**1. Introduction**

New Zealand manuka honey is harvested from *Leptospermum scoparium* (Myrtaceae) through‐ out the country. Internationally, this honey has received considerable attention and value due to its unique health‐related benefits. Major destination markets include Hong Kong and China, Japan, the European Union, United Kingdom, the United States and Australia. Over 80% of the total honey exported from New Zealand is now pre‐packaged, hive numbers in the country have almost doubled in the last 10 years [1] and the value of the manuka honey industry is now estimated in the vicinity of NZ\$150 million.

Codex Alimentarius [2] defines that a honey must be derived wholly or predominantly from a particular floral source and display the corresponding organoleptic, physico‐chemical and microscopic properties for a floral attribution to be made. Within New Zealand, a number of surplus nectar‐producing common plant species exist with similar distributions and flower‐ ing times as *L. scoparium*. Consequently, manuka honey may contain different levels of dilu‐ tion by other floral types, as honey produced in a natural environment containing a range of plant species is unlikely to be monofloral because of bee behaviour in the forage field [3].

Historically, New Zealand honeys have been classified by physico‐chemical analysis and melissopalynology. Melissopalynology is a common technique internationally for describ‐ ing honeys; however, in New Zealand *Kunzea ericoides* often flowers simultaneously with *L. scoparium*, and the pollen grains of these species are virtually indistinguishable in a honey medium [4]. To overcome this, a classification structure was built upon the unique non‐per‐ oxide antibacterial activity that manuka honey exhibits, yet this system did not take into the account of honey's floral composition.

Honey is a complex supersaturated sugar solution containing approximately 80% sugars and a unique combination of other compounds suspended in water. The sugar proportion is prin‐ cipally the monosaccharide fructose and glucose, and the non‐sugar proportion includes a range of bee‐ and plant‐derived compounds such as organic acids, proteins, amino acids, phenolic acids, flavonoids, pollen and waxes [5]. This chemical composition varies between honey types, geographical origin and climate may additionally alter the constituents [5], and furthermore honey processing techniques and age may also be influential [6].

Manuka honey contains a diverse array of compounds that range from unique carbohydrate metabolites to phenolics, flavonoids and volatiles. Many of these have received attention [7– 11], and clearly, this honey carries a number of distinct compounds that may be diagnostic for classification. For example, 2‐methoxyacetophenone (**Figure 1**) [7, 11] and 2‐methoxybenzoic acid [10, 11] have been proposed as floral markers for manuka honey.

In addition, dihydroxyacetone (DHA) and methylglyoxal (MGO) (**Figure 1**) are solely derived from *L. scoparium* nectar in New Zealand honeys [12, 13]. Dihydroxyacetone is present in *L. scoparium* nectar, converting non‐enzymatically and irreversibly to MGO in the acidic envi‐ ronment of a ripened honey solution [13]. This conversion is non‐stoichiometric [14, 15] due to the presence of side reaction pathways in the honey. However, the concentrations of these compounds are not stable throughout a manuka honey's shelf‐life and therefore neither are suitable as reliable chemical markers [6].

Fluorescence: A Novel Method for Determining Manuka Honey Floral Purity http://dx.doi.org/10.5772/66313 97

**1. Introduction**

96 Honey Analysis

New Zealand manuka honey is harvested from *Leptospermum scoparium* (Myrtaceae) through‐ out the country. Internationally, this honey has received considerable attention and value due to its unique health‐related benefits. Major destination markets include Hong Kong and China, Japan, the European Union, United Kingdom, the United States and Australia. Over 80% of the total honey exported from New Zealand is now pre‐packaged, hive numbers in the country have almost doubled in the last 10 years [1] and the value of the manuka honey

Codex Alimentarius [2] defines that a honey must be derived wholly or predominantly from a particular floral source and display the corresponding organoleptic, physico‐chemical and microscopic properties for a floral attribution to be made. Within New Zealand, a number of surplus nectar‐producing common plant species exist with similar distributions and flower‐ ing times as *L. scoparium*. Consequently, manuka honey may contain different levels of dilu‐ tion by other floral types, as honey produced in a natural environment containing a range of plant species is unlikely to be monofloral because of bee behaviour in the forage field [3]. Historically, New Zealand honeys have been classified by physico‐chemical analysis and melissopalynology. Melissopalynology is a common technique internationally for describ‐ ing honeys; however, in New Zealand *Kunzea ericoides* often flowers simultaneously with *L. scoparium*, and the pollen grains of these species are virtually indistinguishable in a honey medium [4]. To overcome this, a classification structure was built upon the unique non‐per‐ oxide antibacterial activity that manuka honey exhibits, yet this system did not take into the

