determination of chloramphenicol in animal tissues and urinine lcma versus gcms

Analytica Chimica Acta 483 (2003) 125–135

Determination of chloramphenicol in animal tissues and urineLiquid chromatography–tandem mass spectrometry versus

gas chromatography–mass spectrometry

Alex Gantverga,∗, Isaac Shishania, Michael Hoffmanb,1

a Maxxam Analytics Inc., 5540 McAdam Road, Mississauga, Ont., Canada L4Z 1P1b 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 andbovine muscle and urine. The method included ethyl acetate extraction of CAP followed by a two-step C18 solid-phase cleanup; 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 comparedto that shown by gas chromatography–mass spectrometry (GC–MS) in electron impact mode. Even in a “dirty” matrix, suchas urine, the estimated CAP detection limit for LC–MS/MS detection was 0.02�g kg−1 while the corresponding value forGC–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 antibioticthat has widely been used since the 1950s to treatfood-producing animals. In 1994, the use of CAP wasbanned by the European Union (EU) due to the poten-tial health risk posed by its traces in food[1]. Alongwith continued improvement of analytical method sen-sitivity, the EU has been lowering the detection limitof prohibited drugs in meat to ensure the highest safetystandards for food products. The year 2001 the detec-tion limit set by EU for CAP detection in urine andtissues 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 determi-nation of CAP in biological matrices, based on analyt-ical techniques such as planar[2], liquid [3,4] and gaschromatography[5,6]. Enzyme-linked immunosor-bent assay (ELISA)[7,8] and radioimmunoassay[9]are also widely used for CAP determination. The highsensitivity of an electron capture detector (ECD) toCAP 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

126 A. Gantverg et al. / Analytica Chimica Acta 483 (2003) 125–135

methods: 1�g kg−1 CAP was detected in shrimptissue using a GC-ECD method[5].

The focus of our research, however, has been onmethods allowing the confirmation of CAP in tissues.The most common method for confirmation of CAP isGC–mass spectrometry (MS) with negative ion chem-ical ionization (CI) mode. This technique has demon-strated excellent sensitivity down to 0.1�g kg−1 inmuscle tissues, although in urine the results wereless sensitive due to the matrix interference[10–12].GC–MS in electron impact (EI) mode, though slightlyless sensitive, has the advantage of obtaining spec-tra reproducible between different instruments andstored in electronic libraries. The sensitivity achievedin fish muscles with CAP detection by GC–MS in EImode was 5�g kg−1 [13]. The general drawback ofusing GC methods for CAP detection has been thenecessity to derivatize CAP in order to improve itschromatographic properties. Liquid chromatography(LC) methods, however, do not require a derivatiza-tion step and LC–MS sensitivity approaches that ofGC–MS. The limit of quantification for CAP in urinemeasured by LC–MS method with an electrosprayion source was 3�g kg−1 [14]. Another publicationalso demonstrates the possibility of using LC–MS inelectrospray negative ion mode to detect the CAPm/z321 molecular ion[15].

Tandem mass spectrometry (MS/MS) is a more so-phisticated technique allowing a very effective isola-tion of analyte ions from the noise-producing matrix.However, no publications were found utilizing thistechnique for CAP detection. The objectives of thisresearch were to develop an effective extraction pro-cedure of CAP from matrices such as horse, beef, andpork urine and muscle tissue, and to select the mostefficient CAP confirmation technique at the regulatorylevel of 2�g kg−1.

2. Experimental

2.1. Reagents and chemicals

Methanol, ethyl acetate, formic acid, hexane, ace-tonitrile, diethyl ether, sodium chloride, sodium sul-fate, sodium dihydrogen phosphate and dihydrogenpotassium phosphate were analytical grade suppliedby VWR. CAP,�-glucuronidase (fromHelix pomatia)

and dithiothreitol were purchased from Sigma. TMSderivatization reagent BSTFA+ 10%TMCS in 1-mlampoules was obtained from Pierce. Solid-phase ex-traction (SPE) columns (Bond Elut-C18, 500 mg, 6 ml)were purchased from Varian.

