# Polyphenolic Content and Sensory Characteristics of New Zealand Honey Ice Cream
## 2 Materials and Methods
### LC-MS Analysis of Honey Ice Cream Samples  
LC-MS was conducted to determine the polyphenolic content in honey ice cream. A 10 g sample of honey ice cream was defatted three times using 10 mL of hexane. The mixture was then centrifuged at 4000 rpm for 20 min at 4 \({}^{\circ}\)C, and the supernatant was discarded each time. Next, the protein was precipitated by adding 12 mL of methanol and vortexing for 15 s. The mixture was stored at \(-\)20 \({}^{\circ}\)C for one hour and then centrifuged at 3950 rpm for 5 min at 4 \({}^{\circ}\)C. The supernatant was then transferred to another Falcon tube. Subsequently, 12 mL of a 9:1 ratio mixture of methanol and formic acid was added to the remaining precipitate. The mixture was vortexed for 15 s and stored at \(-\)20 \({}^{\circ}\)C for another hour. The samples were centrifuged again at 3950 rpm for 5 min at 4 \({}^{\circ}\)C, and the resulting supernatant was combined with the previously stored supernatant. The combined supernatants were centrifuged at 4000 rpm for 30 min at 20 \({}^{\circ}\)C. Then, 5 mL of the liquid obtained from this process was evaporated under a stream of air to reduce the volume to one-fifth of its original volume. Finally, the concentrate was reconstituted with 3 mL of methanol containing 2% acetic acid. Phenomenex Strata XA Cartridges (Phenomenex, Auckland, NZ, USA) were used to isolate the polyphenols. The cartridges were conditioned with 3 mL of methanol (Fisher Chemicals, Fair Lawn, NJ, USA) followed by 3 mL of Milli-Q water (pH = 7). The supernatant was then loaded into the cartridge, and 4 mL of Milli-Q water (pH = 7) was added. The phenolic compounds retained in the cartridge were eluted using 5 mL of methanol:formic acid (1:9 \(v/v\)) (Fisher Chemicals, Fair Lawn, NJ, USA). The eluate was evaporated under a fume hood using an evaporator. Subsequently, the eluate was dried to approximately 0.75 mL, reconstituted with 2 mL of methanol, and mixed with 2% acetic acid (Fisher Chemicals, Fair Lawn, NJ, USA). Before the LC-MS analysis, all samples were filtered using a 0.22 nm filter (Millipore, Bedford, MA, USA). Commercial standards of polyphenols (rutin, quercetin, kaempfer, luteolin, gallic acid, benzoic, caffe, p-coumaric, pinobanksin, chrysin, epicatechin, catechin, apigenin-7-o-glucose, pinocombrin, hydroxybenzoic acid, homovanillic acid, and quinic acid) acquired from Sigma Aldrich (New South Wales, Australia) were used to determine the concentration of polyphenols present in the honey samples. LC-MS analysis was conducted using an Agilent 1260 Infinity Quaternary LC System (Agilent, Santa Clara, CA, USA). The system consisted of the following components: 1260 quaternary pump (model number: G1311B), 1260 infinity ALS sampler (model number: G1329B), 1260 infinity TCC column component (model number: G1316A), and the 1260 infinity diode array detector (DAD) (model number: G4212B) that were connected to a 6420 triple quadrupole LC/MS system with electrospray ionisation (ESI) source (model number: G1948B). The MS ionisation source conditions were as follows: capillary voltage of 4 kV, drying gas temperature of 300 \({}^{\circ}\)C, drying gas flow of 10 L/min, and nebulizer pressure of 40 psi. The negative ionisation mode was performed using multiple reaction monitoring (MRM) for quantitative analysis. The MRM transitions with collision energy and cell accelerator voltage are summarised in Table 1, showing the retention times and chemical formulas of the different polyphenic compounds. The Agilent Poroshell EC-C18 (2.1 \(\times\) 150 mm, 2.7 \(\upmu\)m) column was used for LC-MS analysis. The mobile phases consisted of water containing 0.1% (\(v/v\)) formic acid (A) and  
acetonitrile containing 0.1% (\(v/v\)) formic acid (B). The flow rate was 0.30 mL/min, and the column temperature was maintained at 40 \({}^{\circ}\)C. The initial gradient condition was 95:5 (A:B) and was held for 5 min. From 5 to 8 min, the proportion of B was increased to 10% and held for 5 min.