Determination of chloramphenicol in animal tissues and urine

Analytica Chimica Acta 483 (2003) 125–135

Determination of chloramphenicol in animal tissues and urine Liquid chromatography–tandem mass spectrometry versus gas chromatography–mass spectrometry Alex Gantverg a,∗ , Isaac Shishani a , Michael Hoffman b,1 b

a Maxxam Analytics Inc., 5540 McAdam Road, Mississauga, Ont., Canada L4Z 1P1 United States Department of Agriculture, 901 D Street #344, Washington, DC 20024, USA

Received 19 June 2002; accepted 4 December 2002

Abstract A very sensitive method was developed for detection and confirmation of chloramphenicol (CAP) in equine, porcine and bovine muscle and urine. The method included ethyl acetate extraction of CAP followed by a two-step C18 solid-phase clean up; recovery was >80%. Extracted CAP was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in negative atmospheric pressure chemical ionization mode. LC–MS/MS gave superior sensitivity and selectivity compared to that shown by gas chromatography–mass spectrometry (GC–MS) in electron impact mode. Even in a “dirty” matrix, such as urine, the estimated CAP detection limit for LC–MS/MS detection was 0.02 ␮g kg−1 while the corresponding value for GC–MS was only 2 ␮g kg−1 . © 2002 Elsevier Science B.V. All rights reserved. Keywords: Chloramphenicol; Antibiotics; Urine; Muscle; Mass spectrometry

1. Introduction Chloramphenicol (CAP) is an effective antibiotic that has widely been used since the 1950s to treat food-producing animals. In 1994, the use of CAP was banned by the European Union (EU) due to the potential health risk posed by its traces in food [1]. Along with continued improvement of analytical method sensitivity, the EU has been lowering the detection limit of prohibited drugs in meat to ensure the highest safety standards for food products. The year 2001 the detection limit set by EU for CAP detection in urine and tissues was 2 ␮g kg−1 . ∗ Corresponding author. Tel.: +1-905-890-2455; fax: +1-905-890-2456. E-mail address: [email protected] (A. Gantverg). 1 Tel.: +1-905-890-2555; fax: +1-202-690-6544.

There are several publications devoted to determination of CAP in biological matrices, based on analytical techniques such as planar [2], liquid [3,4] and gas chromatography [5,6]. Enzyme-linked immunosorbent assay (ELISA) [7,8] and radioimmunoassay [9] are also widely used for CAP determination. The high sensitivity of an electron capture detector (ECD) to CAP is employed in most gas chromatography (GC)

0003-2670/03/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(02)01566-0

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methods: 1 ␮g kg−1 CAP was detected in shrimp tissue using a GC-ECD method [5]. The focus of our research, however, has been on methods allowing the confirmation of CAP in tissues. The most common method for confirmation of CAP is GC–mass spectrometry (MS) with negative ion chemical ionization (CI) mode. This technique has demonstrated excellent sensitivity down to 0.1 ␮g kg−1 in muscle tissues, although in urine the results were less sensitive due to the matrix interference [10–12]. GC–MS in electron impact (EI) mode, though slightly less sensitive, has the advantage of obtaining spectra reproducible between different instruments and stored in electronic libraries. The sensitivity achieved in fish muscles with CAP detection by GC–MS in EI mode was 5 ␮g kg−1 [13]. The general drawback of using GC methods for CAP detection has been the necessity to derivatize CAP in order to improve its chromatographic properties. Liquid chromatography (LC) methods, however, do not require a derivatization step and LC–MS sensitivity approaches that of GC–MS. The limit of quantification for CAP in urine measured by LC–MS method with an electrospray ion source was 3 ␮g kg−1 [14]. Another publication also demonstrates the possibility of using LC–MS in electrospray negative ion mode to detect the CAP m/z 321 molecular ion [15]. Tandem mass spectrometry (MS/MS) is a more sophisticated technique allowing a very effective isolation of analyte ions from the noise-producing matrix. However, no publications were found utilizing this technique for CAP detection. The objectives of this research were to develop an effective extraction procedure of CAP from matrices such as horse, beef, and pork urine and muscle tissue, and to select the most efficient CAP confirmation technique at the regulatory level of 2 ␮g kg−1 .

