Introduction:
Chloramphenicol (CHL) is a broad-spectrum antibiotic. It is active against many gram-positive and gram-negative bacteria. CHL is extremely toxic and often evokes severe side effects (a toxic effect on the hematopoietic system and a decrease in the number of red blood cells). The use of CHL is prohibited in veterinary medicine in many countries. CHL is an undesirable component in food products because it can cause allergic reactions, irritation of the mucous membranes of the mouth and pharynx. Children are most sensitive to the drug. Therefore, it is necessary to monitor the content of this antibiotic in milk intended for baby food. The purpose of this study is the synthesis and characterization of tracers with different spacer lengths between CHL and fluorescence dye and development of a fluorescence polarization immunoassay (FPIA) for chloramphenicol with high sensitivity in milk.

Methods:
The amino derivatives of chloramphenicol with different spacer length (CAM-Cn-NH2) were obtained from CHL. Then, these derivatives were labeled by fluorescein isothiocyanate (FITC), purified and characterized. The FPIA methods were developed for determination of CHL. The FP signal was measured by portable fluorimeter Sentry-200.

Results:
The quality of immunoreagents is of great importance for the development of highly sensitive FPIA. The fluorescent labeled amino derivatives of CHL with different carbon chain lengths (CAM-Cn-FITC, n=1,2,3,5,8 and CAM-Lys-FITC) were obtained and characterized. The kinetics of binding of conjugates with anti-CAM antiserum was studied. It was shown that all tracers bound to specific antiserum, the time to establish equilibrium was 5–10 min. The resulting conjugates did not bind with nonspecific serum. An FPIA for the determination of CHL was developed and conditions for sensitive analysis were optimized. Calibration dependencies for determining CHL were constructed and the analytical characteristics of the method were determined. It was shown that the highest sensitivity (IC50) and the lowest limit of detection (LOD) were demonstrated by tracers CAM-C5-FITC and CAM-Lys-FITC. For tracer CAM-Lys-FITC IC50 and LOD were 65 and 3 ng/mL, respectively, and for tracer CAM-C5-FITC IC50 and LOD were 78 and 5 ng/mL, respectively. The accuracy of FPIA has been tested by recovery test in water and cow milk. The recovery was 90–110%.

Conclusions:
In this work, new fluorescently labeled chloramphenicol derivatives were synthesized and characterized: CAM-Cn-FITC, n=1,2,3,5,8 and CAM-Lys-FITC. A highly sensitive FP assay for CHL determination in water and milk has been developed. It was shown that conjugates with a chain length of five carbon atoms had the greatest sensitivity.

Conflicts of interest:
The authors declare that they have no conflict of interest in the publication of this article. The authors have no conflicts of interest to report in this work. Abstract was not submitted elsewhere and was first published here.

Funding:
The work was supported by the state task of Lomonosov Moscow State University 123032300028-0.