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VALIDATION OF THE DETERMINATION OF
CHLORAMPHENICOL RESIDUES IN ANIMAL FEED BY
LIQUID CHROMATOGRAPHY WITH ION TRAP DETECTOR
BASED ON 2002/657/EC.
F. Moragues, C. Igualada, N. León, and C. Juan.
Public Health Research Center (CSISP), Consel eria de Sanitat.
Avda. Cataluña, 21. Valencia. Spain.
moragues_fra@gva.es, igualada_car@gva.es, leon_nur@gva.es Introduction
Results and discussion
Fig.1.- Chromatograms of blank porcine and ovine feed (EIC 257 m/z CAP) with internal standard CAP-D5, 12 µg/kg
Chloramphenicol (CAP) is a broad spectrum antibiotic and its use in food producing 022-2601.d: EIC 256.9 -MS2(321.5), Smoothed (13.9,1, GA) 023-2701.d: EIC 256.9 -MS2(321.5), Smoothed (13.0,1, GA) animals is forbidden in many countries such as the USA, Canada, Australia and EU members states. In spite of it, this drug is still illicitly used in animal farming because of its ready 022-2601.d: EIC 261.9 -MS2(326.5), Smoothed (13.9,1, GA) 023-2701.d: EIC 261.9 -MS2(326.5), Smoothed (13.1,1, GA) availability and low cost, as shown in the recent findings occurred in Spain in broiler meat.
In order to effectively monitor the occurrence of CAP residues, specific and very sensitive analytical methods are required, not only for animal products but also for animal feed. However, Fig.2.- Chromatograms of spiked porcine and ovine feed 10 µg/kg (EIC 257 m/z CAP and EIC 262 m/z CAP-D5).
as far as we know, studies concerning the determination of CAP in animal feed by liquid 012-1401.d: EIC 256.9 -MS2(321.5), Smoothed (13.1,1, GA) 015-1701.d: EIC 256.9 -MS2(321.5), Smoothed (14.1,1, GA) chromatography with mass spectrometric detection have not been already developed.
A liquid chromatographic method with ion trap detector was developed for the 012-1401.d: EIC 261.9 -MS2(326.5), Smoothed (13.1,1, GA) 015-1701.d: EIC 261.9 -MS2(326.5), Smoothed (14.2,1, GA) determination of CAP in animal feed.
Fig.3.- Spectrum for CAP of spiked porcine feed 10 µg/kg.
5,5 IP´s
Table 1.- Relative intensities of the monitored ions for CAP at different concentrations.
CAP.-10 µg/kg
CAP.-25 µg/kg CAP.-100 µg/kg
CHLORAMPHENICOL
Precursor ion
Quantification ion
Experimental
Qualification ion
Qualification ion
ORGANIC EXTRACTION
Selectivity/specificity: 35 blank samples of bovine, porcine, ovine,
2 x 5 ml EtAc
ANIMAL FEED
caprine, rabbit and broiler feed were analysed and no interferences in the Agitate 1 min
(Porcine, bovine, equine,
ovine, caprine, rabbit and
Centrifuge 4000 rpm 5 min.
broiler)
Table 2.- Validation results for CAP in animal feed.
Analyte/
Trueness
Repeatability
Within-laboratory
concentration
reproducibility (%)
SPE EXTRACTION C
CAP.-10 µg/kg
CAP.-25 µg/kg
CAP.-100 µg/kg
The method was validated according to the requirements of the 2002/657/EC European LIQUID EXTRACTION
Ion suppression study
Table 3.- Mean ion suppression (%) from different animal feed (n=10) Sample weight Ion suppression (%) Recovery (%)
ORGANIC PHASES
the analysis provided the lower ion suppression effect and, the best actual recovery to achieve a Conclusions
LC system Agilent 1100 series with Ion Trap G2440A. Agilent Technologies®.
LC conditions:
1. A quantitative and confirmatory LC - ESI (-) – MS/MS method for the determination of - Column:50 x 3.0 mm i.d., 5 µm Hypurity C8 Thermo® chloramphenicol in animal feed was developed and validated.
2. The method was validated according to the Commission Decision 2002/657/EC.
MS conditions: ESI (-) and MRM mode
3. The validation results were very satisfactory.
- Tune: nebulizer (40psi); dry gas (8 l/min); dry temperature (350ºC) skim 1 (-23.0 V); skim2 (- 6.0 V); 4. The estimated CCα and CCβ were 10 and 12 µg/Kg, respectively.
5. By monitoring three product ions 5,5 IP´s were achieved, which provided the method with a Precursor ion
Fragmentation Amplitude
Product Ions
very high selectivity/specificity and gave a great confidence to the results.
6. The method will contribute to carry out a more complete regulatory control programme by applying not only in animal products but also in animal feed.

Source: http://www.csisp.gva.es/documents/csisp/Posters/VALIDATION%20OF%20THE%20DETERMINATION%20OF%20CHLORAMPHENICOL.pdf

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