Analytica Chimica Acta 529 (2005) 249256

Determination of chloramphenicol residues in rainbow trouts by gas chromatographymass spectometry and liquid chromatographytandem mass spectometry
o Castilhoc , Fernando Ramosc, , L ucia Santosa , Jorge Barbosab , M. Conceic a a Carlos A. Fontes Ribeiro , M. Irene Noronha da Silveirac
c a Faculty of Medicine, University of Coimbra, Portugal LNIV, National Laboratory of Veterinary Research, Portugal Faculty of Pharmacy, University of Coimbra, Rua do Norte, 3000, 295 Coimbra, Portugal b

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

Abstract A methodology for the identication and quantication of chloramphenicol (CAP) residues was developed and validated. The method is 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/z 242. Conrmatory method consists on liquid chromatographytandem mass spectrometry (LCMS/MS), in ionspray-negative mode, after the same 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 were acquired in the negative multiple reaction monitoring (NMRM) mode, by monitoring the transitions 321 > 152 and 321 > 194. The method gave a decision limit (CC) and a detection capability (CC) of 0.267 and 0.454 g kg1 , respectively. The described methodology was applied on 40 samples of rainbow trout, collected in supermarkets of the Centre Region of Portugal, in order 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 antibiotic, effective against a wide range of microorganisms, that has widely been used since the 1950s to treat foodproducing animals, mainly because of its ready availability and low cost. However, CAP has been rapidly associated to serious toxic effects, especially bone marrow depression, particularly severe when it assumes the form of the doseindependent, and fatal, aplastic anaemia [1,2].

Corresponding author. Tel.: +351 239 859994; fax: +351 239 827126. E-mail address: fjramos@ci.uc.pt (F. Ramos).

The well-known risk of irreversible bone marrow disorders and carcinogenic properties of CAP [3], and the absence of safe residue levels, has determined European Union (EU) prohibition for veterinary use, in 1994 [4,5], and no maximum residue limit (MRL) has been established for this antibiotic. Despite this legal ban, CAP has been found in several animal-derived foods, mainly aquaculture products, and other foodstuffs like honey, especially originated from Asiatic countries. Along with continued improvement of analytical method sensitivity, the European Commission considers that it is necessary to provide for the progressive establishment of minimum required performance limits (MRPL) of analytical 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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Fig. 1. Structural formula of chloramphenicol.

been established, and in particular for those substances, like CAP, whose use is not authorised or specically prohibited in the Community. So, Commission Decision 2003/181/EC [6] established a MRPL for CAP of 0.3 g kg1 . For conrmatory purposes at trace levels, Commission Decision 2002/657/EC [7] requires that conrmatory methods provide information on the chemical structure of the analyte and, so, mass spectrometry is the generally accepted technique. There are several recently published analytical methods for determination of CAP in various food matrixes, such as equine, porcine and bovine muscle [8], shrimp [9], chicken, turkey, pork, beef and sh in dry powdered form [10] and porcine tissues [11], but none of them reporting to sh muscle. In this study, an analytical method for screening and conrmation of CAP residues in rainbow trout muscle was developed. Gas chromatographymass spectrometry (GCMS) was carried out to screen rainbow trout muscle samples, and liquid chromatographytandem mass spectrometry (LCMS/MS) was applied to conrm suspect samples. GCMS, in negative chemical ionisation (NCI) mode is the more recommended and used technique for CAP determination, because of its better sensitivity [12-15], when compared with GCMS in electron impact (EI) mode [16]. However, GCMS in EI mode was the only GC technique available in our laboratory and, therefore, the one that was used in the present study. Developed methodology was applied on 40 rainbow trout samples, of aquaculture origin, collected in supermarkets of the Centre Region of Portugal, in order to evaluate the presence of CAP residues.

