*3.3. The Fluorescence Spectra Profiles of Adulterated Honey*

Previous studies have examined fluorescence spectra as a means of distinguishing between pure honey and sugar syrup as well as between honeys from different botanical and geographical origins [23]. In light of published articles describing the features of emission spectra, we found that the incorporation of the FDP intensity with these known spectra allowed us to develop profiles for the identification of sugar syrup adulterants in honey.

We blended beet syrup, cassava syrup, malt syrup, rice syrup and HFCS and incrementally added the mixture (0, 10%, 20%, 30% ... 90%) to a blend of all five honeys to assess the correlation between the amount of added syrup and FDP intensities. The apex wavelengths and areas of the fluorescence emission spectra, as well as FDP intensities (Table 2), revealed that as the proportion of syrup increased, a significant blueshift (with the honey peak at 468 nm and the syrup peak at 442 nm) occurred, and the FDP areas decreased in a dose-dependent gradient. Thus, the FPD was positioned at 740 nm, and measurement of its intensity combined with the apex wavelength of the fluorescence emission spectra could be readily used to discriminate between pure and adulterated honey samples. As Figure S1 shows, the *R*<sup>2</sup> between the amounts of adulterated syrup and FDP intensities was 0.9873 (*p* < 0.01). Thus, the FDP intensity can strongly reflect the amount of syrup added.

**Table 2.** Fluorescence spectra information of artificially adulterated honey.


Note: lowercase letters indicate a significant difference (*p* < 0.01).

Commonly, the addition of 30% or higher syrup can make substantial profits. In this study, the minimum amount of syrup added was 10%, and compared with the honey sample, there was a significant difference (*p* < 0.01) (Table 2). We also found that a small deviation in the apex wavelength could be used as an auxiliary qualitative indicator for the relative purity of a given honey sample. Specifically, the apex wavelength shifted from 470 nm to 460 nm, and the FDP intensity decreased from 990 to 610 with the dilution of acacia honey to 30% syrup (Figure 3).

To verify the accuracy of the method, we chose acacia honey samples from different sources and different collection times to compare the intensities of the FDP. Among these acacia honey samples, there were no significant differences (*p* > 0.05) (for detailed information, see Table S1). Thus, the samples with the addition of at least 10% syrup could be identified by significance analysis (*p* < 0.01) compared with natural honey samples.

**Figure 3.** Typical fluorescence spectra of adulterated acacia honey with 30% syrup ((**a**): pure honey; (**b**): acacia honey adulterated with 30% syrup).
