The Detection of C4 Sugars in Honey

Published in Hivelights Vol 12 #1, February 1999

by Carla Barry
Laboratory Services Division
Canadian Food Inspection Agency

The issue of honey adulteration has been addressed by the Canadian Food Inspection Agency in 1998. Laboratory testing of honey and honey adulterated with cane product (called "honey analog") and corn syrup has shown that honey adulteration can be readily detected.
The CFIA analytical testing uses three methods of analysis for detection of honey adulteration
· Carbon Isotope Ratio Mass Spectrometry
· Retrograde sugars by Gas Chromatography
· Oligosaccharides by HPLC -PAD

The Carbon Isotope Ratio is accepted as a standard method of analysis by the Association of Official Analytical Chemists. Any addition of corn or cane protein to honey is readily identifiable using this method. Since carbon is present in nature in two forms, carbon 13 and carbon 12 isotopes, the ratio of these two isotopes of carbon can be used to determine which type of photosynthetic pathway was used to make food through CO2 fixation.
The Carbon dioxide fixation process in plants occurs in one of three ways:
C3 or Calvin cycle used by maple, beet, and most fruit
C4 or Hatch Slack cycle used by corn and cane plants
CAM or Crassulacean Acid Metabolism used by pineapple plants

The ratio of the carbon isotopes found in plants in these 3 categories falls into a well defined range.
C3 plants : -22 to -30 parts per thousand
C4 plants : -8 to -11 parts per thousand
CAM plants: -11 to -13.5 parts per thousand

Carbon Ratio Analysis of Honey and of Protein in Honey
Nearly all protein in honey originates from the bee in the form of enzymes that ripen the nectar. Therefore the carbon ratio of the honey and that of the protein isolated from that honey should be similar.

Detection of Cane Sugar Addition
The addition of cane sugar and corn syrups to honey will change the carbon ratio of the honey but not of the protein. Therefore the difference between the carbon ratio value of the protein and that of the honey provides an unequivocal objective measure of the honey's C4 (cane or corn) sugar content. The AOAC International Method accepts that a difference equal to -1 parts per thousand indicates addition of C4 sugars. This means that the addition of cane sugar to honey can be detected to level as low as 7% .