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.