A RAPID METHOD FOR THEDETERMINATION OF CHLORAMPHENICOL

RESIDUES IN BLACK TIGER SHRIMP

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Gordon Kearney, Anthony NewtonWaters Corporation, Atlas Park, Simonsway, Manchester, UK.

INTRODUCTION

The global production of fish, as a food item, hasgreatly increased over the last few decades and isnow at the highest levels on record. Currently, morethan 15% of world protein consumption is providedby aquatic species, both captured and farmed. Thegrowth in global human population means that thedemand for fish will continue to rise, creatingpressures on already over fished wild stocks ofcommercially caught species. Because of thisdemand, starting in the mid 1980s, there has been asteady increase in the quantity of fish produced byaquaculture, especially in Far Eastern and SouthAmerican countries1.

Antibiotics are used in aquaculture, as in otherfarming methods, in order to prevent and treatbacterial diseases that would otherwise lead toincreased animal mortality, reduced growth andlower yield at harvest. In the recent past there hasbeen, in some countries, a tendency to overuseantibiotic treatments in order to compensate fordeficiencies in aquacultural practice. Theindiscriminate use of antibiotic compounds has led to concerns that it may cause increasing levels ofdrug resistance in organisms pathogenic to humans.Also, some of these compounds may have long termtoxic effects when regularly consumed as residues inthe diet2.

Waters® Micromass® Quattro micro™ API triple-quadrupole mass spectrometer

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One of the most commercially lucrative speciesfarmed in the Far East, and increasingly in SouthAmerica, is the black tiger shrimp Penaeus monodon,Figure 1, which is exported in quantity to manyWestern nations.

Recent shipments of shrimp, from various Far Easterncountries, into Europe, Canada and the United Statesof America, were found to be contaminated withresidues of chloramphenicol Figure 2, a broad-spectrum antibiotic used to treat bacterial infections inhumans. Chloroamphenicol is a cause of a conditionknown as idiosyncratic aplastic anemia, a depressionof the ability of bone marrow to produce blood cells.Approximately one in thirty thousand individuals havea hypersensitivity to chloramphenicol that may causethis aplasia irrespective of exposure level3. For thisreason, no Acceptable Daily Intake (ADI) can bedetermined and the drug has been listed in annex IVof the European Union Council regulation2377/90/EEC. This means that it is prohibited foruse in animals destined for human consumption andany produce contaminated with chloramphenicolresidues may not be sold. Equivalent regulationsapply in other Western countries.

There is no Maximum Residue Level (MRL) set forchloramphenicol in food of animal origin and anydetection and confirmation of the compound will leadto condemnation of produce. For the protection ofconsumers it is necessary to have as sensitive ananalytical method as possible in order to detect itsunlawful use. Many thousands of metric tonnes ofshrimp are exported to the West every year and arapid, sensitive and confirmatory method is requiredto determine the presence and quantity ofchloramphenicol in these exports. This paperdescribes an extraction and LC/MS/MS method forthe quantification and confirmation ofchloramphenicol in raw black tiger shrimp.

METHOD

Extraction

A quantity of peeled, de-veined, raw shrimp meat isfinely chopped. 10 g shrimp meat is weighed into a50 mL polypropylene test tube with screw cap lid and2 mL water is added. Shrimp meat containsapproximately 80% water. 10 mL acetone is addedand the tube contents are macerated for 1 min usingan Ultra Turrax homogeniser. The tube is thencentrifuged at 6000 rpm for 5 mins. An 8 mL aliquotof the supernatant is transferred to a secondpolypropylene tube. 10 µL formic acid is added tothe second tube and the contents mixed. 5 mL ethylacetate is added and the tube is shaken for 1 min.The upper organic phase is transferred to a 14 mLcapacity glass vial and the aqueous phase is re-extracted with 5 mL ethyl acetate. The extracts arecombined, the vial is placed on a heated block at 40 °C and the solvent is evaporated under a streamof nitrogen. 1 mL aqueous 0.1% acetic acid is addedto the vial together with 1 mL hexane. The vial isvortexed for 30 s and the hexane is carefullyremoved with a pipette. The aqueous sample isfiltered through a 0.45 µm membrane filter into a 2 mL glass sample vial. The vials are spiked withdeuterated internal standard prior to analysis. Usingthis method 1 mL analytical sample is equivalent to 4 g shrimp meat.

