## 3 Results and Discussion
### Honey Analysis  
Table 5 shows the TPC (expressed as mg gallic acid equivalent/100 g sample) of four different commercial honeys measured using the FC assay in different pH conditions. The results demonstrate the strong pH-dependence of the assay and highlight the importance of carrying out the analysis in near neutral pH (7.9) to minimise any interference of reducing sugars and to maximise the reactivity associated with the various phenolics present in the honey samples; as can be seen from the data presented in Table 5, the absolute  
Figure 6: (**A,B**). Comparison of the absorbances of 30 \(\mathrm{\mu g/mL}\) (**A**) and 110 \(\mathrm{\mu g/mL}\) (**B**) concentrations of gallic acid spiked and blanked with 0.4 g/mL artificial honey solution when treated with FC reagent and 0.75% Na\({}_{2}\)CO\({}_{3}\) (pH 7.9) and measured after 2 h of incubation time (blue), after 2 h and 15 min of incubation time (red), and after 2 h and 30 min incubation time (green). Significance levels were determined using a one-way ANOVA (\(p<0.0001\)) and Tukey post hoc comparison method was used to further compare the results, with different symbols indicating the level of significance (ns = not significant, \({}^{\star}=p<0.05\), and \({}^{\star\star}=p<0.005\)). \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Honey/Sugar**} & \multicolumn{3}{c}{**TPC mg GAE/100 g Honey**} & \multicolumn{2}{c}{**\% Difference**} \\ \cline{2-5}  & **pH 1.8** & **pH 7.9** & **pH 10.8** & **pH 7.9 and 10.8** \\ \hline TAB & 10.61 \(\pm\) 0.14 & 18.55 \(\pm\) 0.08 & 23.12 \(\pm\) 0.41 & 19.77 \\ MIX & 19.86 \(\pm\) 0.22 & 34.60 \(\pm\) 0.27 & 53.08 \(\pm\) 0.35 & 34.82 \\ MAN & 24.64 \(\pm\) 0.43 & 54.00 \(\pm\) 1.44 & 84.9 \(\pm\) 0.62 & 36.40 \\ JAR & 28.56 \(\pm\) 0.16 & 56.03 \(\pm\) 0.57 & 79.51 \(\pm\) 0.21 & 29.53 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Total Phenolics Content (expressed in mg gallic acid equivalent/100 g sample) for different commercial honeys obtained by the FC Assay in different pH conditions. value of TPC can be more than triple for a honey, depending on the pH of the assay conditions. As has been demonstrated in this study, higher absorbance readings and thus higher calculated TPCs are found in strongly basic conditions (pH 10.8) due to the contributions of reducing sugars. Thus, in this instance, the TPC of the honeys is overestimated which is further reflected by the % difference of the calculated TPC for each samples when assayed with FC using the modified versus the traditional version of the assay. Comparing TPC data for acidic and slightly basic conditions, where sugar interference has been found to be below the limit of quantification (see Table 5), illustrates the different levels of reactivity associated with phenols and phenolates. To allow comparative analyses of the TPC between honey and other, less sugar-rich food items, which are traditionally analysed at basic pH where the various phenolic compounds are accounted for in their phenolate state, for the analysis of honey, the FC assay should be carried out at pH 7.9 to maximise absorbance readings from its phenolic constituents. By employing the modified assay conditions, Jarrah (JAR) honey was found to contain 56.03 \(\pm\) 0.57 and Manuka (MAN) honey 54.00 \(\pm\) 1.44 mg GAE/100 g, and the TPC of two multifloral honeys (MIX and TAB) was 18.55 \(\pm\) 0.08 and 34.60 \(\pm\) 0.27 mg GAE/100 g, respectively. The TPC values determined in this study fall within previously reported TPC values for these honeys since Western Australian Jarrah (\(n\) = 6) was reported to have a TPC between 46.26 and 56.09 mg GAE/100 g with a mean of 50.58 \(\pm\) 3.76, and for Australian and New Zealand Manuka honeys (\(n\) = 29), TPC values ranged between 22.60 and 66.30 mg GAE/100 g with a mean of 35.40 \(\pm\) 10.54. The TPC of Western Australian multifocal honeys (\(n\) = 12) ranged from 17.60 to 45.90 with a reported mean TPC of 35.1 \(\pm\) 10.9 mg GAE/100 g [59; 60; 61].