:

1. Di-sodium hidrogenophosphate solution, 0.1 M, preparedby dissolving 4.26g in 300 ml Milli-Q water.

2. Phosphoric acid solution,0.1 M,using 1.36 mlphosphoricacid 85% and completing the volumeofa 200 ml volumet-ric ask with Milli-Q water.

The mixture of the total volume of both solutions results in abuffer solution with a pH of 6.0 0.2.

2.4. Mobile phase used in LCMS/MS

Mobile phase used in LC system was constituted by twophases:

A Water (1000 ml) + glacial acetic acid (1 ml).B Water (1000 ml) + acetonitrile (900ml) + glacial

acetic acid (1 ml).

Fig. 4. Chromatograms obtained for a negative sample (extracted ions: m / z 225 (top) and m / z 230 (bottom)).

Both solutions were ltered, by vacuum, using a 0.2 mmembrane lter.

2.5. Samples

Rainbow trout ( Oncorhynchus mykiss ) samples (n = 40),of aquaculture origin, were collected, between February andJuly 2003, in Portuguese supermarkets.

Fish muscle was separated from the rest, and then sub-sampled in portions of 10.0 0.1 g, and stored at 20 C inairtight containers until analysis.

2.6. Sample preparation

2.6.1. ExtractionThe sample tissue, spiked with 50 l of Internal Standard

(CAP-d5 1 g kg 1), was homogenized with phosphatebuffer (pH 6.0, 0.1 M), using a Stomacher , and thencentrifuged at 2600 100 g for 5 min. Supernatant wasdecanted into a new glass tube and defatted with 10 ml of n-hexane. Extraction of CAP from this primary extract was

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obtained by homogenisation with 10 ml ethyl acetate, byrst thoroughly hand shaking the mixture, followed by cen-trifugation, for 2 min. The upper organic phase was collectedand, subsequently, dried under nitrogen stream at 45 5 C.

2.6.2. Solid phase extraction (SPE)

Dried samples were reconstituted using 5 ml of puriedwater. The C18 columns were activated, subsequently, with1 ml methanol and 1 ml water, using the vacuum pump at theminimum pressure. After passing the entire sample throughthe column, it was washed with 2.5 ml water and 2.5 ml of a mixture methanol/water (10:90). The columns were well

Fig. 5. LCMS/MS mass spectrum of CAP: (A) MS1 (B) MS/MS.

dried,rst athigh vacuum pressure andthenbycentrifugationat 2600 100 g for 5 min. CAP was, nally, eluted with2.5 ml of methanol.

2.7. Gas chromatography

Samples dried after elution from the C18 columns werereconstituted in 2 100 l methanol, and transferred to GCvials. Methanol was then evaporated, under nitrogen streamat 45 5 C, and dry residue reconstituted in 50 l derivati-sation reagent. Tightly closed vials were incubated at 65 5 C, for 30 min.

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A splitless injectionof 2 lwasmadeat250 C over1 min.The initial oven temperature was held at 110 C for 2minrising to 300 C at 17 Cmin 1, where it was held for 5 min.The detector temperature was 280 C. The MS detector wasoperated in theelectron impact(EI) ionisation mode, anddatawere acquired in selective ion monitoring (SIM) mode: m / z

208, m / z 225 and m / z 242 for CAP, and m / z 230 for CAP-d5.

2.8. Liquid chromatography

Suspect samples, dried after elution from the C18columns,werereconstituted in 100 l mobile phase, vortexedand transferred into LC vial.

Fig. 6. LCMS/MS mass spectrum of CAP-d5. (A) MS1 (B) MS/MS.

Samples (60 l) were injected in the LC system with acolumn temperature of 40 C, a ow rate of 1 ml min 1 andthe following gradient conditions:

Time (min) A (%) B (%)

01 90 10114 55 451416 10 901618 10 901819 90 10

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Fig. 7. LCMS/MS chromatograms of rainbow trout sample extract, NMRM mode: blank sample (a), positive sample (1.99 g/kg 1 CAP) (b).

The MS detector was operated with an electrospray ion-isation (ESI) interface. An m / z 321 ion was selected as aparent and m / z 152 and m / z 194 as daughter ions. The datawere acquired in the negative multiple reaction monitoring(NMRM), monitoring the transitions: 321 > 152 and 321 >194.

2.9. Validation

For method validation, relevant characteristics were de-termined, in order to verify its compliance with the criteriafor the proper identication according to the European Com-mission Decision 2002/657/EC [7].

