C4 Sugars - AOAC

Test Description

NOTE: The C4 AOAC test is considered the gold standard for C4 sugars testing, whilst more expensive than the lower cost C4 Screen test, the AOAC test provides much higher levels of accuracy and should be used when a high level of precision is required.

C4 sugar analysis is carried out to ascertain if a honey has been adulterated by the addition of sugar. Sugars in plants are produced by two distinct biochemical processes (C3 and C4 pathways). Nectar contains C3 sugars that are collected by honey bees and converted into honey. Cane sugar and high fructose corn syrup consist of C4 sugars than may be used for feeding of honey bees when the availability of nectar is limited. The Food Standards of Australia, New Zealand and Codex Alimentarius (FAO & WHO) define honey as a natural substance that must not have anything added or removed from it [1,2]. It is internationally accepted that the C4 sugar content of natural (unadulterated) honey must not be greater than 7%.

To determine the C4 sugar content of honey, a method (AOAC 998.12) was developed by the Association of Official Analytical Chemists in 1977 [3]. It is based on the observation that the ratio of the carbon isotopes 13C to 12C is different in C3 and C4 sugars which can be determined by an analytical method known as isotope ratio mass spectrometry (IRMS). The carbon isotope ratio is lower in C3 sugars than C4 sugars. This method was adopted and used as an internationally accepted C4 sugar adulteration test all around the world. The principle of this method is that the ratio of the carbon isotopes 13C to 12C in an unadulterated honey is very similar to the carbon isotope ratio found in the protein extracted from the same honey where protein is used as an internal reference. If the difference in the isotope ratio of whole honey and protein is greater than 1‰ (per thousand), it indicates that the C4 sugar content of honey is greater than 7%.

C4 AOAC Testing

The AOAC 998.12 C4 sugar method stipulates measurements in triplicate (6 separate instrument readings in total). This is the method used by Gribbles Labnet when undertaking the AOAC C4 testing. Other labs also use the AOAC method, but only take duplicate measurements, which may result in greater test variation. AOAC testing requires more time to complete which also means the testing cost is higher. When compared to the C4 Screen, the AOAC 998.12 method reduces test variability to just 1% (for example, the range of uncertainty for a result of 6% could fall between 5% to 7%) and it allows the assessment of the consistency of results. It is recommended to use the AOAC C4 sugar test for high value manuka honey destined for export.

Test Methodology

LC-MS/MS In-house procedure.

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Standard Turnaround Time

1 working day following receipt of sample.


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