Figure 1. Black Tiger Shrimp.

Figure 2. Structure of chloramphenicol.

Chromatography

Chromatographic separation was carried out using an Waters®

Alliance® System with the 2795 Separations Module and a 4.6x 50 mm Symmetry® C8 3.5 µm HPLC column. A 3.9 x 20 mmSymmetry C8 3.5 µm pre-column was placed upstream of themain analytical column. Mobile phase A was 0.1% formic acidin water and mobile phase B was 0.1% formic acid inacetonitrile. The sample injection volume was 50 µL and themobile phase flow rate was 1 mL/min. The chromatographicgradient that was applied is shown in table 1.

Mass Spectrometry

Chloramphenicol was determined using a Waters® Micromass®

Quattro micro API triple-quadrupole mass spectrometer with anelectrospray ion source in negative polarity. Initial experimentswere conducted to compare the sensitivity and selectivity ofSingle Ion Recording (SIR) MS with Multiple Reaction Monitoring(MRM) MS/MS acquisitions. Figure 3 shows chromatogramsfrom SIR and MRM analyses of chloramphenicol spiked intoblank shrimp matrix. The MRM method is highly selective forchloramphenicol, removing much of the baseline chemical noiseand giving a much higher S:N value than that obtained usingthe SIR method. Therefore, an MRM method was chosen for thedetermination of chloramphenicol in this study.

Table 2 shows the MRM transitions monitored, together with theirdwell times, cone voltages and fragmentation collision energies.

Chromatograms corresponding to these 4 transitions are shownin Figure 4.

The abundance ratio of the quantification to the 1st confirmatorytransition is 1.06. The abundance ratio of the quantification tothe 2nd confirmatory transition is 1.53. The presence ofchloramphenicol is considered confirmed if the observedabundance ratios do not deviate by more than 20% from theseexpected values4.

A series of matrix-matched calibration standards, spiked matrixsamples, matrix blanks and recovery samples were analysed inorder to determine method accuracy, linearity, precision,repeatability and recovery. The limit of determination (LoD) wasalso estimated. Internal standard was spiked at 0.5 µg/Kg in allsamples. Calibration standards were made up at 0.025, 0.5,0.2, 0.4, 0.6 and 0.8 µg/Kg. Spiked matrix samples weremade up at 0.1 and 0.5 µg/Kg. Recovery samples were spikedat 0.1 µg/Kg prior to extraction.

Table 1. Chromatographic Gradient.

Figure 3. SIR compared to MRM for chloramphenicol in shrimp matrix.

Transition Precursor Ion (m/z)

Product Ion (m/z)

Dwell Time (s)

Cone Voltage (V)

Collision Energy (eV)

Quantification

321 152 0.08 30 18

1st Confirmatory

321 257 0.08 30 12

2nd Confirmatory

323 152 0.08 30 18

Internal Standard

326 157 0.08 30 18

Table 2. MRM parameters.

Time / min 0 mins 0.1 mins 6.0 mins 6.1 mins 7.1 mins 7.2 mins 12 mins % B 0 % B 25 % B 56 % B 100 % B 100 % B 0 % B 0 % B

Standard 0.05 µg/Kg

2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60Time1

100

%

1

100

%

1

100

%

0

100

%

05Sep_05 Sm (Mn, 1x2) MRM of 4 Channels ES- 326 > 157.1

7.74e33.17

05Sep_05 Sm (Mn, 1x2) MRM of 4 Channels ES- 323 > 152

1.56e33.20

05Sep_05 Sm (Mn, 1x2) MRM of 4 Channels ES- 321 > 257

1.44e33.20

05Sep_05 Sm (Mn, 1x2) MRM of 4 Channels ES- 321 > 152

2.31e33.20

Quantification Chromatogram

2nd Confirmatory Chromatogram

1st Confirmatory Chromatogram

Internal Standard

Figure 4. MRM chromatograms.