CAP stock solution (100�g ml−1) was prepared inmethanol every 3 months and stored below−10◦C.CAP working solution (0.2�g ml−1) was made by di-luting stock solution with ethyl acetate. This solutionwas prepared biweekly. Equine, bovine and porcinesamples of muscles and urine were procured from sev-eral farms in Canada and the United States.

2.2. Muscle tissue extraction

Tissue sample (5.0 g) was weighed in a 50-ml plastictube. Selected blank samples were spiked with knownquantities of CAP working solution. A 5.0 g of sodiumsulfate and 10 ml of 0.5 g l−1 dithiothreitol solutionin ethyl acetate were added to the tube. Dithiothreitol(Cleland’s reagent) was used to disrupt any proteinbinding of CAP.

The sample tissue was homogenized with ultraturrax and the tissue residue in the homogenizer wasrinsed with an additional 5 ml of dithiothreitol solu-tion. After vortexing, the sample was centrifuged at2000 rpm for 5 min. The supernatant was decantedinto a new 50-ml plastic tube. The remaining sam-ple precipitate was rinsed with an additional 5 mlof dithiothreitol solution and centrifuged. The su-pernatants were combined and placed in the freezerbelow−10◦C for at least 1 h.

Precipitated fat was removed by centrifuging at3500 rpm for 5 min at−5◦C. The supernatant wasdried on the nitrogen dryer at 40◦C. Excessive dryingand exposure to the atmosphere should be avoided toprevent loss of CAP. The residue was reconstituted in4 ml of 4% sodium chloride solution and 2 ml of hex-ane. The hexane fraction of the centrifuged sampleswas discarded. The wash step of the aqueous fractionwas repeated with 2 ml of hexane.

A C18 column was equilibrated with 5 ml ofmethanol, 5 ml of chloroform, 5 ml of methanol and5 ml of water using the vacuum pump at the minimumpressure. After passing the entire sample through thecolumn it was rinsed with two 2-ml aliquots of waterfollowed by 2 ml of methanol:water (20:80, v/v). Thesample was dried for 1 min at high vacuum pressure,

A. Gantverg et al. / Analytica Chimica Acta 483 (2003) 125–135 127

CAP was eluted with 3 ml of methanol:water (50:50,v/v) and the eluate was collected under gravity inglass tubes.

In the second C18 step, the eluate was diluted with7 ml water and applied to a C18 column conditionedthe same way as the first one. After washing thecolumns with 1 ml of methanol:water (20:80, v/v) thecolumn was dried and eluted with 4 ml of methanol.Obtained samples were dried on the nitrogen dryerat 40◦C under a mild stream of nitrogen. Calibrationstandards were prepared in 10 ml-tubes by introduc-ing 10–200�l aliquots of CAP working solution.Calibration standard samples were dried under thesame conditions as the samples.

2.3. Urine extraction

A 5-ml aliquot of centrifuged urine was transferredto a 50-ml plastic tube. Selected samples were spikedaccording to the same procedure as for muscle tis-sue analyses. After incubation of all samples with1 ml of 0.2 M phosphate buffer (pH 6.0) and 20�l of�-glucuronidase at 40◦C for 90 min, they were ex-tracted with 10 ml of ethyl acetate by gentle invertingthe tubes in a mechanical rotator for 15 min. Vortex-ing was found to be unsuitable due to excessive foamformation.

The ethyl acetate layer of sample the centrifuged at3500 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 at2000 rpm at−5◦C for 10 min and the urine layer wasdiscarded. The extract, dried under nitrogen, was re-constituted in a mixture of 1 ml of 4% sodium chlo-ride solution and 100�l of diethyl ether, sonicatedand transferred into a 10 ml-glass tube. The sampleremaining in the plastic tubes was added to the glasstube with a mixture of 2 ml of 4% sodium chloridesolution and 1 ml of hexane. The organic layer of thecentrifuged sample was discarded and the aqueous ex-tract applied to a C18 SPE column as described formuscle tissue.

