**3. Examination of non‐fluorescent markers in commercial honeys**

#### **3.1. 2‐Methoxyacetophenone and methyl syringate**

2‐Methoxyacetophenone has been described as a marker compound for manuka honey pre‐ viously using HS‐SPME‐GC/MS [7, 11]. For the first time, this chapter reports the analysis of this compound in honey solutions using HPLC‐DAD. The concentration of Leptosperin correlated strongly (*R*<sup>2</sup> = 0.8722) with 2‐methoxyacetophenone concentration (**Figure 5A**) rein‐

**Figure 5.** Correlation between Leptosperin and (A) 2‐methoxyacetophenone and (B) methyl syringate in commercial manuka honey samples.

forcing the previous publications regarding the use of this compound as a potential marker for manuka honey.

Methyl syringate concentration is elevated in manuka and kanuka honeys in New Zealand [7, 10]. The linear correlation (**Figure 5B**) of Leptosperin and methyl syringate concentrations is poor (*R*<sup>2</sup> = 0.1704). This may be a reflection of kanuka content in manuka honeys; the kanuka honey is expected to contribute additional elevated levels of methyl syringate. Therefore, methyl syringate is not a reliable marker for manuka honey.

#### **3.2. Dihydroxyacetone and methylglyoxal**

Dihydroxyacetone is the precursor compound for MGO in ripening and maturing manuka honey. Dihydroxyacetone concentration in manuka honey can vary for three reasons: first, the DHA concentration in nectar harvested from the varieties of *L. scoparium* is significantly different, and this may vary as much as twofold [38], second, floral dilution will reduce the amount of DHA being incorporated into a honey during ripening and third, this precursor undergoes chemical reactions in the maturing honey solution [6, 15].

Conversely, MGO is absent in nectar. The MGO concentration increases rapidly during a manuka honey's first couple of years as the chemical conversion of DHA to MGO proceeds. However, the rate of conversion declines as the DHA pool is exhausted. Furthermore, in honeys that are reaching the end of their five‐year shelf‐life, it has been demonstrated that the MGO concentration begins to decline as insufficient DHA remains to sustain the MGO concentration [6].

These changes in DHA and MGO concentration are best demonstrated in elevated tempera‐ ture storage experiments which promote the chemical reactions. Thirteen honeys stored for a little over 3 months at 37°C demonstrate these effects (**Figure 6A**). In this time, DHA decreased by about 18% and MGO increased by 46%, and these concentration shifts continue as the honey matures. Conversely, decreased storage temperature will significantly reduce

**Figure 6.** (A) The relative concentrations of DHA and MGO expressed as a percentage of the initial concentration in 10 manuka honeys stored at 37°C and (B) the relationship of DHA and MGO to the fluorescent marker Leptosperin in commercial manuka honeys.

the rate of chemical reactions in the honey; therefore, the concentration of DHA and MGO in a manuka honey can be considerably influenced by processing and storage conditions.

forcing the previous publications regarding the use of this compound as a potential marker

**Figure 5.** Correlation between Leptosperin and (A) 2‐methoxyacetophenone and (B) methyl syringate in commercial

Methyl syringate concentration is elevated in manuka and kanuka honeys in New Zealand [7, 10]. The linear correlation (**Figure 5B**) of Leptosperin and methyl syringate concentrations is

honey is expected to contribute additional elevated levels of methyl syringate. Therefore,

Dihydroxyacetone is the precursor compound for MGO in ripening and maturing manuka honey. Dihydroxyacetone concentration in manuka honey can vary for three reasons: first, the DHA concentration in nectar harvested from the varieties of *L. scoparium* is significantly different, and this may vary as much as twofold [38], second, floral dilution will reduce the amount of DHA being incorporated into a honey during ripening and third, this precursor

Conversely, MGO is absent in nectar. The MGO concentration increases rapidly during a manuka honey's first couple of years as the chemical conversion of DHA to MGO proceeds. However, the rate of conversion declines as the DHA pool is exhausted. Furthermore, in honeys that are reaching the end of their five‐year shelf‐life, it has been demonstrated that the MGO concentration begins to decline as insufficient DHA remains to sustain the MGO

These changes in DHA and MGO concentration are best demonstrated in elevated tempera‐ ture storage experiments which promote the chemical reactions. Thirteen honeys stored for a little over 3 months at 37°C demonstrate these effects (**Figure 6A**). In this time, DHA decreased by about 18% and MGO increased by 46%, and these concentration shifts continue as the honey matures. Conversely, decreased storage temperature will significantly reduce

methyl syringate is not a reliable marker for manuka honey.

undergoes chemical reactions in the maturing honey solution [6, 15].

