Rapid and Reliable HPLC Method for the Simultaneous Determination of Dihydroxyacetone, Methylglyoxal and 5-Hydroxymethylfurfural in Leptospermum Honeys

Abstract

A reliable determination of dihydroxyacetone, methylglyoxal and 5-hydroxymethylfurfural is essential to establishing the commercial value and antimicrobial potential of honeys derived from the Leptospermum species endemic to Australia and New Zealand. We report a robust method for quantitation of all three compounds in a single HPLC run. Honey samples (n = 6) that are derivatized with o-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine were quantitated against a stable anisole internal standard. Linear regression analysis was performed using calibration standards for each compound (n = 6) and results indicated a high degree of accuracy (R2 = 0.999) for this method. The reliability of some commercial methylglyoxal solutions were found to be questionable. Effective quantitation of methylglyoxal content in honey is critical for researchers and industry, and the use of some commercial standards may bias data. Two accurate methylglyoxal standards are proposed, including a commercial standard and a derivative that can be prepared within the laboratory.

Correlation of the antibacterial activity of commercial manuka and Leptospermum honeys from Australia and New Zealand with methylglyoxal content and other physicochemical characteristics

“…The methylglyoxal (MGO), dihydroxyacetone (DHA) and hydroxymethylfurfural (HMF) content of all manuka and Leptospermum honeys was determined using a previously published method [ 13 ] and the corresponding (theoretical) non-peroxide activity (NPA) was then calculated from the quantified level of MGO. Temperature-adjusted Refractive Index and Brix values were determined simultaneously by spreading a sample of each honey over the entire surface of the reading window of a digital refractometer (Hanna Instruments, Smithfield, RI, USA) as per the instrument manual.…”

Monitoring the Release of Methylglyoxal (MGO) from Honey and Honey-Based Formulations

“…A 5-point calibration curve ( Figure 2 ) for the quantification was obtained by calculating the ratio of the peak area of MGO (RT 14.49 min) to the peak area of the HA internal standard (RT 9.03 min) in the chromatogram ( Figure 3 ) [ 22 ]. The equation derived from the linear regression analysis of the calibration was used to quantify the baseline MGO and also the release profile of MGO from MGO-spiked artificial honey, pure Manuka honey and the five commercial formulations containing Manuka honey.…”

A New Cost-Efficient Technique for Analysis of Nectar Sugars and Dihydroxyacetone in Australian Leptospermum Using Liquid Chromatography–Tandem Mass Spectrometry

“…Aqueous and nectar standards were established with 12 spiked concentrations of DHA, glucose, fructose, and sucrose (mg·L –1 ) of 4545, 2272, 1136, 568, 284, 142, 71, 35, 17, 8.8, 4.4, and 2.2. This concentration range was chosen to encompass the natural analyte ranges found in L. scoparium nectar. , All aqueous and nectar standards had a final ribose internal standard concentration of 454 mg·L –1 . Leptospermum standards were made with L. scoparium nectar diluted 10-fold, to further minimize any potential matrix effects.…”

“…This concentration range was chosen to encompass the natural analyte ranges found in L. scoparium nectar. 6,15 All aqueous and nectar standards had a final ribose internal standard concentration of 454 mg•L −1 . Leptospermum standards were made with L. scoparium nectar diluted 10-fold, to further minimize any potential matrix effects.…”