**5.3. UPLC and fluorescence methods**

**4. Conclusions and applications**

to this analysis.

108 Honey Analysis

**5. Analytical techniques**

**5.1. Fluorescence spectroscopy**

fluorescence units (RFU).

**5.2. HPLC**

Fluorescence analysis is a novel technique to determine manuka honey authenticity. Two unique compounds have been found in manuka honey, Leptosperin and Lepteridine, and these compounds are responsible for the MM1 and MM2 fluorescence described in the honey. In New Zealand, Leptosperin and Lepteridine are present only in *L. scoparium* nectar and therefore, these compounds are reliable chemical markers for manuka honey. However, the concentrations of these compounds do not predict DHA and MGO concentrations in a honey. Fluorescence spectroscopy is a rapid technique with high throughput, and relatively simple fluorescence screening assessments are gaining increasing attention in food processing sys‐ tems. Technology for assessing fluorescence is developing rapidly and handheld fluorometers are available. A handheld fluorometer could be used in the field by beekeepers, alternatively

Fluorescence assessment of manuka honey is an independent method separate from liquid chromatography coupled to detectors such as DAD or mass spectrometry. The use of two sets of wavelengths in combination, which can be screened simultaneously, adds robustness

Therefore, analysis of the MM1 and MM2 wavelengths is an efficient way of screening New Zealand honeys to ensure that attribution of floral source is appropriate and manuka honeys are wholly or predominantly sourced from *L. scoparium*, and these honeys that do not display

Honey fluorescence was analysed by scanning fluorescence spectroscopy according to methods described previously [36]. Honey samples were diluted with distilled water to 2% w/v, and loaded as 100 μL aliquots into a flat‐bottom microplate (OptiplateTM‐384, black). Fluorescence measurements were carried out on a Gemini EM Dual‐Scanning Microplate Spectrofluorometer (Molecular Devices Inc., Sunnyvale, CA, USA) operated with the SoftMax® Pro software. A fluorescence top read setting with automatic calibration and sen‐ sitivity at an ambient temperature was adopted for analysis at both MM1 and MM2 marker wavelengths. Fluorescence intensity was expressed as arbitrary units, in this case relative

Leptosperin, Lepteridine, methyl syringate and 2‐methoxyacetophenone concentrations were quantified on a Dionex UltimateTM 3000 reverse‐phase HPLC system (Thermo Fisher

in market by retailers and may be of use to regulatory authorities.

characteristics of manuka honey are not inappropriately labelled.

Scientific, New Zealand) with diode‐array detection (DAD).

Honey samples (0.5 g) were weighed into a polypropylene extraction tube and solubilised in 9.5 mL of 10% acetonitrile containing 0.1% formic acid in Type 1 water by shaking and ultra‐ sonic agitation. After centrifugation to remove particulates, an aliquot was diluted a further fivefold for analysis by ThermoFisher Ultimate‐3000 UPLC with an RS fluorescence detector ( ex264nm–em365nm), using a Waters XSelect HSS T3 C18 column (2.1 × 30 mm, 2.5 μm particle size). Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in methanol. The elution gradient started at 8% B (92% A) and increased to 100% B over 10 min before equilibration in 92% A for 3 min. Leptosperin was quantified against a synthetic standard.

For gross honey fluorescence analysis, 250 μL of the 20‐fold diluted extract used for the Leptosperin analysis was added to the well of a fluorescence‐grade 96‐well plate and the gross fluorescence of each sample measured at MM1 using a SpectraMax i3 (Molecular Devices LLC, Sunnyvale CA, USA).

## **5.4. DHA and MGO**

Concurrent analysis of DHA and MGO was carried out on a Dionex UltimateTM 3000 reverse‐ phase UPLC‐DAD system (Thermo Fisher Scientific, New Zealand) following derivatisation with *O*‐(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA). Honey samples were prepared in distilled water at a 1:20 w/v ratio. The samples were thoroughly mixed and incu‐ bated at 50°C for 1 hour to allow complete dissolution of any sugar crystals.

The derivatisation procedure was carried out according to methods developed by Kato et al. [41] with some modifications. A 2% stock solution of PFBHA was prepared in 0.1 M citrate buffer adjusted to pH 4 with NaOH (1 M). A working solution of PFBHA derivatising reagent was prepared consisting of 7:2:1 LC‐MS grade acetonitrile:distilled water:PFBHA stock solu‐ tion, and added to the honey samples at a 5:1 v/v ratio. The PFBHA:honey mixture was incu‐ bated at 50°C for 1 hour and cooled to room temperature.

A 5 μL aliquot of the derivatised sample or standard was injected into the UPLC‐DAD system. Separation was carried out by gradient elution on a Hypersil GOLD column (100 × 2.1 mm, 1.9μμm particle size) at a constant flow rate of 0.700 mL/min. The mobile phase consisted of 0.1% v/v aqueous formic acid (Solvent A) and LC‐MS grade acetonitrile (Solvent B), and the gradient elution programme was as follows: initial (B 20%, held 0.6 min), 1.3 min (B 70%), 3 min (B 100%, held 0.5 min) and 4 min (B 20%). The column was thermostatically controlled at 50°C. Dihydroxyacetone was monitored at 214 nm and MGO at 246 nm.

Data acquisition and peak integration were performed with Thermo Fisher ScientificTM Dionex™ Chromeleon™ 7.2 CDS software. Honey DHA and MGO were quantified using external calibration curves generated from the DHA and MGO working standards by linear regression of peak area against concentration.