Honey is a complex supersaturated sugar solution containing approximately 80% sugars and a unique combination of other compounds suspended in water. The sugar proportion is prin‐ cipally the monosaccharide fructose and glucose, and the non‐sugar proportion includes a range of bee‐ and plant‐derived compounds such as organic acids, proteins, amino acids, phenolic acids, flavonoids, pollen and waxes [5]. This chemical composition varies between honey types, geographical origin and climate may additionally alter the constituents [5], and

Manuka honey contains a diverse array of compounds that range from unique carbohydrate metabolites to phenolics, flavonoids and volatiles. Many of these have received attention [7– 11], and clearly, this honey carries a number of distinct compounds that may be diagnostic for classification. For example, 2‐methoxyacetophenone (**Figure 1**) [7, 11] and 2‐methoxybenzoic

In addition, dihydroxyacetone (DHA) and methylglyoxal (MGO) (**Figure 1**) are solely derived from *L. scoparium* nectar in New Zealand honeys [12, 13]. Dihydroxyacetone is present in *L. scoparium* nectar, converting non‐enzymatically and irreversibly to MGO in the acidic envi‐ ronment of a ripened honey solution [13]. This conversion is non‐stoichiometric [14, 15] due to the presence of side reaction pathways in the honey. However, the concentrations of these compounds are not stable throughout a manuka honey's shelf‐life and therefore neither are

furthermore honey processing techniques and age may also be influential [6].

acid [10, 11] have been proposed as floral markers for manuka honey.

industry is now estimated in the vicinity of NZ\$150 million.

account of honey's floral composition.

suitable as reliable chemical markers [6].

**Figure 1.** Chemical structure of Leptosperin, Lepteridine, 2‐methoxyacetophenone, methyl syringate, dihydroxyacetone and methylglyoxal.

Internationally, classifying honeys by chemical signature or key components has received increasing attention over the last 20 years. European honeys have been thoroughly investi‐ gated [16] confirming earlier work on, for example, rosemary [17] and heather [18, 19] honeys.

Further investigation on the phenolic and flavonoid profile of manuka honey has revealed two unique compounds. First, a nectar‐derived glycoside of methyl syringate has been described [20, 21]. Whilst this compound is present in the wider *Leptospermum* genus throughout Australasia, it is restricted to *L. scoparium* in New Zealand and therefore is potentially a suit‐ able floral marker. Consequently, methyl syringate 4‐O‐*β*‐D‐gentiobiose in manuka honey, named Leptosperin<sup>1</sup> (**Figure 1**), has been analysed by high‐performance liquid chromatog‐ raphy (HPLC), mass spectrometry, immunochemistry and immunochromatography [21–24].

More recently, analysis showed the presence of another unique compound in *L. scoparium* nectar and honey. In this case, the compound was described as a pteridine derivative 3,6,7‐tri‐ methyllumazine, and named Lepteridine (**Figure 1**) [25]. This compound has also been quan‐ tified by HPLC in manuka honey [26]. Both Leptosperin and Lepteridine have been reported to be chemically stable over prolonged storage in honey [21, 22, 26].

Methyl syringate (**Figure 1**) has also been shown to be present at elevated concentrations in manuka honey. However, previous studies indicate that this compound does not correlate with non‐peroxide activity [20, 21]. Additionally, methyl syringate concentration has also been reported as elevated in kanuka honeys (*K. ericoides*) and is higher than that reported in manuka honeys [7, 10]. Accordingly, methyl syringate may not be a suitable chemical marker for manuka honey.

<sup>1</sup> Leptosperin was initially named 'leptosin' [20] but was later renamed to avoid confusion with the marine fungus‐ derived leptosins [21].

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 [32, 33].

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 may inform identification of floral source reliably.

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 that was proportional to the dilution.

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 confirmed that these compounds are the primary fluorophores.

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, are additionally quantified.
