Hydroxymethylfurfural Testing of Honey

Make your all doubt clear on freshness honey with HMF testing

HMF (5-hydroxymethylfurfuraldehyde) is one of the organic compounds that is formed by the dehydration of reducing sugars, and this compound is generally not present in the fresh honey; therefore, it is used as a marker molecule of the honey’s status. The significance of hydroxymethylfurfural (HMF) testing in honey goes beyond quality assurance to include health and regulatory compliance. HMF testing is vital for ensuring honey quality such as overheating of honey, poor storage or how much old age of honey, which is further we can ensure about consumer safety, accurate labelling, and adherence to domestic and international standards.

Methods for Testing HMF in Honey

There are several methods are commonly used for HMF testing, including:

1. Spectrophotometric Method
   • Apparatus and Reagents: Spectrophotometer, Carrez solution-I, Carrez solution-II, Sodium bisulfite solution. Dilute 1 + 1 for dilution of reference solution if necessary.                                                                                                              
   • Procedure: Weigh 5 g of honey accurately in a small beaker, then transfer 25 mL of H₂O to a 50 mL volumetric flask. Mix in 0.50 mL of Carrez solution I (15 g K₄Fe (CN)₆×3H₂O and dilute to 100 mL with H₂O), then 0.50 mL of Carrez solution II (Dissolve 30g Zn(CH₃COO)₂×2H₂O and dilute to 100 mL with H₂O), and dilute to volume with H₂O. A drop of alcohol can be added to reduce foam. Filter through paper, discarding the first 10 millilitres of filtrate. Transfer 5 mL of filtrate to each of two test tubes. Add 5.0 mL of H₂O to one tube (test solution) and 5.0 mL of NaHSO₃ to the other. Mix thoroughly with a full Vortex mixer, and then compare the test solution’s A value to the reference at 284 and 336 nm in 1cm cells. If A is greater than 0.6, dilute the test solution with H₂O and the reference solution with 0.1% NaHSO₃ solution to the same extent, and adjust A for dilution. (Reference: AOAC Official Method 980.23)

   • Calculation formula:

     Hydroxymethylfurfural (HMF) mg / 100 g honey = (A ₂₈₄ – A ₃₃₆) x14.97 x5/ g Test Sample

      Factor = 14.97

     A ₂₈₄ = Absorbent at 284                                  

     A ₃₃₆ = Absorbent at 336

2. High-Performance Liquid Chromatography (HPLC) : HPLC is a more precise and accurate method for determining HMF. Honey samples are prepared and injected into an HPLC system, which separates and quantifies HMF. HPLC is often more sensitive and capable of detecting lower HMF concentrations than spectrophotometry. It is appropriate for regulatory testing and the preferred method in many food laboratories.

3. Liquid Chromatography-Mass Spectrometry (LC-MS) : LC-MS combines HPLC separation with mass spectrometry detection. This method detects HMF with high specificity and sensitivity, even at trace levels. It is primarily used in research or detailed analysis due to its high sensitivity and cost.

4. Enzymatic and Colorimetric Kits : Some laboratories use commercial kits that allow for the enzymatic or colorimetric detection of HMF in honey. These kits are quick and easy to use, making them ideal for basic screening.

5. Interpretation of Results : HMF content in honey is typically regulated. For example, the European Union limits HMF levels in honey to 40 mg/kg (except for tropical honey, which is limited to 80 mg/kg) and as per FSSAI the value should not be more than 80 mg/kg (reg no 2.8.3). High HMF values indicate overheating or prolonged storage, which could indicate adulteration or substandard quality.

Why we should go for HMF testing

The significance of hydroxymethylfurfural (HMF) testing in honey extends beyond quality assurance to health and regulatory compliance. HMF testing is critical for ensuring honey quality, consumer safety, accurate labelling, and compliance with domestic and international standards. You must look on the significance of HMF testing.

• Quality Assessment: HMF is produced in honey by the degradation of sugars, particularly fructose, and increases with heat exposure and long-term storage. Fresh, unheated honey contains low levels of HMF, so testing can reveal whether it has been overheated or stored for an extended period of time. Low HMF levels indicate fresh and minimally processed honey, whereas high levels indicate older or heat-treated honey, potentially indicating a decrease in quality.
• Avoiding Heat-Degraded Products: High levels of HMF may indicate overheating, which reduces the nutritional value of honey while also introducing by-products that can be harmful in large quantities. HMF itself can be toxic at high doses, so testing is required to ensure safe consumption.
• Avoidance of Potentially Carcinogenic Substances: While HMF in food is generally low risk, it can transform into more harmful compounds, like furfural derivatives, if honey is further heated or stored poorly. Testing helps ensure the product remains within safe limits.
• Identifying Adulterated Honey: Some honey producers add sugars or syrups with high HMF levels during processing, or they heat honey to blend or liquefy it for easier packaging. As a result, high HMF levels can indicate adulteration, aiding in the detection and prevention of honey fraud.
• Validation of “Raw” Honey Claims: Honey marketed as “raw” or “unprocessed” should have a low HMF level. Testing backs up label claims and helps consumers identify genuine raw honey, promoting transparency and trust.
• Meeting Regulatory Limits: Regulatory bodies such as the European Union, the United States Food and Drug Administration (FDA), and the Codex Alimentarius Commission (Alinorm 01/25, 2000) set HMF limits in honey for 80 mg/kg and the European Union (Directive 110/2001) established HMF limits to 40 mg/kg.
• Quality Control for Export: Testing for HMF is especially important for honey exporters to ensure that their products meet international standards and can be sold in global markets. Failure to meet these standards may result in product rejections, penalties, or recalls.
• Maintaining Natural Taste and Aroma: Honey with high HMF levels may have a different flavour profile, often tasting burnt or caramelized. Testing ensures that the honey’s natural flavour and aroma are preserved, which increases consumer satisfaction and product consistency.

How can we assist you with your HMF testing in Honey?

Cultivator Phyto Lab Pvt. Ltd. is a benchmark of excellence in honey testing, providing specialised services for a diverse range of honey products. Our ultra-modern laboratory, outfitted with cutting-edge technology, is backed up by a team of highly skilled scientific professionals dedicated to providing precise and reliable results. Beyond standard Hydroxymethylfurfural (HMF) testing, we employ a wide range of advanced testing parameters to ensure strict quality control, authenticity, and safety of honey products. With a focus on compliance and collaboration, we are committed to assisting our partners in meeting global standards and maintaining consumer confidence.