DETECTION AND QUANTITATION OF RESIDUES OF COMMONLY USED ANTIBIOTICS IN RAW MILK
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Date
2018
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR
Abstract
The present study was undertaken to detect and quantitate the residues of
commonly used antibiotics in raw milk in Thrissur district of Kerala. The cross
sectional survey was conducted among the farmers to assess their knowledge and
awareness level on the use of antibiotics. A total of 440 milk samples were collected
from ULF and FRDS, Mannuthy, private farms in Thrissur and individual households
during the period from August 2017 to July 2018. All the samples collected were
subjected for screening of antibiotic residues by microbial inhibition assay (MIA).
The positive samples were analysed by Charm assay to determine the group and level
of antibiotic residues. The amount of residues of oxytetracycline, enrofloxacin and
cloxacillin in positive samples was quantitated using High Performance Liquid
Chromatography (HPLC).
The survey among the farmers showed that 78 per cent of people were aware
of the antibiotic use and 43 per cent of people had knowledge about the withdrawal
period. The most commonly used antibiotics were oxytetracycline (54 per cent),
ßlactams (44 per cent), enrofloxacin (36 per cent), sulphonamides (14 per cent) and
gentamycin (12 per cent).
Of 440 milk samples screened by MIA, the antibiotic residues were detected
in 7.41, 18.57 and 13.33 per cent of samples from ULF and FRDS, private farms and
individual households respectively. The overall occurrence rate of antibiotic residues
observed was 13.18 per cent. On analysing 58 positive samples in MIA by Charm
Rapid One Step Assay (ROSA), it was found that 2.95 per cent of samples were
positive for tetracyclines residues, 3.64 per cent of samples for enrofloxacin residues
and 2.27 per cent of samples for ß-lactams residues.
The HPLC conditions, sample extraction and analytical methods for detection
and quantitation of oxytetracycline, enrofloxacin and cloxacillin residues were
optimized and validated. The analysis of oxytetracycline was carried out on C18
column using a mobile phase of 0.03 M Oxalic acid : Acetonitrile : Methanol (70: 15
: 15) delivered at a flow rate of 1 ml/min. Photodiode array detector was set at 354
nm for oxytertracycline detection. The mobile phase consisting of 0.1 M
Orthophosphoric acid (pH adjusted to 2.5 with triethylamine): Acetonitrile: Methanol
(80:17:3) was used for enrofloxacin detection on C18 column. Flow rate of 0.8
ml/min and detection wavelength of 278 nm were found to be optimal.
Chromatography conditions of cloxacillin consisted mobile phase of 0.1 per cent
Trifluroacetic acid: Acetonitrile (50:50), flow rate of 1 ml/min and detection
wavelength of 210 nm. The retention time noticed for oxytetracycline, enrofloxacin
and cloxacillin was 5.6, 7.7 and 7.48 min respectively. A good linearity with
coefficient of determination greater than 0.99 was obtained for all antibiotics.
Of the 13 samples tested by HPLC for oxytetracycline, 12 samples showed
oxytetracycline residues with a mean concentration of 1450.20 ± 182.09 ng/ml.
Enrofloxacin was detected in all the 16 samples tested by HPLC with highest
concentration of 709.28 ng/ml. Cloxacillin could be dectected only in two samples
out of ten samples tested by HPLC with mean concentration of 118.77 ± 29.76 ng/ml.
The presence of antibiotic residues in edible tissues of animals can cause adverse
effects in human health and also lead to development of antimicrobial resistance
which is one of the most serious global public health threats in this century. Thus,
judicious and proper use of antibiotics, strict adherence to withdrawal period and
maintenance of treatment records are the necessary steps to prevent the occurrence of
antibiotic residues in milk. Control of antibiotic residues in foods of animal origin
needs strict monitoring and surveillance studies so as to discard the food products that
are not safe for human consumption.
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