**3.2. Dihydroxyacetone and methylglyoxal**

= 0.1704). This may be a reflection of kanuka content in manuka honeys; the kanuka

for manuka honey.

manuka honey samples.

concentration [6].

poor (*R*<sup>2</sup>

102 Honey Analysis

Leptosperin and Lepteridine have also been quantified in honeys stored at elevated tempera‐ tures for over 400 days and neither compound demonstrated any significant deviation from the initial concentration [22, 26].

For manuka honey harvested from hives in a particular well‐defined region where the DHA concentrations in the nectar is relatively constant, there is a relatively strong correlation between MM1 fluorescence and DHA concentration [39]. This is because the honeys are of a uniform age and therefore exclude ageing differences, the DHA potential of the nectar is relatively similar as the harvested *L. scoparium* population is very discrete and genetically linked, and any reduction of DHA in the honey can be attributed to floral dilution from other nectar sources. Consequently, the floral dilution alone acts upon the fluorophores such as Leptosperin, resulting in a relatively stronger correlation between DHA and Leptosperin in the honey.

However, when commercial manuka honey samples are considered the effect of both differ‐ ent initial DHA concentrations in nectar, and ageing‐driven chemical reactions, which may occur at different rates due to temperature influences, are very difficult to separate from flo‐ ral dilution. The concentrations of Leptosperin, DHA and MGO (**Figure 6B**) for commercial manuka honeys demonstrate the relatively poor correlation that exists in commercial honeys of different provincial provenance, unknown age and storage conditions.

The concentrations of DHA and MGO relative to Leptosperin (**Figure 7**) in the 71 field honeys shown in **Figure 2B** reinforce the insignificant relationship between these compounds and a consistent floral marker. The regional data groups are scattered and there is no significant linear correlation as it is likely that the genetic linkage within these large regional areas is insufficient to overcome variability between DHA potential in *L. scoparium* nectar. Similarly,

**Figure 7.** The relationship of (A) DHA and (B) MGO to the fluorescent marker Leptosperin in field honeys throughout New Zealand.

MGO displays a poor correlation with Leptosperin, where differences in ageing profile as well as initial DHA potential are magnified.

Therefore, despite both DHA and MGO being unique to manuka honey in New Zealand, nei‐ ther of these compounds are reliable predictors of floral authenticity in commercial manuka honey samples. Both DHA and MGO should be present in genuine manuka honey; however, the concentration of these compounds does not correlate with stable nectar‐derived chemical markers.

#### **3.3. Commercial manuka honey samples summary**

The concentrations of Leptosperin, Lepteridine, 2‐methoxyacetophenone, methyl syringate, DHA and MGO, along with MM1 and MM2 fluorescence and Unique Manuka Factor (UMF) are presented in **Table 1**. UMF is the non‐peroxide antibacterial grading system devised 25 years ago that measured bioactivity [40] and did not take into the account of floral authenticity.

The significant manuka chemical markers vary in concentration up to more than 1 order of magnitude in the examined honey samples. The harvested *L. scoparium* variety and flo‐ ral dilution by other plant species contribute to this range. However, three of the anal‐ ysed honeys, Samples #1, #2 and #3, demonstrated chemically low or marginal results for Leptosperin and Lepteridine, and likewise had particularly low concentrations of DHA and MGO.