Puried water was obtained with a Milli-Q apparatus (Millipore, Bedford, MA, USA). The following devices were used: a Mettler Toledo AG285 balance (Greifensee, Switzerland), a Selecta Meditronic centrifuge (Barcelona, Spain), an Edmund B uhler 7400 horizontal shaker (T ubingen, Germany), a Stomacher Homogenizer (Lameris, Utrecht, Netherlands), a CD7400 WPA pHmeter (Cambridge, UK), a vortex mixer (Retsch, Haan, Germany), a Stuart Scientic vacuum evaporator (purchased from Reagente 5, Porto, Portugal), 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 500 mg/3 ml) were purchased from Macherey-Nagel. A Hewlett Packard (HP) equipment (Soquimica, Lisbon) comprising an HP5890 series II GC chromatograph, an HP6890 autosampler, an HP5972 MSD detector, an HP Vectra VL2 4/50 computer and an HP Deskejet 520 printer was used. A Permabond OV125 m 0.25 mm i.d., 0.25 m lm thicknesses (Macherey-Nagel) was used as CG column. The LCMS/MS system consisted of an Agilent 1100 LC equipment (Palo Alto, USA), an MS/MS detector, from Applied Biosystems Model Sciex API 2000 (Foster City, USA), with TurboIon Spray ionisation source, an Eclipse XDBC18, 2.1 mm 150 mm, 5 m Agilent LC column and a Zorbax Eclipse XDB-C8, 2.1 mm 12.5 mm, 5 m Agilent pre-column. Helium N55 and nitrogen N45 were supplied by Sogafer (Coimbra, Portugal). 2.2. Standard solutions CAP stock solution (1 mg ml1 ) was prepared by weighing accurately 50.0 mg of CAP into a 50.0 ml volumetric ask, diluted with methanol, and stored at 4 C, protected from light. An intermediate solution was prepared in order to achieve a standard solution containing 50 g ml1 , by diluting the previous stock solution with methanol. Taking this solution, suitable CAP working standard solutions were prepared, by diluting with the referred solvent. CAP-d5 standard solution was prepared by diluting the initial 1 mg ml1 solution in a proper volume of methanol, in order to obtain a 50 g ml1 solution.
Table 1 Validation data (n = 9)a Criteria CC (g kg1 ) CC (g kg1 ) Recovery Fortication level(% S.D.) 1.0 g kg1 1.5 g kg1 2.0 g kg1 Within-repeatability (% S.D.) Within-reproducibility (% S.D.)
a

2. Experimental 2.1. Materials and reagents Chloramphenicol (CAP) was obtained from Sigma Aldrich (Steinheim, Germany). CAP-d5, obtained from Cambridge Isotope Laboratories (Andover, USA), was used as internal standard. GC derivatisation reagent [HMDS (hexamethyldisilazane):TMCS (trimethylchlorosilane):Pyridine; 2:1:10] was obtained from Macherey-Nagel (D uren, Germany). All other reagents were provided by Merck (Darmstadt, Germany), except for acetonitrile, ethyl acetate and phosphoric acid 85%, obtained from Carlo Erba (Milan, Italy).

Data 0.267 0.454

117.15 0.12 107.72 0.06 92.80 0.03 5.19 0.21 9.29 0.12

nnumber of samples.

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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 mixing the two following solutions: 1. Di-sodium hidrogenophosphate solution, 0.1 M, prepared by dissolving 4.26 g in 300 ml Milli-Q water. 2. Phosphoric acid solution, 0.1 M, using 1.36 ml phosphoric acid 85% and completing the volume of a 200 ml volumetric ask with Milli-Q water. The mixture of the total volume of both solutions results in a buffer 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 two phases: A Water (1000 ml) + glacial acetic acid (1 ml). B Water (1000 ml) + acetonitrile (900 ml) + glacial acetic acid (1 ml).

Both solutions were ltered, by vacuum, using a 0.2 m membrane lter. 2.5. Samples Rainbow trout (Oncorhynchus mykiss) samples (n = 40), of aquaculture origin, were collected, between February and July 2003, in Portuguese supermarkets. Fish muscle was separated from the rest, and then subsampled in portions of 10.0 0.1 g, and stored at 20 C in airtight containers until analysis. 2.6. Sample preparation 2.6.1. Extraction The sample tissue, spiked with 50 l of Internal Standard (CAP-d5 1 g kg1 ), was homogenized with phosphate buffer (pH 6.0, 0.1 M), using a Stomacher , and then centrifuged at 2600 100 g for 5 min. Supernatant was decanted into a new glass tube and defatted with 10 ml of n-hexane. Extraction of CAP from this primary extract was

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

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obtained by homogenisation with 10 ml ethyl acetate, by rst thoroughly hand shaking the mixture, followed by centrifugation, for 2 min. The upper organic phase was collected and, subsequently, dried under nitrogen stream at 45 5 C. 2.6.2. Solid phase extraction (SPE) Dried samples were reconstituted using 5 ml of puried water. The C18 columns were activated, subsequently, with 1 ml methanol and 1 ml water, using the vacuum pump at the minimum pressure. After passing the entire sample through the column, it was washed with 2.5 ml water and 2.5 ml of a mixture methanol/water (10:90). The columns were well

dried, rst at high vacuum pressure and then by centrifugation at 2600 100 g for 5 min. CAP was, nally, eluted with 2.5 ml of methanol. 2.7. Gas chromatography Samples dried after elution from the C18 columns were reconstituted in 2 100 l methanol, and transferred to GC vials. Methanol was then evaporated, under nitrogen stream at 45 5 C, and dry residue reconstituted in 50 l derivatisation reagent. Tightly closed vials were incubated at 65 5 C, for 30 min.

