Developing reference standards for measuring cyanogenic glycosides in flaxseed

The Oilseeds Program conducts research on Canadian oilseeds including canola, rapeseed, flaxseed, soybean and mustard. We assess the effects of grading factors on oilseed quality and analyze how various components of oilseeds, such as free fatty acids, affect their quality and the products made from them. Another important part of our work is assessing and developing methods used to analyze oilseed quality. This includes validating our reference methods to international standards, developing near infrared spectroscopy models to predict oilseed quality and pioneering new methods for analyzing minor compounds in oilseeds. The Oilseeds Program also evaluates the quality of oilseed samples from the Harvest Sample Program and export shipments.

Cyanogenic glycosides in plants

Although some compounds can occur at low levels in oilseeds, they may still limit their use in food and feed applications. One such group of compounds that are of interest to us in flaxseed are cyanogenic glycosides. There are about 75 known cyanogenic glycosides in more than 2500 species of plants. These compounds are considered to be antinutrients and their concentrations are regulated in some food products, such as cassava flour. In some species they are found in every part of the plant (e.g., cassava and flax) while in others, only certain structures contain cyanogenic glycosides (e.g., apple seeds). The role of cyanogenic glycosides in plants is not completely understood but it has been suggested that they provide plants with resistance to predators such as herbivores, nematodes or fungi.

The role of reference standards

Two cyanogenic diglycosides, linustatin and neolinustatin, are known to occur in sound (undamaged) flaxseeds. Two cyanogenic monoglycosides, linamarin and lotaustralin, have been found in seedlings, developing plants, flowers, damaged seeds and immature seeds. We use gas chromatography to identify and measure levels of cyanogenic glycosides in flaxseed by comparing the chromatographic profiles of flaxseed extracts with those of highly purified reference standards. The high cost and unavailability of some cyanogenic glycoside standards often makes the quantification of these compounds difficult, however, so we investigated the feasibility of developing a rapid and cost-effective method for producing pure standards for use in the routine analysis of cyanogenic glycosides in flaxseed.

Producing purified cyanogenic glycosides for routine analysis

A review of the scientific literature revealed that germinated flaxseeds contain all of the four cyanogenic compounds we wanted to investigate. We tested germinated flaxseeds and determined that 72 hours in a germination tray was sufficient time for the production of cyanogenic glycosides. Several samples and composites were tested to find samples that would produce cyanogenic glycosides in amounts high enough to be used as reference standards (Figure 1). We extracted the cyanogenic glycosides using the method that we previously developed, separated them using preparative high performance liquid chromatography (Prep-HPLC) (Figure 2), collected the HPLC fractions and identified the fractions containing the cyanogenic glycosides (Figure 3). The last step was to analyze the concentrated fractions corresponding to each cyanogenic glycosides using gas chromatography-mass spectrometry to determine if they were suitable for use in routine analyses (Figure 4). Our results show that we were able to produce purified cyanogenic glycosides in their natural form to complement the commercially available standards for all our routine analyses of flaxseed.