Monday, February 3, 2014

INTRODUCTION

Honey is a yellowish-brown sticky sweet food made by bees from the nectar of flowers. Honey gets its sweetness from the sugars fructose and glucose. However, there are many kinds of bacteria and mycotoxins that infect honey bees from moulds that infect flowers and to prevent this or even pesticides from flowers, beekeepers use different types of antibiotics to cure the diseases. The antibiotics are also used to promote growth and increase honey production to meet commercial targets. These antibiotics have acute and chronic toxic effects on human health and also reduce the efficacy and quality of honey. The half life of these antibiotic residues are relatively long and can cause toxin infections in the consumers.

 


Antibiotics


Antibiotics are used to treat infections caused by bacteria. The antibiotics focused on in this experiment are penicillin G, streptomycin, tetracycline and gentamycin. The forms of these antibiotics used to prevent bacterial infection in bees are different than the ones humans are given as they have  arsenic compounds. High amounts of these compounds are dangerous and poisonous to our bodies and can make us resistant to certain antibiotics, therefore our bodies will be unable to fight off future bacterial infections. It can also cause damage to the blood, kidneys, liver, bones and teeth.


The aim of this research paper is to detect if there is any antibiotic residue present in the honey sample and if there is, quantify the amount present in the positive honey samples.
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METHODS AVAILABLE TO DETECT & QUANTIFY ANTIBIOTICS IN HONEY

1. High Performance Liquid Chromatography (HPLC)



HPLC is used to separate the components in a mixture, to identify each component, and to quantify each component. It is used for the analysis of non-volatile samples, where minimal sample pre-treatment is required. It uses a pump which passes liquid solvent through a column containing adsorbent material, known as stationary phase. Each component in the sample reacts differently with the stationary phase which causes different flow rates for the different components and therefore leading to the separation of the components as they flow out of the column. In the scientific field, HPLC has many uses. For example, it is used in the medical field to detect the amount of vitamin D in blood, it is used for legal purposes to detect drugs in urine, it is used in the research field to separate components of biological samples or to separate similar synthetic chemical substances from each other.


2. Ultra Performance Liquid Chromatography (UPLC)



The concept of UPLC is basically the same as HPLC. The difference is that the particle diameter of the packing material in the column has been reduced (<2 μm) and the pressure has been increased (> 689 barr), which achieves fast separation and still preserves high efficiency. The column temperature is also increased to reduce the mobile phase viscosity, permitting for a higher flow rate. Using the van Deemter equation, as the particle size decreases to less than 2.5μm, not only is there a significant gain in efficiency, but the efficiency does not diminish at increased flow rates. Therefore, in more highly equipped and higher budget labs, this method has been used.


3. Thin Layer Chromatography (TLC)



TLC is a technique used to separate non-volatile mixtures. It is usually performed on a sheet of plastic, aluminium foil or glass, which is coated with a  thin layer of stationary phase such as silica gel or aluminium oxide. The samples will be applied to the plate as a small drop, 1.5cm from the edge of the sheet and once this is done, the plate is placed in a solvent mixture, known as mobile phase and is drawn up the plate via capillary action. Due to the different analytes in the different samples, the samples ascend at different rates causing separation to occur.


Selected Methods


In our case study, the selected methods were TLC for confirmation of specific antibiotics in the various honey samples as well as HPLC to quantify the amount of honey present in the various samples.
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MATERIALS & METHODS

Collection of Samples: A total of 100 samples honey samples were collected, 40 branded and 60 non-branded.


Chemical & Reagents used: Standard tetracycline, penicillin, streptomycin and gentamycin, methanol, acetonitrile (ACN), O-phosphoric acid and ethyl acetate. All solvents used were of TLC and HPLC grade, including the deionized water. They were filtered through a 0.45μm filter membrane and degassed by an ultrasonic cleaner for 20 minutes.


Extraction procedure for TLC: 5g of sample were extracted with a mixture of ethyl acetate:water (80:20) by centrifugation at 3,000rpm for 10 minutes and the supernatant was used for spotting on the TLC plate.


TLC Analysis of Antibiotic: The spot of each sample were loaded standard on TLC plate with the help of micro syringes by an automatic TLC spotter. Then placed in the mobile phase of methanol:water (95:5) and the detection was carried out by comparing the Rf value of sample with that of standard.