2. Experimental 2.1. Reagents and chemicals Methanol, ethyl acetate, formic acid, hexane, acetonitrile, diethyl ether, sodium chloride, sodium sulfate, sodium dihydrogen phosphate and dihydrogen potassium phosphate were analytical grade supplied by VWR. CAP, ␤-glucuronidase (from Helix pomatia)

and dithiothreitol were purchased from Sigma. TMS derivatization reagent BSTFA + 10%TMCS in 1-ml ampoules was obtained from Pierce. Solid-phase extraction (SPE) columns (Bond Elut-C18, 500 mg, 6 ml) were purchased from Varian. CAP stock solution (100 ␮g ml−1 ) was prepared in methanol every 3 months and stored below −10 ◦ C. CAP working solution (0.2 ␮g ml−1 ) was made by diluting stock solution with ethyl acetate. This solution was prepared biweekly. Equine, bovine and porcine samples of muscles and urine were procured from several farms in Canada and the United States. 2.2. Muscle tissue extraction Tissue sample (5.0 g) was weighed in a 50-ml plastic tube. Selected blank samples were spiked with known quantities of CAP working solution. A 5.0 g of sodium sulfate and 10 ml of 0.5 g l−1 dithiothreitol solution in ethyl acetate were added to the tube. Dithiothreitol (Cleland’s reagent) was used to disrupt any protein binding of CAP. The sample tissue was homogenized with ultra turrax and the tissue residue in the homogenizer was rinsed with an additional 5 ml of dithiothreitol solution. After vortexing, the sample was centrifuged at 2000 rpm for 5 min. The supernatant was decanted into a new 50-ml plastic tube. The remaining sample precipitate was rinsed with an additional 5 ml of dithiothreitol solution and centrifuged. The supernatants were combined and placed in the freezer below −10 ◦ C for at least 1 h. Precipitated fat was removed by centrifuging at 3500 rpm for 5 min at −5 ◦ C. The supernatant was dried on the nitrogen dryer at 40 ◦ C. Excessive drying and exposure to the atmosphere should be avoided to prevent loss of CAP. The residue was reconstituted in 4 ml of 4% sodium chloride solution and 2 ml of hexane. The hexane fraction of the centrifuged samples was discarded. The wash step of the aqueous fraction was repeated with 2 ml of hexane. A C18 column was equilibrated with 5 ml of methanol, 5 ml of chloroform, 5 ml of methanol and 5 ml of water using the vacuum pump at the minimum pressure. After passing the entire sample through the column it was rinsed with two 2-ml aliquots of water followed by 2 ml of methanol:water (20:80, v/v). The sample was dried for 1 min at high vacuum pressure,

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CAP was eluted with 3 ml of methanol:water (50:50, v/v) and the eluate was collected under gravity in glass tubes. In the second C18 step, the eluate was diluted with 7 ml water and applied to a C18 column conditioned the same way as the first one. After washing the columns with 1 ml of methanol:water (20:80, v/v) the column was dried and eluted with 4 ml of methanol. Obtained samples were dried on the nitrogen dryer at 40 ◦ C under a mild stream of nitrogen. Calibration standards were prepared in 10 ml-tubes by introducing 10–200 ␮l aliquots of CAP working solution. Calibration standard samples were dried under the same conditions as the samples. 2.3. Urine extraction A 5-ml aliquot of centrifuged urine was transferred to a 50-ml plastic tube. Selected samples were spiked according to the same procedure as for muscle tissue analyses. After incubation of all samples with 1 ml of 0.2 M phosphate buffer (pH 6.0) and 20 ␮l of ␤-glucuronidase at 40 ◦ C for 90 min, they were extracted with 10 ml of ethyl acetate by gentle inverting the tubes in a mechanical rotator for 15 min. Vortexing was found to be unsuitable due to excessive foam formation. The ethyl acetate layer of sample the centrifuged at 3500 rpm was transferred to a new 50-ml plastic tube. Extraction was repeated with 5 ml of ethyl acetate, the combined ethyl acetate extract was centrifuged at 2000 rpm at −5 ◦ C for 10 min and the urine layer was discarded. The extract, dried under nitrogen, was reconstituted in a mixture of 1 ml of 4% sodium chloride solution and 100 ␮l of diethyl ether, sonicated and transferred into a 10 ml-glass tube. The sample remaining in the plastic tubes was added to the glass tube with a mixture of 2 ml of 4% sodium chloride solution and 1 ml of hexane. The organic layer of the centrifuged sample was discarded and the aqueous extract applied to a C18 SPE column as described for muscle tissue.

