DETERMINATION OF THE OXIDATIVE
STABILITY OF OMEGA-3 OIL USING
ELECTROCHEMICAL IMPEDANCE
SPECTROSCOPY
DETERMINACIÓN DE LA ESTABILIDAD OXIDATIVA
DEL ACEITE OMEGA-3 UTILIZANDO ESPECTROSCOPIA
DE IMPEDANCIA ELECTROQUÍMICA
Brenda Patricia Rodríguez Villanueva
Universidad de Puerto Rico, Puerto Rico
Sandra Milena Rodríguez Villanueva
Universidad de Puerto Rico, Puerto Rico
Roberto Del Cristo Martínez Mejía
Universidad de Puerto Rico, Puerto Rico
Henry Nuñez Coavas
Universidad del Atlántico, Colombia
pág. 8307
DOI: https://doi.org/10.37811/cl_rcm.v8i4.12993
Determination of the Oxidative Stability of Omega-3 Oil Using
Electrochemical Impedance Spectroscopy
Brenda Patricia Rodríguez Villanueva
1
brenda.rodriguez1@upr.edu
https://orcid.org/0000-0001-7117-1681
Universidad de Puerto Rico
Puerto Rico
Sandra Milena Rodríguez Villanueva
sandra.rodriguez2@upr.edu
https://orcid.org/0000-0001-9512-1873
Universidad de Puerto Rico
Puerto Rico
Roberto Del Cristo Martínez Mejía
roberto.martinez6@upr.edu
https://orcid.org/0000-0003-1998-8728
Universidad de Puerto Rico
Puerto Rico
Henry Nuñez Coavas
henrynunez@mail.uniatlantico.edu.co
https://orcid.org/0000-0002-7319-1640
Universidad del Atlántico
Colombia
ABSTRACT
One of the main challenges in the supplements and food industry is maintaining the oxidative stability
of Omega-3 rich oils, which are highly susceptible to oxidation. The oxidation of these oils not only
affects their quality and flavor but can also diminish their health benefits, such as reducing inflammation
and supporting brain function. This study evaluates the oxidative stability of Omega-3 oil using
electrochemical impedance spectroscopy (EIS), a technique that offers a faster and non-destructive
alternative compared to traditional chemical methods. Oil samples were subjected to autoxidation at
room temperature and analyzed over a frequency range of 3 kHz to 100 kHz, while peroxide levels were
measured using traditional bromatological techniques for comparison. The results revealed a strong
linear correlation (R² = 0.99) between peroxide levels and imaginary dielectric permittivity,
highlighting the potential of EIS to effectively characterize the oxidative stability of Omega-3 oil. This
finding is significant because EIS allows for faster and non-destructive evaluation, facilitating real-time
monitoring of oxidation in Omega-3 oils, which could be particularly beneficial for manufacturers
seeking to ensure the quality of their products more efficiently. Additionally, the use of EIS can reduce
costs and analysis times, improving the efficiency of quality control processes. However, possible
disadvantages include the need for specialized equipment and precise calibration of the method for
different types of oils. Future research should focus on further optimizing and validating this method
under different storage and processing conditions, as well as exploring its applicability to other types
of edible oils and Omega-3 derived products to ensure their quality and safety. Moreover, it would be
beneficial to investigate the integration of EIS into automated production lines for continuous
monitoring, which could revolutionize the way Omega-3 oil quality is assured in the industry.
Keywords: omega-3, oxidative stability, electrochemical impedance spectroscopy
1
Autor principal.
Correspondencia: brenda.rodriguez1@upr.edu
pág. 8308
Determinación de la Estabilidad Oxidativa del aceite Omega-3 Utilizando
Espectroscopia de Impedancia Electroquímica
RESUMEN
Uno de los principales desafíos en la industria de los suplementos y alimentos es mantener la estabilidad
oxidativa de los aceites ricos en Omega-3, que son altamente susceptibles a la oxidación. La oxidación
de estos aceites no solo afecta su calidad y sabor, sino que también puede disminuir sus beneficios para
la salud, como la reducción de la inflamación y el apoyo a la función cerebral. Este estudio evalúa la
estabilidad oxidativa del aceite Omega-3 utilizando espectroscopia de impedancia electroquímica
(EIE), una técnica que ofrece una alternativa más rápida y no destructiva en comparación con los
métodos químicos tradicionales. Se expusieron muestras de aceite a autoxidación a temperatura
ambiente y se analizaron en un rango de frecuencias de 3 kHz a 100 kHz, mientras que los niveles de
peróxido se midieron mediante técnicas bromatológicas tradicionales para proporcionar una base de
comparación. Los resultados revelaron una fuerte correlación lineal (R² = 0.99) entre los niveles de
peróxido y la permitividad dieléctrica imaginaria, destacando el potencial de la EIE para caracterizar
eficazmente la estabilidad oxidativa del aceite Omega-3. Este hallazgo es significativo porque la EIE
permite una evaluación más rápida y no destructiva, facilitando el monitoreo en tiempo real de la
oxidación en los aceites Omega-3, lo que podría ser particularmente beneficioso para los fabricantes
que buscan asegurar la calidad de sus productos de manera más eficiente. Además, el uso de EIE puede
reducir costos y tiempos de análisis, mejorando la eficiencia de los procesos de control de calidad. Sin
embargo, se mencionan posibles desventajas, como la necesidad de equipos especializados y la
calibración precisa del método para diferentes tipos de aceites. Futuras investigaciones deberían
centrarse en optimizar y validar aún más este método en diferentes condiciones de almacenamiento y
procesamiento, así como explorar su aplicabilidad en otros tipos de aceites comestibles y productos
derivados de Omega-3, para garantizar su calidad y seguridad. Asimismo, sería beneficioso investigar
la integración de EIE en líneas de producción automatizadas para un monitoreo continuo, lo que podría
revolucionar la forma en que se asegura la calidad de los aceites Omega-3 en la industria.