Box‐ and whisker‐plots and histograms (**Figure 8**) demonstrate the statistical distribution of Leptosperin and Lepteridine in the commercial honey set. Both datasets are positively skewed, with mean values greater than the median, and this confirms that more manuka honey in this set has greater concentrations than the proposed lower limits for Leptosperin and Lepteridine.


MGO displays a poor correlation with Leptosperin, where differences in ageing profile as

**Figure 7.** The relationship of (A) DHA and (B) MGO to the fluorescent marker Leptosperin in field honeys throughout

Therefore, despite both DHA and MGO being unique to manuka honey in New Zealand, nei

ther of these compounds are reliable predictors of floral authenticity in commercial manuka honey samples. Both DHA and MGO should be present in genuine manuka honey; however, the concentration of these compounds does not correlate with stable nectar‐derived chemical

The concentrations of Leptosperin, Lepteridine, 2‐methoxyacetophenone, methyl syringate, DHA and MGO, along with MM1 and MM2 fluorescence and Unique Manuka Factor (UMF) are presented in **Table 1**. UMF is the non‐peroxide antibacterial grading system devised 25 years ago that measured bioactivity [40] and did not take into the account of floral

The significant manuka chemical markers vary in concentration up to more than 1 order of magnitude in the examined honey samples. The harvested *L. scoparium* variety and flo

ral dilution by other plant species contribute to this range. However, three of the anal

ysed honeys, Samples #1, #2 and #3, demonstrated chemically low or marginal results for Leptosperin and Lepteridine, and likewise had particularly low concentrations of DHA

Box‐ and whisker‐plots and histograms (**Figure 8**) demonstrate the statistical distribution of Leptosperin and Lepteridine in the commercial honey set. Both datasets are positively skewed, with mean values greater than the median, and this confirms that more manuka honey in this set has greater concentrations than the proposed lower limits for Leptosperin

‐

‐

‐

well as initial DHA potential are magnified.

**3.3. Commercial manuka honey samples summary**

markers.

New Zealand.

104 Honey Analysis

authenticity.

and MGO.

and Lepteridine.

Fluorescence: A Novel Method for Determining Manuka Honey Floral Purity http://dx.doi.org/10.5772/66313 105

**Table 1.** Chemical composition, fluorescence, and non‐peroxide antibacterial activity (UMF) of 17 commercial manuka honey samples.

**Figure 8.** Statistical distribution of Leptosperin and Lepteridine in commercial manuka honeys.

The relationship of Leptosperin and Lepteridine with the associated fluorescence for the com‐ mercial honeys is illustrated (**Figure 9**). For the purposes of this discussion, the minimum accepted concentration of Leptosperin is 100 mg/kg, and Lepteridine 4 mg/kg, these concen‐ trations relate to MM1 (2000 RFU) and MM2 (500 RFU) in a honey matrix. RFU is an arbitrary unit and varies between fluorometers. These lower acceptable levels of these four parameters are illustrated in **Figure 9**.

Honey samples 1 and 2 do not meet the criteria for chemical concentration of either Leptosperin or Lepteridine, and fluorescence profile of both honeys did not meet the lower threshold. Honey sample 3 Leptosperin and Lepteridine concentration was 102 and 3.37 mg/kg, respec‐ tively. The fluorescence signature of this sample was slightly less than the threshold, and most probably was not wholly or predominantly harvested from *L. scoparium*.

When assessing honeys that are close to the lower threshold, it is appropriate to consider mul‐ tiple characteristics and accordingly honey samples 4 and 6 are considered accepted despite the MM2 fluorescence being in the order of 450 RFU rather than 500 RFU.

Therefore, in this commercial set of manuka honeys, three out of 17 samples did not display the fluorescence characteristics or contain the concentrations of the key markers that would be expected to be encountered in an authentic manuka honey. Rapid assessment by fluorescence would have identified these three samples as requiring a full analytical workup, and allowing the balance of samples to be retailed as manuka honey.

**Figure 9.** Commercial manuka honey samples Leptosperin and Lepteridine concentrations in relation to MM1 and MM2 fluorescence.

**Figure 8.** Statistical distribution of Leptosperin and Lepteridine in commercial manuka honeys.

106 Honey Analysis
