OXIDATION/ANTIOXIDANTS

oils or fish oil capsules, and this can ultimately lead to the oil becoming rancid,” according to a white paper prepared by the GOED and the US Council for Responsible Nutrition (CRN).

“When fatty acids react with oxygen from the surrounding air, the chemical bonds in the fatty acid molecules break down to form a variety of oxidative products such as fatty acid peroxides, alcohols and aldehydes.

“Some specific oxidation products resulting from the lipid peroxidation of highly unsaturated fatty acids include 4-hydroxy-2-hexenal (4-HHE), 4-hydroxy2-nonenal (4-HNE), and a wide variety of isoprostanes, the presence of which are often measured as signs of oxidative stress in clinical trials,” the white paper says.

Multiple factors contribute to the rate at which lipids oxidise, including exposure to oxygen, light, heat and the degree of unsaturation of the fatty acids.

“Highly unsaturated lipids, like EPA and DHA, are more prone to oxidation and generally require special handling to avoid off-flavours from developing. These include the use of antioxidants to slow the rate of oxidation, limiting exposure to ambient air during manufacturing, refining oils in a vacuum, and using blanketing storage containers with inert gases such as nitrogen that displace oxygen.”

The white paper says these strategies are widely applied in the manufacturing of omega-3 products.

Measuring oxidation

There are three primary analytical methods used to measure oxidation in omega-3 oils – the peroxide value (PV), the para-anisidine value (pAV) and total oxidation value (TOTOX).

Peroxide value (PV) is a measure of how much peroxide is present in oil, according to the GOED white paper.

“When polyunsaturated fatty acids oxidise, the first compounds created are peroxides, so PV is a measure of primary oxidation. This method is used in a wide variety of oils but while the PV initially increases as oil oxidises, it can actually decrease as the peroxides are consumed during further oxidative reactions. Therefore, a low PV is not necessarily an indicator of high quality oils.”

This means that measures of secondary oxidation are also often used to determine the true ‘freshness’ of an oil.

Secondary oxidation products are formed from the initially-formed peroxides during further steps in the oxidative process, and include chain-shortened aldehydes and alcohols.

The p-anisidine value (pAV) is a colourimetric method where the absorbance of a specific wavelength of light – 350nm – by a solution of oil and acetic acid is measured after paraanisidine is added.

“This method primarily measures the presence of 2-alkenals and 2,4-alkadienals, secondary oxidation products that react with para-anisidine in acidic conditions to turn yellow, absorbing that specific wavelength of light.”

The white paper says there is significant variance in pAV results between various types of oils and the GOED only recommends the test for EPA and DHA oils in triglyceride or ethyl ester form that do not contain natural pigments or added ingredients, other than antioxidants.

“The p-Anisidine test is not appropriate for measuring secondary oxidation in omega-3 oils that have a strong colour or contain added flavourings,” the white paper says.

“Oils like krill oil or virgin salmon oils naturally contain levels of carotenoids, such as astaxanthin, which interfere with the p-Anisidine assay and yield invalid results. In addition, challenges in properly dissolving polar lipid oils like krill oil can preclude reliable measurements of pAV.

“Flavourings consist of a variety of compounds. In many fruit-derived flavours, the desirable odours, taste and colours are carried by compounds containing aldehydes. Since pAV measures the presence of aldehydes, these flavourings can often interfere with pAV results when added to oils.”

Total oxidation value (TOTOX) is a third way to measure oxidation and is a calculation combining PV and pAV. It was conceived as a way to give a complete picture of oxidation by including primary and secondary oxidation measurements, according to the white paper.

“However, although a convenient measure of oxidation, TOTOX also has its limitations. If one of its components – PV or pAV – cannot be reliably determined, for example, because the oil is flavoured or strongly coloured, TOTOX cannot be calculated."

This could be the case with flavoured oils, krill oils and virgin salmon oils.

Analytical tests

In 2002, omega-3 industry representatives established the Council for Responsible Nutrition (CRN) Voluntary Monograph, now known as the GOED Voluntary Monograph.

This set limits for EPA and DHA oils lower than other edible oils.

The peroxide limit for refined vegetable oils in most countries is 10meq/kg and 20meq/kg for extra virgin olive oil, the white paper says. The British Pharmacopoeia, European Pharmacopoeia and Australian regulatory authorities also have limits for refined omega-3 oils at 10meq/kg.

The GOED monograph’s limits for EPA and DHA oils is:

Peroxide value (PV): 5 meq/kg p-Anisidine value (pAV): 20

Total oxidation value (TOTOX): 26

“The more than 2,000 test results available from scientific literature, thirdparty testing labs, and GOED’s industry testing programme show that more than 94% of products meet the stricter GOED limits for peroxide value and nearly 98% meet the limit for p-anisidine value,” the white paper concludes. ● This article is based on the white paper, 'Oxidation in Omega-3 Oils: An Overview', prepared by the Global Organization for EPA and DHA Omega-3s and the Council for Responsible Nutrition