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tubes were incubated in the oven at 65 ± 5 ◦ C for 30 min. Derivatized samples were transferred into GC vial inserts. An Agilent GC 6890 instrument with autosampler and 5973 MS detector were used. A HP-5MS 30 m × 0.25 mm i.d., 0.25 ␮m film thickness column (Agilent Technologies) was used with helium as the carrier gas at the constant flow rate of 1 ml min−1 . A splitless injection of 2 ␮l was made at 250 ◦ C over 0.75 min. The initial oven temperature was held at 110 ◦ C for 2 min rising to 250 ◦ C at 17 ◦ C min−1 and then to 300 ◦ C at 10 ◦ C min−1 where it was held for 5 min. The detector temperature was 280 ◦ C. The MS detector was operated in the electron impact (EI) ionization mode, ionization energy 70 eV. Data were acquired in single ion monitoring (SIM) mode with the following ions, m/z: 225, 208, 242. The dwell time was 100 ms. 2.5. LC–MS/MS analysis Samples dried after their elution from the C18 column were reconstituted in 100 ␮l of mobile phase, vortexed and transferred into LC vial inserts. The LC– MS/MS system comprised of Shimadzu LC-10AD pump with autosampler and Sciex API III Plus triple quadrupole MS detector. A Hypersil-BDS-C8 5 ␮m 5 × 0.46 cm (Chromatography Sciences Co.) column was used with the mobile phase 50:50 (v/v) acetonitrile:0.025 M ammonium acetate in 0.3% formic acid. The flow rate was 1 ml min−1 , the chromatographic run time 4 min, the injection volume 10 ␮l and the column temperature 30 ◦ C. The MS detector was operated in the negative ion mode with an atmospheric pressure chemical ionization (APCI) source also known as a heated nebulizer. The APCI source was heated to 500 ◦ C. Nitrogen was used as a curtain gas and argon as a collision gas. A m/z 321 ion was selected as a parent and m/z 152, 257 and 194 as daughter ions. The data were acquired in the multiple reaction monitoring (MRM) mode: 321 → 152; 321 → 257; 321 → 194. The dwell time was 400 ms.

2.4. GC–MS analysis 3. Results and discussion Samples dried after elution from the C18 column were reconstituted in 50 ␮l of BSTFA:10%TMCS mixture under anhydrous conditions. Tightly closed

This study involved extraction of CAP from muscle and urine matrices of three animal species. It should

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Table 1 CAP standard calibration and recovery from spiked bovine muscle tissue detected by GC–MS Retention time (ion 225) Standard 1 2 4 10

Standard peak counts Ion 225

Ion 208

Ion 242

136252 290972 647785 1953435

41681 91846 221521 663591

9585 19379 35534 106306

Total peak counts

Relative intensity (%) 208/225

12.601 12.604 12.598 12.600

233801 709074

82933 263106

15507 45791

187518 402197 904840 2723332

30.6 31.6 34.2 34.0

7.0 6.7 5.5 5.4

Mean R.S.D. (%)

32.6 5.5

6.2 13.2

332241 1017971 NDb

37.1 37.1 35.5

6.6 6.5

86.0 103.1

6.5

94.5

Mean b

242/225

(µg kg−1 )a

Spike (µg kg−1 ) 2 12.590 4 12.588 Blank NDb

a

Recovery (%)

r = 0.999; slope = 285509; intercept = −158942. ND, not detected at the noise threshold 10.0.

