Quantitative analysis of sugars in Brazilian and Canadian honey samples using 1H NMR spectroscopy

Quantitative analysis of sugars in Brazilian and Canadian honey samples using 1H NMR spectroscopy

*Thays C. Valim1, Rafael Q. Ferreira1, Camilo F. Martinez-Farina2, Ian W. Burton2, FabriceBerrué2, Valdemar L. Junior1, Álvaro C. Neto1

1 Federal University of Espírito Santo, Vitoria, ES, Brazil

2 National Research Council Canada, Halifax, NS, Canada

*thayscvalim@gmail.com

Honey is widely used worldwide for medicinal purposes or as a natural sweetener. It is a complex

matrix consisting of around 200 substances, with approximately 85% of its content comprising sugars.

Glucose and fructose are the major sugars, but honey samples can also contain maltose, sucrose,

nigerose, turanose, kojibiose, and trehalose.1 The ratio between glucose and fructose is important for

verifying the quality of honey and may indicate its ability to crystallize. Additionally, the amount of

sucrose is an important indicator of quality.2

As a powerful technique, NMR has been widely used for food analysis, either for qualitative or

quantitative purposes. Quantitative NMR (qNMR) offers significant advantages compared to other

techniques, especially due to its primary analytical characteristic.3 For this reason, most papers related

to qNMR present a quantification process based on a direct relation between the analyte and an

internal standard. Therefore, the aim of this work has been quantifying the amount of sugars using

1H NMR, compare the results between Brazilian and Canadian honey samples, and validate a

methodology for the quantification process. Methodology validation requires good selectivity,

linearity, precision, accuracy, detection and quantification limit, and suitable robustness for the

analysis.4

The qNMR spectra were acquired using three different equipment: a 9.4 T (400 MHz) Varian/Agilent

spectrometer equipped with a 5 mm broadband probe, an 11.7 T (500 MHz) Bruker Avance III system

featuring both with a 5 mm TXI probe and a 5 mm BBFO probe with auto-tune and match utilizing

90o pulse, and a 16.4 T (700 MHz) Bruker Avance III NMR spectrometer equipped with a 5 mm

CPTCI cryogenically cooled probe. A comprehensive investigation was conducted on the relaxation

delay to ensure that all signals utilized for sugar quantification achieved full relaxation during the

quantification analysis. These preliminary results have affirmed that the sensitivity of the technique

may be affected by the type of probe, analyte concentration, and magnetic field strength.

The trimethylsilylpropionic acid (TMSP) was employed as the internal standard, and all analysis are

being performed with 1 mL of 50 mM NaH2PO4/Na2HPO4 buffered D2O at pH 7.2, as the solvent. As

part of the validation process, a calibration curve was constructed for each analyte using

concentrations ranging from 1 mM to 8.5 mM at six different levels. Statistical analysis and other

performance parameters are ongoing. The validation protocol will be applied to Brazilian and

Canadian honey for comparison to assess any discernible differences.

Acknowledgments: CAPES, CNPq, FAPES, LabPetro and NRC Canada for financial support.

References

1. Ian W. Burton; et al. Molecules, 28 (4), 1656 (2023).

2. Silvia Valverde; et al. Food Chemistry, 387, 132920 (2020).

3. Santosh K. Bharti; Raja Roy. TrAC Trends in Analytical Chemistry, 35, 5-26 (2012).

4. Phillip Borman; David Elder. ICH quality guidelines: an implementation guide, 127-166

(2017).