## 3 Results and Discussion
### Validation of the Modified Folin-Ciocalteu Assay Suited to Honey Analysis  
#### 3.2.1 Specificity  
Specificity reflects the ability of an assay to detect the analyte(s) in the presence of other constituents. In the specific context of honey analysis, the traditional FC assay (pH 10.8) lacks sufficient specificity as, under basic conditions, other reductants (e.g., reducing sugars) and not just phenolics, will react (Figure 1). Therefore, changes were made to the assay protocol to increase its specificity for the analysis of TPC of honey. Figure 3 illustrates the increased specificity of the modified assay at pH 7.9. Earlier, it was described that reducing sugars also have the ability to react with the FC reagent under strongly basic reaction conditions, a finding that has also been confirmed by other researchers [12; 15; 17]. Figure 2 demonstrates that lowering the pH effectively removes this interference which is in accordance with other studies [13; 51]. To confirm the effectiveness of the modified FC assay protocol in minimizing the interference of the sugar matrix, 20% \(m/v\) aqueous solutions of individual sugars and also an artificial honey solution (see Section 2.4 for composition) were also analysed using the modified protocol. Table 3 illustrates that the artificial honey solution produced a detectable reaction in the modified assay, equivalent to 0.59 \(\pm\) 0.02 mg GAE/100 g, whereas among the individual sugar solutions, only maltodextrin (2.82 \(\pm\) 0.16), fructose (2.23 \(\pm\) 0.06), galactose (0.55 \(\pm\) 0.07), and sucrose (0.08 \(\pm\) 0.03) could be detected; however, all were below the established limit of quantification of the assay. Figure 5: Comparison of the standard curves of gallic acid spiked and blanked with 0.4 g/mL artificial honey solution after treatment with FC reagent and 0.75% Na\({}_{2}\)CO\({}_{3}\) (pH 7.9) after 2 h of incubation (blue line) and after 3 h of incubation (red line). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sugar** & **Amount in Floral Honey (\%)** & **TPC (mg GAE/100 g)** & **Remarks** \\ \hline Artificial honey & N.A. & 0.59 \(\pm\) 0.02 & \textless{}LOQ \\ Glucose & \textless{}30.00\% [53] & Not detected & \textless{}LOD \\ Frutose & \textless{}50.00\% [53] & 2.23 \(\pm\) 0.06 & \textless{}LOQ \\ Sucrose & \textless{}5.00\% [54] & 0.08 \(\pm\) 0.03 & \textless{}LOQ \\ Maltose & \textless{}2.00\% [55] & Not detected & \textless{}LOD \\ Xylose & \textless{}0.50\% [56] & Not detected & \textless{}LOD \\ Galactose & \textless{}0.01\% [57] & 0.55 \(\pm\) 0.07 & \textless{}LOQ \\ Lactose & \textless{}0.01\% [57] & Not detected & \textless{}LOD \\ Maltodextrin & N.A. & 2.82 \(\pm\) 0.16 & \textless{}LOQ \\ Mannose & N.A. & Not detected & \textless{}LOD \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Total Phenolic Content of Sugar Solutions. The impact of reducing sugars on the FC assay was explored in a number of previous studies. [PERSON] et al., found that among the tested sugars (glucose, sucrose, fructose, xylose, mannose, rhamnose, and arabinose), glucose and sucrose had the least effect while fructose and xylose had the highest effect [12]. [PERSON] et al. reported significant interference from xylose, while glucose also had a noticeable interference when 11.50% Na\({}_{2}\)CO\({}_{3}\) was used [58]. [PERSON] et al., found that glucose had higher interference compared to fructose, with sucrose showing no reaction in the assay [17]. The findings of this study demonstrate that only fructose, sucrose, and galactose, which are commonly found in honeys, react under the modified assay condition, but that their interference, while not completely hindered, can be minimized by adjusting the pH to 7.9. The artificial honey analyzed in this study contained glucose, fructose, sucrose, and maltose, and its detectable reaction was most likely due to its fructose and sucrose constituents. [PERSON] et al. [17] discovered an additive effect when fructose is combined with gallic acid and analyzed using the FC assay with 6.00% base.