be noted that extraction of muscle samples has to be done immediately after tissue homogenization. This measure would prevent any loss of CAP due to reactivation of endogenous enzymes in vitro, as was observed in muscle, liver and kidney calf tissues [16]. Glucuronidase hydrolysis of urine samples was incorporated in order to restore CAP from its conjugated state with glucuronic acid present in urine and kid-

ney. It is reported that glucuronidase treatment of pig kidney homogenate spiked with CAP improved its recovery by 80% [17]. Derivatized samples were analyzed by GC–MS, non-derivatized samples by LC–MS/MS. Examples of CAP linearity and recovery calculations are shown for bovine muscle tissue (Table 1) and equine urine (Table 2). Standard curves for CAP were linear within

Table 2 CAP standard calibration recovery from spiked equine urine detected by LC–MS/MS Retention time (ion 152) Standard (µg kg−1 )a 1 2.211 2 2.208 4 2.207 8 2.209

Spike (µg kg−1 ) 2 2.195 4 2.194 Blank NDb

a b

Standard peak counts Ion 152

Ion 257

Ion 194

8263 17986 44135 89542

4451 8767 20563 43970

3536 7146 16422 34020

18259 43905

9124 21015

r = 1.000; slope = 21853; intercept = −7249. ND, not detected at the noise threshold 10.0.

7905 16351

Total peak counts

Relative intensity (%)

Recovery (%)

257/152

194/152

16250 33899 81120 167532

53.9 48.7 46.6 49.1

42.8 39.7 37.2 38.0

Mean R.S.D. (%)

49.6 6.2

39.4 6.3

35288 81271 NDb

50.0 47.9

43.3 37.2

97.3 101.3

Mean

99.3

48.9

40.3

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Fig. 1. GC–MS chromatograms of bovine muscle extracts, SIM scan: blank matrix (a), 2 ␮g kg−1 spiked matrix (b), 2 ␮g kg−1 standard without matrix (c). Ions monitored: (top) 225; (middle) 208; (bottom) 242.

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Fig. 2. GC–MS chromatograms of porcine urine extracts, SIM scan: blank matrix (a), 2 ␮g kg−1 spiked matrix (b). Ions monitored: (top) 225; (middle) 208; (bottom) 242.

Fig. 3. LC–MS/MS chromatograms of bovine muscle extracts, MRM mode: blank matrix (a), 2 ␮g kg−1 spiked matrix (b), 2 ␮g kg−1 standard without matrix (c).

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a range of 1–10 ␮g kg−1 for all conducted experiments, irrespective of the selected detection method. The recovery values for muscles and tissues were >80% and did not differ from one species to another. Chromatograms of different species were similar at 2 ␮g kg−1 level of CAP added to the tissues or urine. Typical GC–MS chromatograms for CAP detection in bovine muscle tissue and porcine urine are shown

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on the Figs. 1 and 2. The level of interference detected by GC–MS in the muscle tissue was generally lower than in urine. The results for bovine muscle tissue and porcine urine using LC–MS/MS detection shown in Figs. 3 and 4 indicate that LC–MS/MS method sensitivity to CAP at the same 2 ␮g kg−1 level was much higher for both matrices—muscles and urine.

Fig. 4. LC–MS/MS chromatograms of porcine urine extracts, MRM mode: blank matrix (a), 2 ␮g kg−1 spiked matrix (b).

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Fig. 4. (Continued ).

The method sensitivity calculation was based on the lowest peak count out of three monitored ions. The signal of a CAP-spiked matrix three times higher than the blank matrix noise level was accepted as the limit of detection. The GC–MS response for muscles and urine, spiked with 2 ␮g kg−1 CAP, produced signal-to-noise values close to 3 (Table 3). The LC–MS/MS response was more than a hundred

times greater than the GC–MS ratios for the same concentration of CAP. Urine, perceived as a “dirty” matrix for GC–MS analyses, produced LC–MS/MS signal-to-noise ratio the same or higher as for muscle (Table 3). The CAP detection limit was calculated on the basis of a signal-to-noise value of 3 and the actual signal-to-noise level observed at 2 ␮g kg−1 CAP. The estimated detection limit of CAP for the GC–MS

A. Gantverg et al. / Analytica Chimica Acta 483 (2003) 125–135 Table 3 Signal-to-noise ratios for GC–MS and LC–MS/MS detection methods at 2 ␮g kg−1 CAP added to the matrix Matrix type