2.4. GC–MS analysis

Samples dried after elution from the C18 columnwere reconstituted in 50�l of BSTFA:10%TMCSmixture under anhydrous conditions. Tightly closed

tubes were incubated in the oven at 65± 5◦C for30 min. Derivatized samples were transferred into GCvial inserts.

An Agilent GC 6890 instrument with autosamplerand 5973 MS detector were used. A HP-5MS 30 m×0.25 mm i.d., 0.25�m film thickness column (AgilentTechnologies) was used with helium as the carrier gasat the constant flow rate of 1 ml min−1. A splitless in-jection of 2�l was made at 250◦C over 0.75 min. Theinitial oven temperature was held at 110◦C for 2 minrising to 250◦C at 17◦C min−1 and then to 300◦Cat 10◦C min−1 where it was held for 5 min. The de-tector temperature was 280◦C. The MS detector wasoperated in the electron impact (EI) ionization mode,ionization energy 70 eV. Data were acquired in singleion 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 col-umn were reconstituted in 100�l of mobile phase,vortexed and transferred into LC vial inserts. The LC–MS/MS system comprised of Shimadzu LC-10ADpump with autosampler and Sciex API III Plus triplequadrupole MS detector. A Hypersil-BDS-C8 5�m5 × 0.46 cm (Chromatography Sciences Co.) columnwas used with the mobile phase 50:50 (v/v) ace-tonitrile:0.025 M ammonium acetate in 0.3% formicacid. The flow rate was 1 ml min−1, the chromato-graphic run time 4 min, the injection volume 10�land the column temperature 30◦C. The MS detec-tor was operated in the negative ion mode with anatmospheric pressure chemical ionization (APCI)source also known as a heated nebulizer. The APCIsource was heated to 500◦C. Nitrogen was used as acurtain gas and argon as a collision gas. Am/z 321ion was selected as a parent andm/z 152, 257 and194 as daughter ions. The data were acquired in themultiple reaction monitoring (MRM) mode: 321→152; 321→ 257; 321→ 194. The dwell time was400 ms.

3. Results and discussion

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


Table 1CAP standard calibration and recovery from spiked bovine muscle tissue detected by GC–MS

Retention time(ion 225)

Standard peak counts Total peakcounts

Relative intensity (%) Recovery (%)

Ion 225 Ion 208 Ion 242 208/225 242/225

Standard (µg kg−1)a

1 12.601 136252 41681 9585 187518 30.6 7.02 12.604 290972 91846 19379 402197 31.6 6.74 12.598 647785 221521 35534 904840 34.2 5.5

10 12.600 1953435 663591 106306 2723332 34.0 5.4

Mean 32.6 6.2R.S.D. (%) 5.5 13.2

Spike (µg kg−1)2 12.590 233801 82933 15507 332241 37.1 6.6 86.04 12.588 709074 263106 45791 1017971 37.1 6.5 103.1

Blank NDb NDb 35.5

Mean 6.5 94.5

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

be noted that extraction of muscle samples has to bedone immediately after tissue homogenization. Thismeasure would prevent any loss of CAP due to reac-tivation of endogenous enzymes in vitro, as was ob-served in muscle, liver and kidney calf tissues[16].

