## 3 Results and Discussion  
### Modification of the Folin-Ciocalteu Assay Suited to Honey Analysis  
#### 3.1.1 pH Effect  
To optimise the FC assay conditions for honey analysis, different pH scenarios were trialed using different concentrations of Na\({}_{2}\)CO\({}_{3}\) with the aim to minimise reducing sugar interference and maximise the reaction of phenolics in the assay. The extent of sugar interference was determined by comparing the absorbances of gallic acid samples with gallic acid samples spiked and blanked with artificial honey solution (0.4 g/mL). It was assumed that the absorbance readings of both samples would be very similar if there was no sugar interference. Figure 1 captures the extent of interference of reducing sugars in honey when the assay is carried out with gallic acid as a test sample (blanked with water) as compared to being spiked and blanked with 0.4 g/mL artificial honey solution using 7.50% aqueous  
Na\({}_{2}\)CO\({}_{3}\) (pH 10.8), in line with the traditional FC assay conditions. A significant difference (\(p=<0.0001\)) in the mean (\(n=3\)) absorbance of both the 20 and 40 \(\upmu\)g/mL aqueous gallic acid solutions can be observed with the mean absorbance of the spiked solutions being 49.33% (for 20 \(\upmu\)g/mL) and 48.61% (for 40 \(\upmu\)g/mL) higher. This confirms the findings of previous studies that have reported the interference of reducing sugars when using the FC reagent at high pH [12; 15; 17]. The literature also reports the use of acidic assay conditions for the FC method [24; 29; 51], which can be achieved by replacing the 7.50% Na\({}_{2}\)CO\({}_{3}\) solution with water due to the acidic nature of the FC reagent. The assay of 20 \(\upmu\)g/mL and 40 \(\upmu\)g/mL gallic acid solutions and gallic acid solutions spiked and blanked with artificial honey under acidic conditions (pH 1.8, achieved by replacing Na\({}_{2}\)CO\({}_{3}\) with water) resulted in very similar absorbance values, indicating an absence of or negligible sugar interference (Figure 2). Statistically, no significant difference in the mean (\(n=3\)) of the absorbances of 20 \(\upmu\)g/mL  
Figure 1: (**A**–**C). (**A**)—Comparison of the standard curve of gallic acid blanked with water (blue line) and of gallic acid spiked and blanked with 0.4 g/mL artificial honey solution (red line); (**B**,**C) show the absorbances of 20 \(\upmu\)g/mL and 40 \(\upmu\)g/mL concentrations of gallic acid blanked with water (blue) and spiked and blanked with 0.4 g/mL artificial honey solution (red). Both assays used FC reagent and 7.50% Na\({}_{2}\)CO\({}_{3}\) (pH 10.8). Significance levels were determined using a two-tailed \(t\)-test and is indicated as **** = \(p<0.0001\). gallic solution for both conditions was noted when treated with FC reagent and 0.00% Na\({}_{2}\)CO\({}_{3}\) solution (\(p\) = 0.1028). However, a significant increase in the absorbance of the 40 \(\upmu\)g/mL gallic acid solution when compared with the same solution spiked and blanked with the artificial honey solution could be noted (\(p\) = 0.0074), but the said increase was only 2.77%. An overall marked decrease in absorbance readings was, however, noted for the samples analysed under acidic conditions (absorbance range 0.020-0.150, see Figure 2) compared to corresponding samples analysed under basic conditions (absorbance range 0.050-0.650, see Figure 1). This reduction in absorbance might be explained by a reduced reaction rate of phenolics compared to the corresponding phenolates. Ultimately, this might lead to an underestimation of the TPC of the honey sample. A pH of about 7.9, achieved by using 0.75% aqueous Na\({}_{2}\)CO\({}_{3}\) solution, was found to provide suitable conditions with most of the phenolic compounds being accounted with very minimal interference from reducing sugars (Figure 3).