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LC/MS/MS Analysis of Chloramphenicol in Shrimp Using Solid Phase Extraction

Philip J. Koerner, Matthew Trass, Liming Peng, and Jeff Layne

Phenomenex, Inc., 411 Madrid Ave.,Torrance, CA 90501 USA

Chloramphenicol (CAP) is a broad spectrum antibiotic that exhibits activity against both gram-positive and gram-negative bacteria. CAP has been commonly used in aquaculture as a disinfectant to prevent diseases, or as a chemotherapeutic agent to control diseases.

CAP has been implicated as a probable causative agent of aplastic anemia and as a possible carcinogen. Therefore, its use in aquaculture and meat-producing animals has been banned in the European Union (EU), Canada, and the United States (USA).

Despite this ban, CAP is still used illegally to treat seafood products because of its broad spectrum activity, ready availability, and low cost.

The present methodology specified in the EU has defined a maximum residue limit (MRL) of CAP at 0.3 ng/g, while China has set an MRL of 0.5 ng/g.

The current official USFDA method uses HPLC coupled with MS/MS detection. We describe here an approach for the analysis of chloramphenicol in shrimp that utilizes liquid-liquid extraction of the shrimp tissue followed by solid phase extraction (SPE) for sample cleanup and concentration, and ultra-fast LC/MS/MS analysis using a Kinetex® core-shell C18 HPLC column.

Introduction

Compound Structure

Chloramphenicol is a hydrophobic compound that is very slightly acidic (due to its amide functionality). Therefore, chloramphenicol is an ideal candidate for SPE using hydrophobic retention followed by reversed phase HPLC.

H

H

O2N

OH HN

OHO

Cl

Cl

Kinetex Core-Shell Technology

2.6 µm Core-Shell

Particle

Performance equivalent to or better

than fully porous sub-2 µm particles

Can be used on conventional HPLC

systems and UHPLC/UPLC® systems

0.35 µm Porous Shell

1.9 µm Solid Core

™

Sample Preparation

Homogenize ~100 g of thawed shrimp using a blender or tissue homogenizer.

Weigh out 5 g of homogenized shrimp and transfer to a 15 mL polypropylene tube.

Add 50 μL of Chloramphenicol-d5 internal standard (ISTD) solution and vortex thoroughly

to ensure adequate distribution of the ISTD throughout the homogenate.

Add 2 mL of water and vortex well to mix.

Add 5 mL of ethyl acetate. Transfer the tube to a mechanical

shaker and shake vigorously for 30 minutes.

Centrifuge at 7000 rpm for 15 minutes.

Transfer the supernatant to a new 15 mL polypropylene test

tube, reserving the tissue pellet.

Add 5 mL of ethyl acetate to the tissue pellet from the previous

step and repeat the extraction process.

Combine the resulting ethyl acetate supernatant with the previously obtained extract.

Dry the combined supernatant extracts under nitrogen gas at 55 ºC.

Reconstitute with 300 μL of methanol and dilute to 10 mL with

water. At this point, the sample is ready for solid phase extraction.

Solid Phase Extraction

The extracted shrimp tissue is further cleaned up and concentrated using SPE

Cartridge: Strata™-X 60 mg / 3 mL

Part No.: 8B-S100-UBJ

Condition: 3 mL Methanol (1-2 mL/min)

Equilibrate: 3 mL Water (1-2 mL/min)Load: 10 mL extracted shrimp tissue sample (1 mL/min)

Wash: 3 mL of WaterDry: >10” Hg for 5-10 minutes to remove residual water

Elute: 3 x 1.0 mL Ethyl acetate (1 mL/min)Dry down: Nitrogen gas at 55 ºC

Reconstitute: 500 μL of Acetonitrile/Water (20:80)

Solid Phase Extraction

MRM conditions:Analyte Q1 Q3 CE

Chloramphenicol (quant) 321.2 152.0 -24

Chloramphenicol (qual) 321.2 257.2 -16

Chloramphenicol-d5 326.0 157.0 -26

AB SCIEX™ API 4000™

Ion source conditions:Ionization: ESI

Polarity: NegativeScan Type: MRM

Gas 1 (GS1): 45Gas 2 (GS2): 50

DP: -50Collision Gas (CAD): 5Temperature (TEM): 450

IS: -4500

HPLC conditions:Column: Kinetex 2.6 µm C18 100A

Dimensions: 50 x 2.1 mm IDPart No.: 00B-4462-AN

Mobile Phase: A: 5 mM Ammonium bicarbonateB: Acetonitrile

Gradient:

Flow Rate: 0.4 mL/minColumn Temperature: Ambient

Injection: 25 μLDetection: API 4000™ MS/MS

Time (min) B (%)