Glucuronidase hydrolysis of urine samples was in-corporated in order to restore CAP from its conjugatedstate with glucuronic acid present in urine and kid-

Table 2CAP standard calibration recovery from spiked equine urine detected by LC–MS/MS

Retention time(ion 152)

Standard peak counts Total peakcounts

Relative intensity (%) Recovery (%)

Ion 152 Ion 257 Ion 194 257/152 194/152

Standard (µg kg−1)a

1 2.211 8263 4451 3536 16250 53.9 42.82 2.208 17986 8767 7146 33899 48.7 39.74 2.207 44135 20563 16422 81120 46.6 37.28 2.209 89542 43970 34020 167532 49.1 38.0

Mean 49.6 39.4R.S.D. (%) 6.2 6.3

Spike (µg kg−1)2 2.195 18259 9124 7905 35288 50.0 43.3 97.34 2.194 43905 21015 16351 81271 47.9 37.2 101.3Blank NDb NDb

Mean 99.3 48.9 40.3

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

ney. It is reported that glucuronidase treatment of pigkidney homogenate spiked with CAP improved its re-covery by 80%[17].

Derivatized samples were analyzed by GC–MS,non-derivatized samples by LC–MS/MS. Examplesof CAP linearity and recovery calculations are shownfor bovine muscle tissue (Table 1) and equine urine(Table 2). Standard curves for CAP were linear within


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 standardwithout matrix (c). Ions monitored: (top) 225; (middle) 208; (bottom) 242.


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).


a range of 1–10�g kg−1 for all conducted experi-ments, 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 at2�g kg−1 level of CAP added to the tissues or urine.Typical GC–MS chromatograms for CAP detectionin bovine muscle tissue and porcine urine are shown

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

on the Figs. 1 and 2. The level of interference de-tected by GC–MS in the muscle tissue was generallylower than in urine. The results for bovine muscletissue and porcine urine using LC–MS/MS detectionshown in Figs. 3 and 4indicate that LC–MS/MSmethod sensitivity to CAP at the same 2�g kg−1 levelwas much higher for both matrices—muscles andurine.


Fig. 4. (Continued ).

The method sensitivity calculation was based onthe lowest peak count out of three monitored ions.The signal of a CAP-spiked matrix three times higherthan the blank matrix noise level was accepted asthe limit of detection. The GC–MS response formuscles and urine, spiked with 2�g kg−1 CAP, pro-duced 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 sameconcentration of CAP. Urine, perceived as a “dirty”matrix for GC–MS analyses, produced LC–MS/MSsignal-to-noise ratio the same or higher as for muscle(Table 3). The CAP detection limit was calculated onthe basis of a signal-to-noise value of 3 and the ac-tual signal-to-noise level observed at 2�g kg−1 CAP.The estimated detection limit of CAP for the GC–MS


Table 3Signal-to-noise ratios for GC–MS and LC–MS/MS detection meth-ods at 2�g kg−1 CAP added to the matrix

Matrix type Muscle Urine

GC–MS LC–MS/MS GC–MS LC–MS/MS

Bovine 3.2 263 2.5 5152.8 338 2.6 417

Porcine 4.3 313 3.0 4923.8 376 2.1 612

Equine 2.9 275 1.4 6062.6 299 1.5 507

Mean 3.3 311 2.2 525R.S.D. (%) 20.1 13.4 29.2 14.1

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

Spike concentration Species Retention time(ion 152)

Peak counts Relative intensity (%)

Ion 152 Ion 257 Ion 194 257/152 194/152

Blank 1 Porcine NDa NDa NDa NDa – –Blank 2 Porcine NDa NDa NDa NDa – –2�g kg−1 spike 1 Porcine 2.19 16061 7698 6257 47.9 39.02�g kg−1 spike 2 Porcine 2.19 21685 9983 7875 46.0 36.32�g kg−1 standard Standard 2.19 17986 8767 7146 48.7 39.7

Mean 2.19 47.5 38.3R.S.D. (%) 0.00 2.9 4.7

Blank 1 Equine NDa NDa NDa NDa – –Blank 2 Equine NDa NDa NDa NDa – –2�g kg−1 spike 1 Equine 2.19 16712 7848 6260 47.0 37.52�g kg−1 spike 2 Equine 2.19 17165 8161 6879 47.5 40.12�g kg−1 standard Standard 2.19 17986 8767 7146 48.7 39.7

