Improving the measurement of cyanogenic glycosides in flaxseed

Cyanogenic glycosides in flaxseed

We analyze minor compounds in oilseeds because they can have physiological importance or can be perceived as negatively affecting health. One group of minor compounds that our lab studies in flaxseed is cyanogenic glycosides. There are about 75 known cyanogenic glycosides in more than 2,500 plant species and they can be found in an entire plant or just in certain structures such as roots or seeds. Although cyanogenic glycosides occur in low levels in flaxseed, they are a concern because hydrogen cyanide (HCN), a well-known toxin, is released when they break down through hydrolysis. HCN is only released if the plant or seed tissues are damaged and the enzymes responsible for hydrolysis come into contact with the cyanogenic glycosides.

The European Union and Japan have set limits on the total amount of cyanogenic glycosides allowed in flaxseed that is used in food products and feed. In Canada and the United States flaxseed is considered safe for consumption and numerous studies have documented its health benefits due to the presence of alpha-linolenic acid (an omega-3 fatty acid), fiber and lignans.

Traditional methods for measuring cyanogenic compounds

Two cyanogenic diglycosides, linustatin and neolinustatin, are found in sound (undamaged) flaxseeds and two cyanogenic monoglycosides, linamarin and lotaustralin, are found in seedlings, developing plants, flowers, damaged seeds and immature seeds (Figure 1). The traditional method for analyzing these compounds measures the amount of HCN produced after the cyanogenic glycosides are hydrolyzed. To be accurate, this method requires the complete hydrolysis of the cyanogenic glycosides and the compete capture of the HCN produced. Hydrolysis may be incomplete, however, if the acids or enzymes being used to cause hydrolysis interact with other compounds or are not specific to the cyanogenic glycosides being hydrolyzed. For example, flaxseed cyanogenic glycosides need two active enzymes in sequence to be hydrolyzed and using only one will lead to almost no HCN production. This traditional method gives highly variable results with very low reproducibility.

A better approach to the analysis of cyanogenic glycosides is to quantify the intact cyanogenic compounds. An accurate high pressure liquid chromatography (HPLC) method for this has been reported in the scientific literature, but the method is very long as several steps are necessary to improve the detection limit.

Analytical improvements

Our team has been working to improve the analysis of cyanogenic glycosides in flaxseeds and to develop an accurate, repeatable and reproducible method to measure them. We first were able to lower the detection limit using gas chromatography (GC) with flame ionization detection (FID) instead of HPLC. We also improved the extraction of cyanogenic glycosides from seeds and then developed a GC-mass spectrometry (GC-MS) method to analyze baked products which further decreased the limit of detection. We also found that one of the cyanogenic monoglycosides was not commercially available in its natural form, so we developed a method to obtain pure compounds to use as reference material, which led to an improvement in the quantification of cyanogenic monoglycosides.

Comparison study with Health Canada

The Oilseeds team was also involved in a project with Health Canada that compared our GC-MS method with a liquid chromatography-mass spectrometry (LC-MS) method used to directly quantify cyanogenic glycosides. Although they can be cost prohibitive, LC-MS methods are often considered to be the best since compounds are analyzed directly without transformation into a gaseous state. They are also very specific since the mass spectra allow compounds to be identified.

The results of the study (Table 1) show that our GC-MS method has very good reproducibility with the relative standard deviation (RSD) of analysis being 0.90%. A comparison of the GC-MS and LC-MS results showed no statistical difference between the two methods. These results indicated that the analysis of intact cyanogenic glycosides should be the method of choice for their quantification.