Muscle GC–MS

Urine LC–MS/MS

GC–MS

LC–MS/MS

Bovine

3.2 2.8

263 338

2.5 2.6

515 417

Porcine

4.3 3.8

313 376

3.0 2.1

492 612

Equine

2.9 2.6

275 299

1.4 1.5

606 507

Mean R.S.D. (%)

3.3 20.1

311 13.4

2.2 29.2

525 14.1

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method in muscle and urine was 2 ␮g kg−1 . The corresponding value for LC–MS/MS was <0.02 ␮g kg−1 . This latter is only an estimate due to the limited number of CAP concentration levels tested. The developed method allows detection as well as confirmation of CAP in animal tissues and urine. Examples of CAP LC–MS/MS confirmation data for various muscle tissues are shown in Table 4. CAP confirmation analyses were based on the retention time and ion ratios of a minimum of three ions specific for the CAP standard spectrum. The relative intensities were the ratios calculated for the m/z 257 or 194 ions over the major m/z 152 ion. Reproducibility of these values obtained by LC–MS/MS was high

Table 4 LC–MS/MS ion ratios for chloramphenicol added to muscle tissue at 2 ␮g kg−1 level Spike concentration

Blank 1 Blank 2 2 ␮g kg−1 spike 1 2 ␮g kg−1 spike 2 2 ␮g kg−1 standard

Species

Porcine Porcine Porcine Porcine Standard

Mean R.S.D. (%) Blank 1 Blank 2 2 ␮g kg−1 spike 1 2 ␮g kg−1 spike 2 2 ␮g kg−1 standard

Peak counts

Relative intensity (%)

Ion 152

Ion 257

Ion 194

257/152

194/152

NDa NDa 2.19 2.19 2.19

NDa NDa 16061 21685 17986

NDa NDa 7698 9983 8767

NDa NDa 6257 7875 7146

– – 47.9 46.0 48.7

– – 39.0 36.3 39.7

47.5 2.9

38.3 4.7

– – 47.0 47.5 48.7

– – 37.5 40.1 39.7

47.7 1.8

39.1 3.6

– – 46.1 43.7 48.7 46.2 5.4

– – 36.1 34.8 39.7 36.9 6.9

2.19 0.00 Equine Equine Equine Equine Standard

Mean R.S.D. (%) Blank 1 Blank 2 2 ␮g kg−1 spike 1 2 ␮g kg−1 spike 2 2 ␮g kg−1 standard Mean R.S.D. (%)

Retention time (ion 152)

NDa NDa 2.19 2.19 2.19

NDa NDa 16712 17165 17986

NDa NDa 7848 8161 8767

NDa NDa 6260 6879 7146

2.19 0.00 Bovine Bovine Bovine Bovine Standard

NDa NDa 2.20 2.19 2.19 2.19 0.26

NDa NDa 20061 23125 17986

NDa NDa 9243 10106 8767

NDa NDa 7233 8041 7146

Analysis requirements Acceptability criteria Calibration curve correlation coefficient Retention time R.S.D. R.S.D. of relative intensity to the base peak a

ND, not detected. Noise threshold 10.0.

>0.990 ±0.5% ±15%

Study results 0.9996 0.00–0.26% 1.8–6.9%

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and consistent irrespectively of the matrix tested. R.S.D. fluctuation of ion relative intensities was in the 1.8–6.9% range for the different species (Table 4) and resembled the ion ratios obtained for CAP standards (Table 2). The GC–MS results at 2 ␮g kg−1 CAP were less consistent owing to the inadequate sensitivity of the method. The identical extraction procedure in our experiments with different detection methods allowed a clear

comparison of the detection capabilities of GC–MS and LC–MS/MS. Assuming that the CAP derivatization yield is >50%, the substantial difference in the detection limits observed between the GC–MS and LC–MS/MS methods could only be attributed to the difference between detector sensitivities. Derivatization of CAP with trimethylsilyl reagents leads to the formation of a silylated molecule and its ion, m/z 466. This ion is usually prominent in the softer ionization

Fig. 5. Mass spectrum of trimethylsilylated CAP. GC–MS, EI mode, scan range 150–500.