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RESULTS

Figure 5 shows chromatograms for the three MRMtransitions, taken from analysis of the calibrationstandard at 0.025 µg/Kg. The observed peak topeak S:N ratio for the most sensitive transition (m/z 321 > m/z 152) is 42:1. By extrapolation, theLoD, defined as the concentration at which S:N is3:1, is approximately 0.002 µg/Kg.

The linearity of the method is demonstrated by thecalibration graph shown in Figure 6.

Method accuracy and precision are demonstrated bythe graph in Figure 7. Six replicate analyses wereperformed on matrix spiked at 0.1 and 0.5 µg/Kg.Mean average observed concentrations were 0.10and 0.50 µg/Kg and relative standard deviationvalues were 2.3% and 2.8% respectively.

Method repeatability is shown by the graph in Figure 8.The response factor is shown for all standards andspiked samples during a batch analysis. The relativestandard deviation of response factor is 5.7%.

Four recovery experiments were performed on shrimpspiked at the 0.1 µg/Kg level. Each sample wasanalysed three times, giving 12 determinations in total.The mean observed concentration was 0.092 µg/Kgwith a relative standard deviation of 8.9%.

During a batch analysis of over 100 injections the ionabundance ratios of the transitions did not deviate bymore than 20% from the expected values, even forthe lowest calibrated level of 0.025 µg/Kg. Thismeans that the method is able to confirm the presenceof chloramphenicol at the lowest calibrated level.During this batch analysis the chromatographic peakretention time, width at half height and asymmetryfactor remained constant.

Compound name: ChloramphenicolCorrelation coefficient: r = 0.999858, r^2 = 0.999715Calibration curve: 2.99162 * x + 0.0106534Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0.000 0.200 0.400 0.600 0.800µg/Kg0.00

2.42

Response

Figure 6. Calibration graph for chloramphenicol inshrimp matrix.

6 Replicate Analyses of Matrix Spikes

0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6

Obs

erve

d C

onc.

Spiked at 0.1µg/KgSpiked at 0.5µg/Kg

Figure 7. Results of 6 replicate measurements at twospiked levels.

Matrix Standard @ 0.025 µg/Kg

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00Time1

100

%

1

100

%

1

100

%

05Sep_17 MRM of 4 Channels ES- 323 > 152

1.07e3S/N:PtP=25.41

1.47 1.96

05Sep_17 MRM of 4 Channels ES- 321 > 257

896S/N:PtP=13.74

0.70 1.65

05Sep_17 MRM of 4 Channels ES- 321 > 152

1.61e3S/N:PtP=42.70

1.61

Figure 5. Chromatograms from lowest calibration standard.

Response Factor During Batch Analysis

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Resp

onse

Fac

tor

Figure 8. Graph showing response factor overmultiple injections at different levels.

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CONCLUSION

A rapid and sensitive analytical method is required inorder to protect consumers from shrimp contaminatedwith chloramphenicol. Multiple MRM transitions allowconfirmatory criteria to be met whilst providingexcellent sensitivity and selectivity. A quick extractionand short sample analysis time mean that this methodmay be applicable in a high throughput environment.

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REFERENCES

1. The State of World Fisheries and Aquaculture, Food and

Agriculture Organisation of the United Nations, 2002.

2. M. Flaherty and C. Karnjanakesorn, Marine Shrimp

Aquaculture and Natural Resource Degredation in Thailand,

Environmental Management, 1995, 19(1), pp 27-37.

3. J. E. Roybal, Analytical Procedures for Drug Residues in

Food of Animal Origin, Eds., S. B. Turnipseed and A. R.

Long, Science Technology System, Sacramento, CA,

1998, pp 227-260.

4. SANCO/1085/2000, Commission of the European

Communities, Brussels, 2000.

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