Methodology used for identification of chlorophyll and other bioactive compounds in Green Honey (GH) and Tualang Honey

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An overview of the methodology in the identification of chlorophyll and other bioactive compounds in both Green Honey (GH) and Tualang Honey (TH). The document outlines the systematic approach used to analyze these compounds in both GH and TH.

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Sample preparation: The green honey (GH) sample was prepared using a standard procedure as described by Alghamdi et al., [1]. A total of 2.5 g of the corresponding honey sample was dissolved in 25 ml of de-ionized water in a beaker. The resulting solution was then filtered through a 0.045 µm nylon filter into a 50 ml volumetric flask. A suitable volume of the filtered solution was then transferred into a Liquid Chromatography-Mass Spectrometry (LC-MS/MS) instrument for further analysis. Instrument: The instrument used in the study is Agilent 6560 Ion Mobility Quadrupole Time-Of-Flight (Q-TOF) LC/MS. LCMS Quadrupole Time-Of-Flight (Q-TOF): The chlorophyll and its derivatives were quantified using an LC system coupled with Quadrupole Time-Of-Flight (Q-TOF) according to a method previously described with a little modification [2, 3]. This study adopted an already developed method of chlorophyll and its derivatives identification. LC parameters are LCMS QTOF mode with a mobile phase A 0.1 formic acid in water, mobile B 0.1 formic acid in acetonitrile injection volume are 1 µl with a flow rate of 0.3 ml/min, a column Zorbax Extend C18 (2.1 X 50mm/1.8micron). HyPURITY C18 column, and a gradient mobile phase of methanol and acetone, operated in negative-positive mode, were used to separate the chlorophylls and chemical compounds in both honey samples within 40 minutes with a flow rate of 1 ml/min. Detection was achieved at A660nm, and identification was based on retention time, mass spectra, and mass-to-charge ratio. Gradient elution for the experiment is given in (Table 1.) Table 1. Gradient elution phase for both targeted and untargeted analysis Time(min) A% B% 0 99 1 2 99 1 12 0 100 15 0 100 16 99 1 20 99 1 MS parameter set for the experiment are positive ion polarity, Gas temp 225 OC, Drying gas flow 12 L/min, Nebulizer 45 psi, Sheet Gas flow 12 L/min, Vcap 3500 V, MS TOF Fragmentor 150 V, and Oct 1 RF Vpp 750 V.

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