C3 and C4 Sugar Adulterants Testing

1. Elemental Analysis – Isotope Ratio Mass Spectrometry (EA-IRMS)

• Determines the δ¹³C values of honey and its protein fraction to identify potential adulteration with C4 sugars.

2. Liquid Chromatography – Isotope Ratio Mass Spectrometry (LC-IRMS)

• Enables separation and isotopic analysis of individual sugar components for more detailed profiling.

3. Δδ¹³C Analysis (Difference between honey and protein fraction δ¹³C values)

• Helps detect the presence of extraneous C4 plant-derived sugars such as high-fructose corn syrup (HFCS) and cane sugar.

These methodologies align with FSSAI guidelines and international standards for honey authenticity testing, ensuring accurate detection of adulteration while upholding quality and regulatory compliance. Following are the methods that are applicable:

1. 13 C Stable Carbon Isotope Ratio Method (SCIRA) or EA/IRMS: – Limit of C4 Syrup Detection 7% originally developed in the 1970’s and the only AOAC Official method – 998.12 C4 for Plant Sugars in Honey. This method can detect only corn or sugar cane derived syrups (C4 sugars), it cannot detect syrups made from beets, rice, wheat or other plants (C3 sugars). Using EA-IRMS values of honey protein and bulk honey have been determined.

2. 13 C EA/LC-IRMS (C3/C4 sugar detection):- This test was developed and validated in 2004-2007 to detect the presence of added sugars both from C4 plants (e.g. corn and cane syrup) and C3 plants (e.g. rice, wheat, beet syrup). It has been used commercially for honey adulteration analysis since 2007. The first ISO 17025 accreditation was achieved in 2008. • This method differs from the AOAC SCIRA method in that it makes use of a liquid chromatography to separate each major sugar and evaluate them individually. This improves the sensitivity and makes possible the detection of some C3 sugars. • In 2015-2017, this method was used in an EU Commission monitoring program to check honey products for adulteration on the European market (~2300 samples, ~14% non-compliance). Using LC- IRMS  C values of glucose, fructose, di-, tri-, and oligosaccharides have been determined

The LC IsoLink LC-IRMS is the first high sensitivity interface connecting high performance liquid chromatography (HPLC) with Isotope Ratio MS for the reproducible and accurate on- line determination of 13 C/ 12 C isotope ratios. All organic compounds eluting from an HPLC column are analysed while maintaining the chromatographic resolution. In the LC IsoLink LC- IRMS the sample is oxidized within the aqueous solvent eluting from the HPLC, afterwards the generated CO 2 is separated from the liquid phase. This process is quantitative and fractionation-free. The oxidation reagent consists of two solutions, the oxidizing agent and phosphoric acid. Both are pumped separately and added to the mobile phase of HPLC. Within this mixture all individual organic compounds eluting from the HPLC column are oxidized quantitatively into CO 2 when passing through a heated reactor. In a downstream separation unit the CO 2 is removed from the liquid phase and entrenched into a stream of He. The individual CO 2 peaks are subsequently dried in an on-line gas Drying unit and then admitted to the Isotope Ratio MS via an open split interface. The δ13C value is the 13C/12C ratio of the sample related to the 13 C/ 12 C ratio of a reference material to ensure international compatibility of data sets. δ13C = (( 13 C/ 12 C) Sample / ( 13 C/ 12 C) Reference – 1) × 1000 for a rough estimation, δ 13 C relates to atom% divided by 1000.

The EA IsoLink IRMS System uses the principle of Dumas combustion for δ 13 C analysis. Approximately 100-200 μg of honey are loaded into a tin capsule, which are dropped from the Autosampler into a reactor filled with chromium oxide and cobaltous/cobaltic oxide here it is combusted in the presence of pure oxygen to form CO 2 for analysis. A reactor can analyse 800-1000 honey and protein samples before a replacement of reactors and trap material is required. The CO 2 produced from combustion of the bulk honey and of the protein fraction is analysed for the δ 13 C by IRMS. The elemental analyser (EA) is interfaced to the IRMS via ConFlow Interface, which also performs automated referencing and dilution. The EA IsoLink System is operated through the Software Suite. The proteins in the honey sample are extracted following the AOAC 998.12 which specifies mixing 15 g of honey with 3 ml of water and heating to 80 °C. The proteins precipitate after addition of acid and tungstic solution within about 2 minutes. The supernatant is repeatedly decanted after centrifuging and rinsing with water. After drying, 100-200 μg of the protein sample are loaded into a tin capsule for analysi