Omega-3 fatty acids in dietary supplements – TOTOX, PV and AV indicators and how the capsule shell protects DHA and EPA against oxidation

Omega-3 fatty acids are among the fastest-degrading ingredients in dietary supplements. Oxidation of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) leads to the formation of hydroperoxides, followed by aldehydes, ketones and other decomposition products that alter the taste, smell and quality of the finished product. This is a process that occurs regardless of whether the manufacturer measures it or not.

The problem is that the mass of omega-3 declared on the label may be formally correct, while the oxidative quality of the oil remains overlooked. The number of milligrams of DHA and EPA says nothing about the extent to which the oil has oxidized before reaching the consumer. Meanwhile, oxidative decomposition products not only reduce sensory qualities but also decrease the actual content of intact fatty acids.

Three numbers make it possible to objectively describe the oxidative state of the oil enclosed in a capsule: peroxide value (PV), anisidine value (AV) and the TOTOX value, which combines the two previous parameters. Below, we explain exactly what each of these parameters measures, how storage conditions accelerate oxidation and why the choice of capsule shell material is a real tool for protecting oxidative stability.

DHA and EPA – why polyunsaturated fatty acids oxidize quickly

Polyunsaturated fatty acids (PUFA) contain multiple double bonds separated from one another by a single methylene group. The hydrogen atom located in this group, in the so-called bis-allylic position, is particularly weakly bound and susceptible to abstraction by free radicals. The more such positions there are in the molecule, the more easily the oxidation reaction begins (Mori, 2004).

Lipid autoxidation proceeds in three stages, which are well described in the literature (Kuennemann, 2025):

• Initiation – heat, UV light or metal ions (Fe2+, Cu2+) abstract a hydrogen atom from the bis-allylic position, resulting in the formation of a lipid radical;
• Propagation – the lipid radical reacts with molecular oxygen (O2), forming a lipid hydroperoxide (LOOH), i.e. the primary oxidation product that drives the subsequent chain reaction;
• Termination – radicals react with one another, producing secondary oxidation products: aldehydes, ketones and alcohols. It is aldehydes, measured as the anisidine value (AV), that are typical termination products.

This mechanism explains the key difference between the two main omega-3 acids. DHA (22:6n-3) has six double bonds, while EPA (20:5n-3) has five. A greater number of double bonds means more bis-allylic positions susceptible to initiation, which is why DHA oxidizes faster than EPA (Mori, 2004; Kuennemann, 2025). Unsaturated fatty acids of fish origin also oxidize much faster than saturated or monounsaturated fatty acids, and the rate of this process in vitro depends on oxygen availability, temperature and the presence of pro-oxidants such as iron ions (Mori, 2004).

Three oxidation indicators: PV, AV and TOTOX – what each one measures

The oxidative state of fish oil is assessed using three complementary indicators. None of them alone provides a complete picture, so only their combined interpretation makes it possible to reliably assess oil quality (Kolanowski, 2010; Kuennemann, 2025).

PV – peroxide value

PV measures the concentration of primary oxidation products, i.e. lipid hydroperoxides (LOOH), and is expressed in meq O2/kg of oil. A low PV indicates oil freshness or the absence of primary oxidation. However, this indicator has an important limitation: PV first rises and then may fall, because peroxides decompose into secondary products. As a result, the oil may become increasingly oxidized while the measured PV decreases. For this reason, PV alone is not sufficient to assess quality.

AV – anisidine value

AV measures the concentration of secondary oxidation products, primarily aldehydes, especially 2-alkenals. Unlike PV, the anisidine value increases monotonically as oxidation progresses and does not decrease. As a result, AV better reflects the entire oxidative history of the oil than PV, which describes only the momentary state.

TOTOX – total oxidation value

TOTOX is calculated using the formula TOTOX = 2 x PV + AV. This indicator combines the two previous parameters, taking into account both the current state of oxidation (PV) and its history (AV). TOTOX is the parameter recommended by GOED (Global Organization for EPA and DHA Omega-3) as the leading measure of the oxidative quality of fish oil, because it provides the most complete picture of the advancement of the oxidation process (Kuennemann, 2025).

The limits recommended by GOED for finished oil are: PV no more than 5 meq O2/kg, AV no more than 20 and TOTOX no more than 26 (GOED, 2021). These are currently the only widely used industry limits for the oxidative quality of omega-3 (Kuennemann, 2025).

Worth knowing: what GOED limits mean in practice

GOED limit values (PV no more than 5, AV no more than 20, TOTOX no more than 26) are minimum industry standards, not a legal standard. They are not mandatory in the EU under any regulation concerning dietary supplements – a manufacturer declares compliance with GOED voluntarily. Exceeding these values is, however, a clear warning sign: heavily oxidized oil develops a rancid smell, loses its taste qualities and may potentially change its composition, because decomposition products appear in place of DHA and EPA. The study by Mason and Sherratt (2020) showed significant variability in EPA and DHA content between different production batches of one popular fish oil dietary supplement, which may be linked to a lack of oxidative control on the production line or inappropriate packaging. The conclusion is unequivocal: oxidative quality should be monitored throughout the entire shelf life, not only during production.

How the course of oxidation depends on storage conditions

The rate of omega-3 oil oxidation is not a fixed characteristic of the raw material. It is determined by a set of environmental factors acting on the oil during production, packaging and storage. Their control translates directly into the TOTOX measurement result after 12 and 24 months (Kuennemann, 2025; Mori, 2004).