0.00 5

2.00 5

2.01 95

4.50 95

Results

Conditions: As notedAnalytes: 1. Chloramphenicol 321.2/152.0, 321.2/257.2

2. Chloramphenicol-d5 326.1/157.0

Conditions: As notedAnalytes: 1. Chloramphenicol 321.2/152.0, 321.2/257.2

2. Chloramphenicol-d5 326.1/157.03. Impurity

Figure 2. Extracted sample 0.01 ng/g of shrimp

Figure 1. Chloramphenicol analysis 5 ng/mL standard

Inte

nsity

, cp

s

Inte

nsity

, cp

s

Inte

nsity

, cp

s

Inte

nsity

, cp

s

0.5 1.0 1.5 2.0 2.5 3.0 3.5

0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

0.05.0e41.0e51.5e52.0e52.5e53.0e53.5e54.0e54.5e55.0e5

0.05.0e41.0e51.5e52.0e52.5e53.0e53.5e54.0e54.5e55.0e5

19302

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

2.8e5

0.5 1.0 1.5 2.0 2.5 3.0 3.5

0.5 1.0 1.5 2.0 2.5 3.0 3.5

1,2 1

21

Ap

p ID

193

02

0.2

1

3

2

0.4 0.6 0.8 1.0 1.2 1.6 1.81.4 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

Ap

p ID

194

53

min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

Ap

p ID

194

53

Figure 3. Chloramphenicol standard curve 0.1 ng/mL - 200 ng/mL in mobile phase

0

50

60

50

40

30RF

20

10

0100

Concentration (ng/mL)

R2 = 0.9999

150 200 250

Figure 4. Extracted sample calibration curve with QC samples (1.0 ng/g and 7.5 ng/g) ranging from 0.01 - 20 ng/g of shrimp

0

50

60

50

40

30RF

20

10

0

-5

100

Concentration (ng/mL)

R2 = 0.9997

200 200 250

Calibration stds QC samples

Figure 1 shows a representat ive ex tracted chloramphenicol sample at a concentration of 0.05 ng/g of shrimp. The chromatogram is very clean due to the selectivity of the solid phase extraction sorbent, which retains chloramphenicol but does not retain any polar impurities.

In addition to a clean baseline, Figure 1 demonstrates a very narrow analyte peak width. The narrow peak width means that the compound can be detected at very low levels which would not be possible using a conventionally

porous HPLC column. In fact, based on the signal-to-noise ratio (115.9) at the lowest level (0.01 ng/g) the limit of quantitation is ≤0.001 ng/g of shrimp (Figure 2).

Figure 4 shows an extracted calibration curve from 0.01 - 20 ng/g of shrimp. The curve is linear with an R2 value of 0.9997. In order to validate the method, QC samples were extracted in replicate (n=6) at two different concentration levels (1.0 ng/g and 7.5 ng/g). The QC samples confirmed the accuracy and reproducibility of the method with RSD values less than 5 %.

Results and Discussion

Conclusions

SPE with Strata-X in conjunction with the outlined sample preparation method is an effective way of concentrating and cleaning up shrimp samples for chloramphenicol analysis.

Analysis of chloramphenicol in shrimp is a very topical method that is being tested more and more frequently. Therefore, a sample cleanup method is important as it

reduces the amount of contamination that the costly LC/MS/MS system is subjected to.

Chromatographically, the Kinetex C18 reversed phase column gives a very narrow chloramphenicol peak, resulting in an easily integrated peak with very low detection limits.

TrademarksKinetex is a registered trademark of Phenomenex in the United States, European Union, and other jurisdictions. Strata-X is a trademark of Phenomenex. API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license

Strata-X is patented by Phenomenex. U.S. patent No. 7,119,145

© 2011 Phenomenex, Inc. All rights reserved.

References

1. J.X. Shen, C.I. Tama and R. N. Hayes, J. Chromatogr. B, 2006, 843, 275-282.

2. R.F. Venn, J. Merson, S. Cole and P. Macrae, J. Chromatogr. B, 2005, 817, 77-80.

3. D.A. Wells, “High Throughput Bioanalytical Sample Preparation – Methods and Automation Strategies”, Progress in Pharmaceutical and Biomedical Analysis, Volume 5, Elsevier, Amsterdam, 2003.

4. E. Chambers, D. Diehl and J. Mazzeo, LCGC Applications Note Book, Sept. 2005, pp. 14-15.

5. An Trinh, The Reporter (Supelco), 2006, Vol. 24.5, pp. 3-5.

TrademarksKinetex is a registered trademark of Phenomenex, Inc. Strata-X is a trademark of Phenomenex, Inc. PerkinElmer and MultiPROBE II are registered trademarks of PerkinElmer, Inc.

DisclaimerComparative separations may not be representative of all applications.Phenomenex, Inc. is in no way affiliated with PerkinElmer, Inc.

Strata-X is patented by Phenomenex, Inc.

© 2010 Phenomenex, Inc. All rights reserved.