Honey Testing

Se, Kuan Wei, et al. Journal of Food Composition and Analysis 80 (2019): 16-32.

Honey is a nutritious and naturally sweet substance produced by honeybees from the nectar collected from various flowers. However, the health benefits and high value of honey have led to fraudulent practices of honey adulteration, where cheaper sweeteners are added directly or indirectly. Strict quality control measures are therefore necessary to ensure the authenticity of honey and protect consumer health.

Detection Methods for Adulterants in Honey

1. Thin-Layer Chromatography (TLC): TLC is a simple technique used to identify the presence of unknown chemical components in natural honey. A better method for detecting adulterants in honey is the use of High-Performance Thin-Layer Chromatography (HPTLC).

2. Stable Carbon Isotope Ratio Analysis (SCIRA): SCIRA is a recognized standard method for honey adulteration and detection, which can be used to differentiate honey from different plant sources.

3. Chromatographic Techniques:

3.1. Gas Chromatography (GC): GC is a highly versatile technique for detecting sugar-based adulterants in honey, as it can directly analyze volatile and semi-volatile compounds (honey aroma).

3.2. Liquid Chromatography (LC): LC is one of the few mature techniques used to identify C3 and C4 sugars in honey.

4. Spectroscopic Techniques:

4.1. Nuclear Magnetic Resonance (NMR) Spectroscopy: Proton NMR combined with statistical modeling can be used to assess the authenticity of monofloral and multifloral honeys from various plant and geographical sources.

4.2. Raman Spectroscopy: Raman spectroscopy provides complementary information to infrared spectroscopy, and when combined with chemometrics, it can perform highly accurate quantitative assessment of different sugar adulterants in honey.

4.3. Infrared (IR) Spectroscopy: IR spectroscopy has become a popular technique for detecting adulterants in honey. It has several notable advantages, such as speed, simple sample preparation, low cost, non-destructive nature, user-friendliness, and suitability for on-site monitoring.

Ploegaerts, Grégory, et al. Journal of Trace Elements and Minerals 4 (2023): 100070.

Although honey is generally considered a pure natural product, an article published by the Belgian consumer association Test-Achats in 2017 showed that over 66% of the tested honeys did not meet the quality standards claimed by the manufacturers. Furthermore, the article reported that the assessment only focused on sugar composition, enzyme activity, product degradation (due to aging or heating), and pollen source, without evaluating potential chemical contaminants such as trace elements.

Nevertheless, the detection of these contaminants is crucial to ensure the safe consumption of honey. Contaminated food not only has a direct impact on health, but even low-level pollution, if consumed regularly, can also be toxic. The Codex Alimentarius, outlined in food safety regulations, stipulates that "honey shall not contain heavy metals that may endanger human health."

Ma, W., Yang, B., Li, J., & Li, X. (2022). Molecules, 27(3), 1056.

Honey is a highly valuable food product, and it is also considered to have medicinal properties such as anti-inflammatory, anti-tumor, and antioxidant effects. Honey from different sources can be used to treat various diseases. Traditional quality control methods for honey are based on its physicochemical properties, such as moisture content, total carbohydrates (glucose and fructose), diastase activity, and even electrical conductivity. Recently, the quantitative analysis of free amino acids (FAA) in honey has gained increasing attention, as it provides information for identifying the plant and geographic origin of honey, and assists in authenticity verification.

A simple, fast, and reliable analytical method has been developed to determine 20 free amino acids (FAA) in honey samples using a dilution injection strategy and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS).

Gialouris, P. L. P., Koulis, G. A., Nastou, E. S., Dasenaki, M. E., Maragou, N. C., & Thomaidis, N. S. (2023). Heliyon, 9(11).

Honey is a natural sweetener produced by honeybees. The volatile components of honey include alcohols, aldehydes, ketones, esters, hydrocarbons, furans, pyrans, terpenes, and benzene derivatives, which impart the unique aroma of honey. Therefore, the volatile components have a significant impact on the quality of honey and the preferences of global market consumers.

Using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), a targeted and suspect screening method was used to determine 53 volatile compounds in honey samples.

Rial-Otero R, Gaspar EM, Moura I, Capelo JL. Talanta. 2007 Feb 15;71(2):503-14.

As honey is a difficult matrix, sample pretreatment is crucial to obtaining reliable results. Over the past decades, various methods have been proposed to reduce sample handling and toxic waste. The extraction of pesticides from honey is typically accomplished through one of the following procedures:

Solvent Extraction (SE): In the SE extraction process, we can distinguish three steps. In the first step, the honey is diluted in water or a water mixture, such as acetone-water or methanol-water, to obtain better sample homogenization before analyte extraction. In the second step, pesticides are extracted using different water-immiscible solvents, depending on the polarity of the pesticides. In the third step, the extracts obtained after sample treatment are cleaned up to avoid the co-extraction of undesirable high-molecular-weight compounds that could contaminate the chromatographic system and reduce interfering compounds.

Supercritical Fluid Extraction (SFE): SFE has been proven to be an efficient and fast method for the separation of pesticides from honey. This procedure takes advantage of the unique properties of supercritical fluids to extract pesticides from solid samples.

Solid-Phase Extraction (SPE): In SPE, the sample is passed through a cartridge or column packed with a solid sorbent, and the pesticides are adsorbed, then eluted with organic solvents.

Matrix Solid-Phase Dispersion (MSPD): MSPD is an extraction and clean-up technique aimed at overcoming the common drawbacks of SE and SPE in extracting analytes from solid or semi-solid samples. Compared to SE, this technique requires less time and solvents while providing similar results. It also avoids the previous step of diluting solid or semi-solid samples to pass through SPE.

Solid-Phase Microextraction (SPME): SPME has become a popular method for the analysis of organic compounds, as it combines sampling and preconcentration in a single step. In this technique, a fused-silica fiber coated with a polymeric film is immersed in the sample. Pesticides are adsorbed onto the stationary phase and then thermally desorbed into the GC injection port.

Stir Bar Sorptive Extraction (SBSE): In SBSE, a stir bar coated with an adsorbent is stirred in the sample for a certain time until the analytes reach equilibrium between the polymer and the aqueous phase, according to their distribution constant. The analytes are then thermally desorbed into the GC injection port or liquid-desorbed for HPLC analysis. The most important advantage of SBSE is the same as SPME, but SBSE can achieve higher recovery rates.