Extraction procedure for HPLC: The samples for HPLC were subjected to a deproteinizing chemical procedure using ACN. A 2 g of honey sample was placed into a 10mL test tube and shaken intensively with 3mL ACN for 1 min. The mixture was centrifuged for 15 min at 5000 rpm. The supernatant was collected and dried under nitrogen stream at 40°C. The residue was redissolved in methanol, filtered through 0.45μm filter membrane and 10 μl was injected to the HPLC system.


HPLC Analysis of Antibiotics Residues: A Hitachi (D-2000 Elite system manager) with a dual pump (L-2130), auto sampler L-2200 and UV-Visible detector L-2420 was used for the quantification of targeted antibiotic residue, in which the separation was achieved using Column oven L-2300 and column Intersil ODS-3 C18 (GL Sciences Inc. Tokyo Japan 5μm, 250mm×4.6 mm). All solvents were filtered through 0.45 μm sartolon polyamide membrane by filtration assembly of (Rocker-300 Model Taiwan) and degassed by ultrasonic cleaner Ceia (Model CP-104 Italy). The determination of these compounds were performed using different mixture of an aqueous mobile phase (A) Acidified water and organic mobile phase (B) methanol/ACN, both with a flow rate of 1 ml/min. The compounds were detected at 210-240 nm. The quantification was achieved by comparison of the peak area of the sample with that of the external standard. The identical chromatogram was quantified by the peak area of sample with that of standard in same retention time.
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RISK ASSESSMENT



Setting:

Laboratory setting

Process:

Handling and processing of various chemicals in High Performance Liquid Chromatography and Thin Layer Chromatography.











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ANALYTICAL RESULTS

TLC Results


As seen in the Table 1 above, it was found out that 6 out of 40 samples of the branded honey and 12 out of the 60 samples of the unbranded honey were positive for antibiotics. Overall in the 100 samples, 5% of samples contained penicillin G, 6% contained streptomycin, 7% contained tetracycline and none of the samples contained gentamycin. TLC was a detection step in the process and therefore, the samples that were positive for the various antibiotics were then used in HPLC for quantification. 

HPLC



As seen in the Table 2 above, from 5 of the samples with the highest positive from the TLC, tetracycline was the highest detected antibiotic which was calculated to be 19.98μg/mL, while penicillin G was 14.59μg/mL and streptomycin was 13.44μg/mL and with no detection in gentamycin as seen in the negative result from the TLC test. The calculations were determined by a previous research which had concluded that the retention time for the various antibiotics were: 5.63 minutes for tetracycline, 2.60 minutes for streptomycin, 10.96 minutes for gentamycin and 13.50 minutes for penicillin G.
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DISCUSSION & CONCLUSION

The problem of detecting antibiotics in honey is that there can be a broad use of various antibiotics by the beekeepers. However, in this research, the antibiotics focused on were penicillin G, streptomycin, tetracycline and gentamycin. In another similar research conducted in Russia, it was found that there were peaks of unidentified residues, possibly other antibiotics that are unidentified. 


As seen in the chart above, tetracycline has been identified as the retention time of 5.63 minutes, and other unidentified peaks at 3.45, 4.63, 14.92 minutes, in the other research conducted in Russia.


It has also been reported that there have been higher traces (even higher amounts than found in this research paper) of tetracycline and streptomycin in other regions of the world, including antibiotics not covered by this research paper such as sulfonamides, ciprofloxacin and chloramphenicol.


In conclusion, our research paper shows that the use of the antibiotics tetracycline, penicillin G and streptomycin are extensively used by beekeepers for curing diseases in bees.
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REFERENCES
















http://www.chromatographyonline.com/lcgc/data/articlestandard/lcgc/242005/164646/article.pdf

http://www.google.com.sg/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&ved=0CD0QFjAD&url=http%3A%2F%2Fwww.drs.illinois.edu%2Fmsds%2FDownloadFile.aspx%3FGuid%3D71062003-5489-4BEE-BEE5-38E74E8A8675&ei=2Ov4UuKuOeOpiAfUzICIAg&usg=AFQjCNGPrHWs8xiRRjSYQ8ceiSNde-QBzg&sig2=QrucoFTfSTmV1U2w1hN4FQ

http://www.sciencelab.com/msds.php?msdsId=9927393

http://www.sciencelab.com/msds.php?msdsId=9927335

https://www.sciencelab.com/msds.php?msdsId=9927227

 





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