**2. Fluorescence markers in manuka honey**

The fluorescence markers in manuka honey were assessed in a number of honey collections, first, field honeys harvested from *L. scoparium* hive sites in New Zealand, and second, a com‐ mercial set purchased from retail distributors in Singapore. The honeys in the purchased set were labelled as manuka honey and therefore should be wholly or predominantly sourced from *L. scoparium*.

#### **2.1. Leptosperin**

Beyond traditional analytical techniques, fluorescence spectroscopy has demonstrated use in analysing a range of food products including honeys [27–30]. Fluorometric methods are reported to be up to 1000 times more sensitive than absorption‐based techniques [31]. Fluorescence spectroscopy provides improved specificity by examining distinct excitation and emission wavelengths and is a rapid, cost‐effective and efficient non‐destructive method

Fluorescence in honeys has been attributed to phenolic and polyphenolic compounds [27–30], amino acids [28–30] and Maillard reaction products [28, 29]. As phenolic and polyphenolic compounds have been described as reliable indicators of botanical and geographical origin of honeys [10, 16, 34, 35]; the fluorescence properties of these intrinsic and unique fluorophores

Recent examination of the fluorescence profiles of the main New Zealand honey types dem‐ onstrated that manuka honey exhibited unique fluorescence characteristics that distinguish it from the other honey types [36]. Manuka honey contained two unique fluorescence signa‐ tures, ex270–em365 nm and ex330–em470 nm, named MM1 and MM2, respectively [36]. Dilution of manuka honey with other New Zealand honey types, which did not fluoresce at the diag‐ nostic wavelengths, resulted in a reduction of the fluorescence signal in the manuka honey

Further work confirmed that Leptosperin was responsible for the MM1 fluorescence signa‐ ture (ex270–em365 nm) [22] and Lepteridine was the principal compound associated with MM2 fluorescence (ex330–em470 nm) in manuka honey [26]. For these compounds, standards were synthesised for Leptosperin [37] and Lepteridine [25], and seeding of honeys experimentally

Consequently, these findings demonstrate manuka honey contains unique fluorophores that may be quantified to establish floral authenticity. As this technique is fluorescence‐based, it provides the opportunity for rapid screening of honey samples to confirm honey labelling is appropriate and complies with the wholly or predominantly ruling in Codex Alimentarius [2]. In this chapter, the fluorescence technique is applied to sets of field‐collected manuka honeys and a set of manuka honeys purchased commercially in 2016. Other compounds of interest in manuka honey, such as 2‐methoxyacetphenone, methyl syringate, MGO and DHA,

The fluorescence markers in manuka honey were assessed in a number of honey collections, first, field honeys harvested from *L. scoparium* hive sites in New Zealand, and second, a com‐ mercial set purchased from retail distributors in Singapore. The honeys in the purchased set were labelled as manuka honey and therefore should be wholly or predominantly sourced

may inform identification of floral source reliably.

confirmed that these compounds are the primary fluorophores.

**2. Fluorescence markers in manuka honey**

that was proportional to the dilution.

are additionally quantified.

from *L. scoparium*.

[32, 33].

98 Honey Analysis

Leptosperin has been shown to be uniquely derived from the *Leptospermum* genus in New Zealand and is present in manuka nectar and honey. This compound is readily quantified by liquid chromatography and mass spectrometry techniques and has been recently shown to be primarily responsible for the fluorescence exhibited by manuka honey at MM1 wavelengths [22]. As Leptosperin has been demonstrated to be chemically stable during extended storage experiments [22], this compound is an ideal candidate as a chemical and fluorescence marker for manuka honey.

Leptosperin is present in manuka honey at a concentration up to approximately 1700 mg/ kg, with a minimum reported concentration of 93 [22] to 126 mg/kg [20], and therefore, it is probable that manuka honey can be expected to carry a minimum of 100 mg/kg. Fluorescence of Leptosperin is readily detected in manuka honey using the reported technique with lower detection limit of 10 ppm.

The field collected manuka honeys (*n* = 28) and the commercial honeys (*n* = 17) exhibited fluorescence that strongly correlated (*R*<sup>2</sup> = 0.9530) with the quantified concentration of Leptosperin (**Figure 2A**), confirming the previous research of this compound. The concen‐ tration of Leptosperin in the commercial samples fell in the lower half of the range recorded for the field samples. The mean concentration of Leptosperin was 423 and 192 mg/kg in the field and commercial samples, respectively (*p* < 0.0001). This is consistent with the previously reported comparison of field and commercial manuka samples [22]. However, two of the commercial samples contained less than 100 mg/kg Leptosperin which is considered to be the lower than acceptable minimum concentration.

Leptosperin and MM1 analysis of an additional field honey collection (*n* = 71) throughout New Zealand (**Figure 2B**) demonstrated that each region contained honeys that were distrib‐

**Figure 2.** (A) Correlation between Leptosperin concentration and MM1 fluorescence in field and commercial manuka honeys. (B) Regional distributions of Leptosperin concentration and MM1 fluorescence, all data correlation shown.

uted throughout the range of recorded concentrations and signal. These observations rein‐ force the earlier findings that Leptosperin is a reliable fluorescence marker in manuka honey and can be used to categorise the national crop.