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mode, such as GC–MS with chemical ionisation [10–12], however, in our study it was not observed in the EI spectrum of CAP (Fig. 5). The high collision energy of EI source causes multiple fragmentation of the CAP molecule and its transformation in the lower molecular weight ions (m/z 208, 225, 242). Detection in the lower ion range is greatly affected by sample extract interference that causes low signal-to-noise ratios. A softer ionization technique using the APCI source preserves CAP in the form of the molecular ion, m/z 321. This ion is separated from the other sample ions and selectively transferred into the second quadrupole before being fragmented. Such a technique generates a sensitive response coupled with very low noise. The pronounced electron affinity of CAP is an additional factor contributing to its efficient detection in the negative ion mode that was used for LC–MS/MS. The described LC–MS/MS method for chloramphenicol detection in the APCI negative mode, allows greater sensitivity than other methods of chloramphenicol detection.

4. Conclusions 1. LC–MS/MS allows detection and confirmation of CAP in equine, porcine and bovine muscle and urine matrices with a detection limit of 0.02 ␮g kg−1 , which represents about a 100-fold improvement in sensitivity over the GC–MS method. 2. The GC–MS technique is unsuitable for CAP detection at levels <2 ␮g kg−1 due to insufficient detector sensitivity and selectivity in the electron impact mode. 3. The high sensitivity of the LC–MS/MS method can be attributed to the following three factors: (a) the softer ionization technique of the LC–MS ion source; (b) the presence of the second stage mass spectrometer acting as a “filter” for interfering ions from the sample extract;

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(c) the pronounced electron affinity of CAP that causes its efficient detection in the negative ion mode. Acknowledgements We thank Prof. Robert Epstein (USDA) for the advice on the CAP extraction procedure. Authors would also like to thank Roger Demers, Sami Jamokha, Gene Aznar and Hari Pal for their technical expertise that contributed to this work and the US Export Group for funding this research. References [1] European Commission, Amending Regulation 1430/94, 1994. [2] J.-P. Abjean, J. AOAC Int. 80 (1997) 737. [3] C. Hummert, B. Luckas, H. Siebenlist, J. Chromatogr. B 668 (1995) 53. [4] S.V.C. De Souza, G. Silva, M.H.G.M. Dinitz, E.V. Dos Santos, J.A. Lima, J.C. Teodoro, Rev. Braz. Toxicol. 13 (2000) 17. [5] A.P. Pfenning, J.E. Roybal, H.S. Rupp, S.B. Turnipseed, S.A. Gonzales, J.A. Hurlbut, J. AOAC Int. 83 (2000) 26. [6] R.L. Epstein, R.B. Ashworth, R.M. Simpson, Am. J. Vet. Res. 47 (1986) 2075. [7] C. Van de Water, N. Haagsma, J. Chromatogr. B 566 (1991) 173. [8] A.V. Kolosova, J.V. Samsonova, A.M. Egorov, Food Agric. Immunol. 12 (2000) 115. [9] D. Arnold, A. Somogyi, J. AOAC Int. 68 (1985) 984. [10] R.L. Epstein, C. Henry, K.P. Holland, J. Dreas, J. AOAC Int. 77 (1994) 570. [11] S. Börner, H. Fry, G. Balizs, R. Kroker, J. AOAC Int. 78 (1995) 1153. [12] L.A. Van Ginkel, H.J. Van Rossum, P.W. Zoontjes, H. Van Blitterswijk, G. Ellen, E. Van der Heft, A.P.J.M. De Jong, G. Zomer, Anal. Chim. Acta 237 (1990) 61. [13] T. Nagata, H. Oka, J. Agric. Food Chem. 44 (1996) 1280. [14] V. Hormazabal, M. Yndestad, J. Liquid Chromatogr. Relat. Technol. 24 (2001) 2477. [15] J. Morgan, M.E. Joyce-Menekse, R.T. Rowlands, I.H. Gilbert, D. Lloid, Rapid Commun. Mass Spectrom. 15 (2001) 1229. [16] P. Sanders, P. Guillot, M. Dagorn, J.M. Delmas, J. AOAC Int. 74 (1991) 483. [17] A.D. Cooper, J.A. Tarbin, W.H.H. Farrington, G. Shearer, Food Additives Contaminants 15 (1998) 637.