Mean 2.19 47.7 39.1R.S.D. (%) 0.00 1.8 3.6

Blank 1 Bovine NDa NDa NDa NDa – –Blank 2 Bovine NDa NDa NDa NDa – –2�g kg−1 spike 1 Bovine 2.20 20061 9243 7233 46.1 36.12�g kg−1 spike 2 Bovine 2.19 23125 10106 8041 43.7 34.82�g kg−1 standard Standard 2.19 17986 8767 7146 48.7 39.7Mean 2.19 46.2 36.9R.S.D. (%) 0.26 5.4 6.9

Analysis requirements

Acceptability criteria Study results

Calibration curve correlation coefficient >0.990 0.9996Retention time R.S.D. ±0.5% 0.00–0.26%R.S.D. of relative intensity to the base peak ±15% 1.8–6.9%

a ND, not detected. Noise threshold 10.0.

method in muscle and urine was 2�g kg−1. The corre-sponding value for LC–MS/MS was<0.02�g kg−1.This latter is only an estimate due to the limitednumber of CAP concentration levels tested.

The developed method allows detection as wellas confirmation of CAP in animal tissues and urine.Examples of CAP LC–MS/MS confirmation data forvarious muscle tissues are shown inTable 4. CAPconfirmation analyses were based on the retentiontime and ion ratios of a minimum of three ions spe-cific for the CAP standard spectrum. The relativeintensities were the ratios calculated for them/z 257or 194 ions over the majorm/z 152 ion. Reproducibil-ity of these values obtained by LC–MS/MS was high


and consistent irrespectively of the matrix tested.R.S.D. fluctuation of ion relative intensities was inthe 1.8–6.9% range for the different species (Table 4)and resembled the ion ratios obtained for CAP stan-dards (Table 2). The GC–MS results at 2�g kg−1

CAP were less consistent owing to the inadequatesensitivity of the method.

The identical extraction procedure in our experi-ments with different detection methods allowed a clear

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

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


mode, such as GC–MS with chemical ionisation[10–12], however, in our study it was not observed inthe EI spectrum of CAP (Fig. 5).

The high collision energy of EI source causes mul-tiple fragmentation of the CAP molecule and its trans-formation in the lower molecular weight ions (m/z208, 225, 242). Detection in the lower ion range isgreatly affected by sample extract interference thatcauses low signal-to-noise ratios. A softer ionizationtechnique using the APCI source preserves CAP inthe form of the molecular ion,m/z 321. This ion isseparated from the other sample ions and selectivelytransferred into the second quadrupole before beingfragmented. Such a technique generates a sensitive re-sponse coupled with very low noise. The pronouncedelectron affinity of CAP is an additional factor con-tributing to its efficient detection in the negative ionmode that was used for LC–MS/MS.

The described LC–MS/MS method for chloram-phenicol detection in the APCI negative mode, allowsgreater sensitivity than other methods of chloram-phenicol detection.

4. Conclusions

1. LC–MS/MS allows detection and confirmationof CAP in equine, porcine and bovine mus-cle and urine matrices with a detection limit of0.02�g kg−1, which represents about a 100-foldimprovement in sensitivity over the GC–MSmethod.

2. The GC–MS technique is unsuitable for CAP de-tection at levels<2�g kg−1 due to insufficientdetector sensitivity and selectivity in the electronimpact mode.

3. The high sensitivity of the LC–MS/MS methodcan 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 spec-

trometer acting as a “filter” for interfering ionsfrom the sample extract;

(c) the pronounced electron affinity of CAP thatcauses its efficient detection in the negativeion mode.

Acknowledgements

We thank Prof. Robert Epstein (USDA) for the ad-vice on the CAP extraction procedure. Authors wouldalso like to thank Roger Demers, Sami Jamokha, GeneAznar and Hari Pal for their technical expertise thatcontributed to this work and the US Export Group forfunding this research.

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determination of chloramphenicol in animal tissues and urinine lcma versus gcms

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