Honey is a widely consumed natural food known for its nutritional value and health benefits. In addition to being a sweetener, honey is recognized for its medicinal properties, including anti-inflammatory, anti-bacterial, antioxidant, and even cancer-preventive effects (Afrin et al., 2020). As demand for honey continues to grow, especially in the United States where over 70% of honey is imported (USDA, 2022), so does the risk of adulteration.

Unfortunately, some imported honey is found to be altered — either by mixing it with cheaper sugar syrups, like corn syrup, or by feeding bees sugar syrup to boost yield. Both practices are considered unethical. In fact, the FDA found that about 10% of imported honey was violative in a recent assessment (FDA, 2021–2022).

Bees collect their nectar mainly from the flowers of C-3 plants and seldom form the flowers of C-4 plants (Cane and Corn). C-3 and C-4 plant sugars have distinct δ¹³C signatures. δ¹³C signature of honey and its protein fraction can help us identify the C-4 adulteration.

To detect such adulteration, we use a well-established technique called Stable Carbon Isotope Ratio Analysis (SCIRA), as described in the AOAC 998.12 method. This approach can identify whether honey contains more than 7% of C-4 plant sugars, such as those derived from corn or cane sugar.

Our Approach

We determine the carbon isotope ratios (δ¹³C) of both the honey and its protein fraction, which is isolated through the repetitive washing method specified in AOAC 998.12. These measurements are performed using an Isotope Ratio Mass Spectrometer (IRMS, Finnigan MAT252) coupled to an Elemental Analyzer (EA, Carlo Erba NC 1500).

By comparing the δ¹³C values of the whole honey and its protein, we can estimate the percentage of C-4 sugar adulteration. A significant difference between these values indicates the presence of added sugars from C-4 plants, helping us ensure the integrity and authenticity of the honey. The equation that AOAC 998.12 method uses to evaluate the authenticity of honey is:

C-4 sugars, % = {(δ¹³C_P – δ¹³C_H) / (δ¹³C_P – (- 9.7))} * 100

Where δ¹³C_P and δ¹³C_H are δ¹³C values, ‰, for protein and honey, respectively. -9.7 is the average δ¹³C value for corn syrup, ‰. According to the method, we report negative values from the calculations as 0% sugar. Product is considered to contain significant C-4 sugars (primarily corn and cane) only at or above a value of 7% sugars.

To establish this method in our lab, we adulterated pure honey with pure corn syrup at different percentages starting from 1 to 20%. We extracted the proteins from those adulterated bulk honeys and analyzed them to evaluate AOAC official method 998.12.

Results

We first evaluated honey authenticity using the known adulterant values. The pure corn syrup that we used as an adulterant had a value of -11.045‰ for δ¹³C, so we substituted the -9.7‰ average C4 value with our value. In an ideal scenario, the δ¹³C of bulk honey and its protein is assumed to be the same. We took this assumption and calculated the % sugars in our lab adulterated honeys. The results are shown in table 1.

Table 1.

In a real-world scenario, natural variation in honey and its protein is observed between 0-1‰. Honey that we used for our adulteration analysis had a difference (δ¹³CP – δ¹³CH) of 0.52‰.

Fig. 2 shows results when we incorporate the natural variation between bulk honey and its protein using a known C4 adulterant δ¹³C value.

Fig. 4 shows the comparison between 1) an ideal authentic honey adulteration, 2) honey with an unknown adulterant but known natural variation and 3) honey with unknown adulterant and unknown natural variation (δ¹³CP – δ¹³CH) between bulk honey and its protein.