• Oxygen (O2) – the main reactant in the oxidation process; any break in the oxygen barrier of the packaging accelerates oxidation;
• Temperature – an increase in temperature rapidly accelerates the rate of oxidation reactions, which is why warm storage drastically shortens oil shelf life;
• Light (UV) – initiates photo-oxidation, especially in the presence of photosensitizers; capsules in transparent bottles are particularly exposed to it;
• Metal ions (Fe, Cu) – pro-oxidants catalyzing the initiation stage; their source may be the raw material, the process environment or packaging materials (Mori, 2004);
• Moisture – indirectly activates pro-oxidative reactions; capsule shells with a higher moisture content increase the risk of oxidation of the oil enclosed in them.

The last of these factors directly links oxidative stability with the choice of capsule material. Kolanowski (2010) showed that fish oil supplements in HPMC capsules produced a lower TOTOX after 12 months of storage than the same oils in gelatin capsules. The researcher associated this effect with the lower moisture content of the HPMC shell, which is 4.5-6.5%, whereas gelatin contains 13-16% water (Yang et al., 2020). The capsule shell together with the outer packaging therefore forms a barrier that is a critical parameter for TOTOX values over the longer term (Kuennemann, 2025).

Worth knowing: comparison of shells for omega-3 supplements

Parameter	Gelatin	HPMC	Pullulan
Moisture content	13-16%	4.5-6.5%	similar to HPMC
Oxygen barrier	low	moderate	high (manufacturer’s data)
Risk of oxidation with omega-3	higher	lower	lowest (manufacturer’s data)
Cross-linking	risk	none	none
Cost	low	medium	higher (3-5x more expensive than HPMC)

Important note: barrier parameters, including oxygen transmission rate (OTR), provided for pullulan by commercial suppliers are manufacturer data, not verified in independent scientific studies for specific conditions (temperature, relative humidity, wall thickness). For formulations that are critical in terms of OTR, it is recommended to conduct in-house oxidative stability tests instead of relying solely on supplier declarations.

Practical methods for protecting DHA and EPA against oxidation in supplements

Protecting omega-3 oil against oxidation does not come down to a single measure, but to a coherent combination of several methods acting on the various factors of the autoxidation mechanism described earlier. The most commonly used solutions include (Kuennemann, 2025):

• Nitrogen headspace flushing – displacing oxygen from inside the capsule or bottle before closure; an effective method of reducing the initial amount of O2 available for the reaction;
• Antioxidants – natural tocopherols (a mixture of alpha- and gamma-tocopherol, i.e. vitamin E) act as free radical scavengers and are added directly to the oil; rosemary extract and ascorbic acid derivatives are also used;
• Packaging – a dark bottle (amber or dyed HDPE) blocks light exposure; aluminum blisters provide an additional barrier for individual capsules; minimizing headspace is also important;
• Temperature – cool storage (4 degrees C or no more than 15 degrees C) drastically slows oxidation; labeling such as “store in a cool, dark place” is helpful.

These methods work best in combination. Nitrogen reduces the amount of oxygen at the outset, antioxidants interrupt chain propagation, dark packaging eliminates photo-oxidation, and low temperature slows the entire kinetics. The choice of capsule shell material, discussed earlier, is another layer of the same strategy: the less moisture the shell introduces and the better its oxygen barrier, the more slowly TOTOX increases during storage.

Plan the production of an omega-3 supplement with Eubioco

The choice of capsule shell material (gelatin, HPMC or pullulan), antioxidant protection system and target packaging should be designed at the research and development stage, before stability testing begins. These decisions influence one another and jointly determine PV, AV and TOTOX values at the end of the shelf life. Eubioco, as a contract manufacturer of dietary supplements operating in accordance with GMP and ISO 22000:2018 standards, supports customers in designing oxidation-sensitive formulations. Contact us to discuss your project: sprzedaz@eubioco.eu

Bibliography

1. Kolanowski, W. (2010). Omega-3 LC PUFA Contents and Oxidative Stability of Encapsulated Fish Oil Dietary Supplements. International Journal of Food Properties, 13(3), pp. 498-511. DOI: 10.1080/10942910802652222. [online] Available at: https://doi.org/10.1080/10942910802652222 [accessed online: 20.05.2026]
2. Mason, P., Sherratt, S. (2020). Variability in Content of Omega-3 Fatty Acids and other Fatty Acids in Multiple Lots of a Widely Used Fish Oil Dietary Supplement. Journal of Clinical Lipidology, 14(4), p. 575. DOI: 10.1016/j.jacl.2020.05.053. [online] Available at: https://doi.org/10.1016/j.jacl.2020.05.053 [accessed online: 20.05.2026]
3. Kuennemann, E. (2025). Quality Assurance of Omega-3 Fatty Acids in Fish Oil Capsules. American Journal of Biomedical Science & Research, 26, pp. 471-482. DOI: 10.34297/ajbsr.2025.26.003457. [online] Available at: https://doi.org/10.34297/ajbsr.2025.26.003457 [accessed online: 20.05.2026]
4. Mori, T. (2004). Effect of fish and fish oil-derived omega-3 fatty acids on lipid oxidation. Redox Report, 9, pp. 193-197. DOI: 10.1179/135100004225005200. [online] Available at: https://doi.org/10.1179/135100004225005200 [accessed online: 20.05.2026]
5. Yang, N., Chen, H., Jin, Z., Hou, J., Zhang, Y., Han, H., Shen, Y., Guo, S. (2020). Moisture sorption and desorption properties of gelatin, HPMC and pullulan hard capsules. International Journal of Biological Macromolecules, 159, pp. 659-666. DOI: 10.1016/j.ijbiomac.2020.05.110. [online] Available at: https://doi.org/10.1016/j.ijbiomac.2020.05.110 [accessed online: 20.05.2026]
6. GOED (Global Organization for EPA and DHA Omega-3). (2021). GOED Oxidation Guidelines. GOED, Salt Lake City, USA. [online] Available at: https://goedomega3.com/goed-monograph [accessed online: 20.05.2026]