The linearity of the LC method response was testednine times during 1 month with four different concentra-tions between 0.0 and 2.0 g kg 1. The values of slope,intercept and correlation coefcient ( r ) varied between aminimum of 0.23, 0.001 and 0.9957, respectively, anda maximum of 0.24, 0.01 and 0.9987, with four datapoints.

Table 1 shows the within-laboratory repeatability and re-producibility, the recoverypercentages, decision limit (CC )and detection capability (CC ) values.

3. Results and discussion

This study involved extraction of CAP from rainbow troutmuscle, and in order to prevent any loss of analyte, due tothe reactivationof endogenousenzymes, extraction wasdoneimmediately after the homogenisation step.

The screening procedure was made by a qualitativeGCMS method, after a derivatisation step of the ana-lyte. The derivatisation reagent used leads to the forma-tion of a silylated molecule of CAP, and its molecular ionm / z 466. This ion is usually prominent in the softer ion-isation mode, such as GCMS with chemical ionisation.However it was not observed in the obtained EI spec-trum of CAP (Fig. 2). The high collision energy of EIsource causes multiple fragmentations of the CAP molecule

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and its transformation in the lower m / z 208, 225 and 242ions.

According to EU criteria for the analysis of veterinarydrug residues [7], a system of identication points (IP) isadopted to dene the number of ions and their correspondingratios that should be measured when using conrmatory MS

techniques.ForGroupA substances (withno MRL), includedin Annex I of Directive 96/23/CE [17], a minimum of 4 IP isrequired for the conrmation. In GCMS techniques, eitherin EI or CI mode, each ion monitored gives 1 IP. In this study,three ions were monitored and therefore 3 IP were obtained,and EU criteria were not fullled.

Using this GCMS method, samples were considered sus-pected when the following criteria werecumulativelyaccom-plished:

The ratio between the chromatographic retentiontime of the analyte and those of the internal stan-dard must correspond to the one of the calibration

solution, with a tolerance of 0.5%. All the selected ions m / z 225, m / z 208 and m / z242must be present on the mass spectrum of thechromatographic peak.

The relative abundance of these ions must cor-respond to that of the standard, with the accept-able deviations described in Commission Decision2002/657/EC.

The ions intensity must be, at least, three timesgreater than the base noise of the MS detector.

According to the criteria described above, 15 of the 40trout samples were considered to have a suspect result.

Figs. 3 and 4 show representative chromatograms obtainedfor a suspect and a negative sample. On both chromatogramsthe most abundant ion of CAP ( m / z 225) and of the internalstandard monitored ion ( m / z 230) were extracted.

The suspect samples were analysed, for conrmatory pur-poses, by a LCMS/MS technique, which presents a highersensitivity, essentially because of three main factors:

a softer ionisation technique of the ion source; the presence of a second mass fragmentation phase,

that isolates the ion of interest of the remaining in-terfering ions;

the absence of a derivatisation step.

CAP and its internal standard were rst analysed by ESI-MS to optimise the MS conditions. The full mass spectra of CAP and itsdeuterated internal standard display several ions,and the most abundant were m / z 321 for CAP and m / z 326for CAP-d5, as shown in Figs. 5 and 6.

For conrmation purposes, LCMS/MS method, by mon-itoring two transition reactions, earning 1.5 IP each, allowsthe obtaining of 4 IP, which fulls the EU criteria.

By LCMS/MS it was possible to conrm nine positiveresults, between the suspect samples, which corresponds to22.5% of the total samples collected, with concentration lev-

els that varied between a minimum of 1.58 g kg 1 and amaximum of 3.94 g kg 1 . Fig. 7 represents LCMS/MSchromatograms of rainbow trout: (a) blank sample and (b)sample with 1.99 g kg 1 of CAP.

The developed methodology allows detection and conr-mation of CAP, at trace levels, in rainbow trout muscle. The

method fullsthe validation parameter requirements, accord-ing to EU criteria for the analysis of veterinary drug residues.

Moreover, the method was applied on 40 samples of rain-bow trout, of aquaculture origin, collected in Portuguese su-permarkets, and positive results were conrmed in 22.5% of the samples, with concentration levels that varied between aminimum of 1.58 g kg 1 and a maximum of 3.94 g kg 1 .

Acknowledgements

The authors are grateful to the Portuguese Foundationfor Science and Technology (CEF-FCT) for supporting thisstudy.

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