The identification and quantification of compounds in the eight honey samples were performed using quantitative ¹H NMR. This technique has demonstrated its effectiveness for the authentication and quality control of food products, particularly honey [7]-[9].

For each sample, 2.5 g of honey was dissolved in 10 mL of distilled water. The resulting solution was filtered to remove any insoluble residues. To minimize chemical shift variations and to standardize the signals of organic acids, the pH of the sample was adjusted to a value of 3.1. Tetramethylsilane (TMS), used as an internal reference for chemical calibration of the spectrum, was then added. Each preparation was finally transferred to a specific NMR tube and then sealed for analysis.

Each of the eight (8) NMR tubes is placed in the NMR spectrometer, which generates an intense magnetic field. Radiofrequency waves are emitted to excite the nuclei of the samples being studied. The signals emitted by the excited nuclei are detected and recorded. Processing these signals generates the NMR spectra [11].

Each of the eight tubes was successively placed in the NMR spectrometer. Under the effect of an intense magnetic field, the nuclei of the hydrogen atoms (protons) of the compounds are excited, detected, recorded and processed to generate the characteristic ¹H NMR spectra of each sample [11].

Sugar composition

¹H NMR analysis identified thirteen compounds in the honey samples, including the three major sugars: fructose, glucose, and sucrose. These constitute the main fraction of honey, representing approximately 70% to 80% of its composition. The fructose (35.6% - 38.9%) and glucose (25.8% - 32.4%) levels measured in the samples are within the limits set by the Codex Alimentarius Commission, which specifies ranges of 32% to 42% for fructose and 26% to 36% for glucose [5][10]. These values confirm the predominance of these two sugars in natural honeys.

The fructose/glucose (F/G) ratio in Table 3, an important indicator of floral origin and crystallization tendency, was calculated for each sample. All samples had an F/G ratio greater than 1.1, characteristic of fructose dominance, typical of natural honeys [5]. A high ratio, such as that of M₁ (1.49), promotes longer storage in liquid form, while a lower ratio, such as that of M₆ (1.16), accelerates crystallization. Furthermore, sample M₇ stands out due to its simultaneously high fructose (38.9%) and glucose (32.4%) content, which could reflect a specific botanical origin.

Table 3. Ratio of fructose to glucose content in honey samples.

Sucrose content and authenticity markers

Sucrose content is a key indicator of honey maturation and authenticity. In a mature and unaltered product, it remains low, as sucrose is hydrolyzed by bee enzymes during honey production in the hive. Regulations, particularly those of the Codex Alimentarius [5], generally set a maximum threshold of 5%. In accordance with this expectation, the majority of the samples analyzed (M₁ to M₅, M₇, M₈) have a very low content (≤0.5%), indicating good maturation (Table 2). In contrast, sample M₆ stands out with a significantly higher concentration (1.9%). This abnormal value may suggest premature harvesting, the possible addition of exogenous sugar, or, to a lesser extent, a specific botanical origin, as some honeydew honeys can naturally contain a higher residual level. These observations corroborate the conclusions of Yeboué etal. [12].

The presence of other characteristic sugars further supports this assessment. Turanose, a rare sugar synthesized during the enzymatic digestion of nectar and absent from commercial sugars, was detected in all samples (0.84% - 2.75%). Its presence thus confirms the authenticity of the honeys analyzed, with variations in content likely linked to their floral origin.

Finally, mannose and dihydroxyacetone (DHA) were identified in trace amounts in all samples. Since DHA is an abundant marker of heather honey, its low concentrations here rule out a dominant botanical origin of this type.

Freshness and Aging Indicator

The 5-HMF (hydroxymethylfurfural) content, which ranges from 0.001% to 0.02% in the samples (Table 2), is a critical indicator of honey’s freshness and heat history. This compound forms naturally during aging, but its formation is significantly accelerated by excessive heating [13]. The Codex Alimentarius standard establishes a maximum limit of 40 mg/kg (or 0.004%) [5]. In this regard, samples M₂ and M₃, with a content of 0.02%, have a concentration five times higher than this limit and twenty times higher than that of M₆. This result strongly suggests that these honeys have undergone intensified heating, potentially for liquefaction or pasteurization, or that they have reached an advanced stage of aging. Conversely, sample M₆ (0.001%) stands out for its remarkable freshness and has clearly not been exposed to significant heat treatment. The other samples (M₁, M₄, M₅, M₇, M₈) show levels within acceptable to good ranges, in compliance with regulatory limits.

Composition in alcohols, acids and other compounds

The presence and concentration of certain alcohols and organic acids are key indicators of honey stability and authenticity. In particular, the detection of ethanol and specific acids can reveal a fermentation process, often induced by excessive water content (>18%) or poor storage conditions [13].

Ethanol, a direct by-product of sugar fermentation by yeast, is a recognized marker of degradation [13]. In this study, the levels ranged from 0.0005% to 0.2%. Samples M₁ (0.2%) and M₆ (0.14%) showed abnormally high concentrations, indicating probable fermentation, which represents a major quality defect. This observation is corroborated by the organic acid profile. Acetic, lactic, and succinic acids, produced during fermentation, are present at particularly high levels in sample M₁, confirming its instability. Notably, sample M₄ stands out with a very high concentration of lactic acid (0.075%), suggesting either a specific lactic acid fermentation or a particular botanical origin. Furthermore, citric acid, the presence of which in trace amounts is normal, is found in standard proportions for all samples, indicating no adulteration with acidic syrups.

Analysis of proline, the main amino acid in pollen honey, provides further insight into authenticity. A sufficient level (generally > 0.05%) indicates maturity and raises suspicion of potential fraud through the addition of refined sugars, which do not contain proline. Here, samples M₂, M₃, and M₈ show zero proline content, which could be a serious warning sign of possible adulteration [10]. The other samples have levels that meet expectations (Table 2), confirming their authenticity based on this criterion.