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

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A splitless injection of 2 l was made at 250 C over 1 min. The initial oven temperature was held at 110 C for 2 min rising to 300 C at 17 C min1 , where it was held for 5 min. The detector temperature was 280 C. The MS detector was operated in the electron impact (EI) ionisation mode, and data were 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 C18 columns, were reconstituted in 100 l mobile phase, vortexed and transferred into LC vial.

Samples (60 l) were injected in the LC system with a column temperature of 40 C, a ow rate of 1 ml min1 and the following gradient conditions: Time (min) 01 114 1416 1618 1819 A (%) 90 55 10 10 90 B (%) 10 45 90 90 10

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

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Fig. 7. LCMS/MS chromatograms of rainbow trout sample extract, NMRM mode: blank sample (a), positive sample (1.99 g/kg1 CAP) (b).

The MS detector was operated with an electrospray ionisation (ESI) interface. An m/z 321 ion was selected as a parent and m/z 152 and m/z 194 as daughter ions. The data were 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 determined, in order to verify its compliance with the criteria for the proper identication according to the European Commission Decision 2002/657/EC [7]. The linearity of the LC method response was tested nine times during 1 month with four different concentrations between 0.0 and 2.0 g kg1 . The values of slope, intercept and correlation coefcient (r) varied between a minimum of 0.23, 0.001 and 0.9957, respectively, and a maximum of 0.24, 0.01 and 0.9987, with four data points.

Table 1 shows the within-laboratory repeatability and reproducibility, the recovery percentages, decision limit (CC) and detection capability (CC) values.

3. Results and discussion This study involved extraction of CAP from rainbow trout muscle, and in order to prevent any loss of analyte, due to the reactivation of endogenous enzymes, extraction was done immediately after the homogenisation step. The screening procedure was made by a qualitative GCMS method, after a derivatisation step of the analyte. The derivatisation reagent used leads to the formation of a silylated molecule of CAP, and its molecular ion m/z 466. This ion is usually prominent in the softer ionisation mode, such as GCMS with chemical ionisation. However it was not observed in the obtained EI spectrum of CAP (Fig. 2). The high collision energy of EI source causes multiple fragmentations of the CAP molecule

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and its transformation in the lower m/z 208, 225 and 242 ions. According to EU criteria for the analysis of veterinary drug residues [7], a system of identication points (IP) is adopted to dene the number of ions and their corresponding ratios that should be measured when using conrmatory MS techniques. For Group A substances (with no MRL), included in Annex I of Directive 96/23/CE [17], a minimum of 4 IP is required for the conrmation. In GCMS techniques, either in 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 suspected when the following criteria were cumulatively accomplished: The ratio between the chromatographic retention time of the analyte and those of the internal standard must correspond to the one of the calibration solution, with a tolerance of 0.5%. All the selected ionsm/z 225, m/z 208 and m/z 242must be present on the mass spectrum of the chromatographic peak. The relative abundance of these ions must correspond to that of the standard, with the acceptable deviations described in Commission Decision 2002/657/EC. The ions intensity must be, at least, three times greater than the base noise of the MS detector. According to the criteria described above, 15 of the 40 trout samples were considered to have a suspect result. Figs. 3 and 4 show representative chromatograms obtained for a suspect and a negative sample. On both chromatograms the most abundant ion of CAP (m/z 225) and of the internal standard monitored ion (m/z 230) were extracted. The suspect samples were analysed, for conrmatory purposes, by a LCMS/MS technique, which presents a higher sensitivity, 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 interfering ions; the absence of a derivatisation step. CAP and its internal standard were rst analysed by ESIMS to optimise the MS conditions. The full mass spectra of CAP and its deuterated internal standard display several ions, and the most abundant were m/z 321 for CAP and m/z 326 for CAP-d5, as shown in Figs. 5 and 6. For conrmation purposes, LCMS/MS method, by monitoring two transition reactions, earning 1.5 IP each, allows the obtaining of 4 IP, which fulls the EU criteria. By LCMS/MS it was possible to conrm nine positive results, between the suspect samples, which corresponds to 22.5% of the total samples collected, with concentration lev-

els that varied between a minimum of 1.58 g kg1 and a maximum of 3.94 g kg1 . Fig. 7 represents LCMS/MS chromatograms of rainbow trout: (a) blank sample and (b) sample with 1.99 g kg1 of CAP. The developed methodology allows detection and conrmation of CAP, at trace levels, in rainbow trout muscle. The method fulls the validation parameter requirements, according to EU criteria for the analysis of veterinary drug residues. Moreover, the method was applied on 40 samples of rainbow trout, of aquaculture origin, collected in Portuguese supermarkets, and positive results were conrmed in 22.5% of the samples, with concentration levels that varied between a minimum of 1.58 g kg1 and a maximum of 3.94 g kg1 .

Acknowledgements The authors are grateful to the Portuguese Foundation for Science and Technology (CEF-FCT) for supporting this study.

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