You are here
Validation of methods of determination residual veterinary of drugs in products of animal origin
The most used drugs in veterinary practice tetracycline and sulfanilamide group and the antibiotic chloramphenicol.
These drugs have a big spectrum of activity and is often used for skin infections, infections of respiratory tract, gastrointesti-
nal and genitourinary diseases. For determination the residual of veterinary drugs use different methods, but the most sensi-
tive and selective method is based on liquid chromatography with mass spectrometric detection (LC/MS/MS). For exactly
results is necessary make validation of the methods.
The goal of our work is systematisation the procedure validation of confirmation methods determination residual of vet-
erinary drugs for permitted and prohibited substances and comparing received results.
The study was conducted at the Research Chemical and Toxicological Department of the State Scientific and Research
Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (SRILDVSE). To determine the tetracicline used
guidelines on "Determination of antibiotics in animal products using liquid chromatomas-spectrometer", and "Determination
of chloramphenicol in foods of animal origin using liquid сhromatomas- spectrometer". Products of the animal origin - the
muscles of poultry and pig and cow were used for the study. Validation was performed according the European Commission
Decision 2002/657/EC of 12 August 2002, which provides implementation of Council Directive 96/23/EC concerning the
performance of analytical methods and the interpretation of results.
The main characteristics of supporting life evaluation methods include: quantitative decision limit (CCa), detection ca-
pability (CCb), accuracy, repeatability, reproducibility, specificity by Commission Decision 2002/657/EC of 12 August 2002.
Before applying the method in the laboratory should be made extra work, to prepare samples of different matrices and prove
that new method there is high effective in terms of time and finance.
Previously were sampled muscle tissue of cattle, pigs and poultry, and was added chloramphenicol, with levels MRPL, it
is 0.3 mg / kg, 1,5 MRPL, 0.45 mg / kg and 2 MRPL, 0.6 mg / kg. Separately were selected muscles of cattle, pigs and poul-
try for determination of tetracycline, and like in firs variant were added tetracycline with level 0.5 MRL or 50 mg / kg MRL
or 100 mg / kg and 1.5 MRL or 150 mg / kg. After research according to test procedures we had results, which are chloram-
phenicol CCa 0.1, recovery 99.8 %, repeatability and reproducibility (CV%) 2,4. Results of tetracycline are CCa 109,6, re-
covery 103 %, repeatability and reproducibility (CV%) 5.7.
As we can see reproducibility and repeatability for chloramphenicol are satisfying the requirements of Directive 2002/657 / EC,
indicating that this ratio must not be above 32% for concentration less than 1 mg / kg. This method also satisfies the recovery
requirements of the above-mentioned document and included in the limit 50% - 120% for concentrations less than 1 mg / kg. CCa
was counting by calibration curve procedure according to ISO 11843. The corresponding concentration at the y-intercept plus 2,33
times the standard deviation of the reproducibility of the intercept equals the decision limit for prohibited substances. About of tetra-
cycline, we have results satisfying the requirements of Directive 2002/657 / EC too, indicating that this ratio must not be above 23%
for concentration less than 100 mg / kg. This method also satisfies the recovery requirements of the above-mentioned document and
included in the limit 80% - 110 % for concentrations less than 100 mg / kg. CCa was counting by calibration curve procedure ac-
cording to ISO 11843. The corresponding concentration at the y-intercept plus 1.64 times the standard deviation of the reproducibil-
ity of the intercept equals the decision limit for allowed substances.
Conclusions and prospects for further research: 1. Was systematic procedure for assessing the suitability of supporting
methods to determine residual amounts of veterinary drugs both for permitted and banned substances.
2. The methods of determination antibiotics and chloramphenicol was validated, parameters MS/MS detection and vali-
dation was set.
3. These methods of determining antibiotics and chloramphenicol according to the results are sensitive and specific and
satisfy all the requirements of European Directive 2002/657 / EC.
4. This method is suitable for the sanitary inspection determination of the products of animal origin and can be used suc-
cessfully in Veterinary Laboratories.
1. Vyznachennja antybiotykiv u produkcii' tvarynnogo pohodzhennja za dopomogoju ridynnogo hromatomas-
spektrometra / [Ju. M. Novozhyc'ka, O. V. Ivanova, O. M. Stupak ta in.]. – K., DNDILDVSE, 2014. – 12 s.
2. Vyznachennja hloramfenikolu v neobroblenyh harchovyh produktah tvarynnogo pohodzhennja za dopomogoju ridyn-
nogo hromatomas-spektrometra / [A.V. Abramov, Ju. M. Novozhyc'ka, O.V. Ivanova, O.M. Stupak ta in.].– K.:
DNDILDVSE, 2008. – 13 s.
3. Metodychni rekomendacii' z validacii' skryningovyh ta pidtverdzhujuchyh metodiv vyznachennja zalyshkovyh
kil'kostej veterynarnyh preparativ metodom gazovoi' ta ridynnoi' hromatografii' / [Ju.M. Novozhyc'ka, O.V. Ivanova,
O.V. Bondarec' ta in.]. – K: DNDILDVSE, 2013. – 33 s.
4. Commission Decision 2002/657/EC of 12 August Implementing Council Directive 96/23/EC Soncerning the Rerfor-
mance of Analytical Methods and the Interpretation of Result, European Comission, Brussels.
5. ISO 11843: 1997 Capability of detection - Part 1: Terms and definitions, Part 2: Methodology in the linear calibration
case Part 2: Methodology in the linear calibration case.
6. Protocol for the Design, Conduct and Interpretation of Method Performance Studies / [Horwitz W., Pellerin F., Sveh-
la G. et al.]. – Pure & Appl Chem, 1995.– Vol. 67. – Р. 331-43.