Palabras clave: omega-3, estabilidad oxidativa, espectroscopia de impedancia electroquímica
Artículo recibido 28 julio 2024
Aceptado para publicación: 15 agosto 2024
pág. 8309
INTRODUCTION
Omega-3 oil is widely used in various pharmaceutical supplements due to its health benefits, including
reducing inflammation and supporting brain function (Montgomery, Conway & Spector, 1992).
However, the high susceptibility of Omega-3 fatty acids to oxidation can degrade the product, affecting
its quality and safety (Ortega-Nieblas, Robles-Burgueño & Vázquez-Moreno, 2001). To ensure it is
suitable for consumption, these oils must meet strict regulations, such as the peroxide limit established
by the United States Pharmacopeia Convention, which must not exceed 5 milliequivalents of peroxide
per kilogram of oil (5 mEq/kg) (Ortega-Nieblas, Robles-Burgueño & Vázquez-Moreno, 2001).
The conventional method for assessing the oxidative stability of Omega-3 oils is the peroxide index,
which, although accurate, is laborious and time-consuming. The need for more efficient methods has
been highlighted in several studies. For example, Shahidi & Zhong (2010) emphasize the importance
of developing rapid and reliable techniques to measure oxidation levels due to the increasing demand
for Omega-3 supplements. Similarly, Frankel (2005) discusses the limitations of traditional methods
and the potential of advanced techniques such as electrochemical impedance spectroscopy (EIS) to
provide real-time monitoring of oxidation.
EIS, already successfully used in other fields such as evaluating the stability of biodiesel (Biernat et al.,
2019) and gasoline (Bocian et al., 2021), offers a rapid and non-destructive methodology. Additionally,
Corugedo et al. (2014) have demonstrated the effectiveness of EIS in diagnosing transformer oils,
suggesting that this technique could be adapted to assess edible oils like Omega-3.
Recent studies have investigated oxidative stability in vegetable oils using innovative techniques. For
example, Rodríguez et al. (2015) evaluated the oxidative stability and shelf life of sacha inchi oil
(Plukenetia volubilis L.) using traditional methods. Although this study does not employ EIS, the
methodology applied and the results obtained are relevant to evaluating oxidative stability in similar
oils. Applying EIS in these cases could improve the accuracy and efficiency of assessments.
On the other hand, Sanaeifar and Jafari (2019) used an integrated system of dielectric spectroscopy and
computer vision to determine the oxidative stability of olive oil. This combination of advanced
techniques provides a detailed and accurate evaluation of oxidation, which could be adapted for Omega-
3 oils, offering an advanced tool for real-time monitoring and improving product quality.
pág. 8310
Moreover, Gashi et al. (2024) evaluated the oxidative thermal stability of fish oil with the addition of
pumpkin seed oil or rosemary extract, demonstrating how natural antioxidants can enhance oxidative
stability. These findings complement studies using EIS to evaluate these effects in Omega-3 oils,
providing a basis for improving the formulation and storage of these oils.
The use of advanced techniques like EIS is not limited to edible oils. Flynn et al. (2018) conducted an
EIS study on the interaction of supported lipid bilayers with free docosahexaenoic acid, demonstrating
the applicability of this technique in various lipid matrices. Similarly, Bocian et al. (2021) have used
EIS to study the oxidative stability of gasoline, developing a chemical stability index that could be
adapted for Omega-3 oils.
Additionally, Rodríguez, Escobar, and Ortiz (2010) developed an oxidative stability test for fish oil,
comparing traditional bromatological techniques with differential scanning calorimetry. Their findings
highlight the potential of advanced analytical techniques in improving the understanding and
assessment of fish oil oxidation, further supporting the relevance of exploring EIS for this purpose.
Finally, Lázaro Mora (2023) has demonstrated how image analysis techniques can monitor the
oxidation of marine oils, suggesting that a combination of EIS and image analysis could offer an even
more robust methodology for evaluating oxidative stability.
The primary objective of this research is to explore the use of EIS as a novel and efficient method for
assessing the oxidative stability of Omega-3 oils. This study investigates the relationship between
peroxide levels and the imaginary dielectric permittivity of Omega-3 oils, validates the accuracy and
reliability of EIS compared to conventional bromatological methods, and demonstrates the potential of
EIS for real-time, non-destructive monitoring of oxidation in Omega-3 oils.
Composition of Omega-3 Fatty Acids
Omega-3 oils are polyunsaturated fatty acids that contain more than one carbon-carbon double bond.
The omega-3 family oils are composed of alpha-linolenic acid (ALA) found in plants, eicosapentaenoic
acid (EPA), and docosahexaenoic acid (DHA) both found in fish. These acids have 3, 5, and 6 carbon-
carbon double bonds in a chain of 18, 20, and 22 carbon atoms respectively, shown in fig. 1.
pág. 8311
Figure 1. Chemical structures of ALA, EPA and DHA respectively
Autoxidation Process
The autoxidation of Omega-3 oils is a significant factor affecting their stability and shelf life. This
process, driven by the interaction of unsaturated fatty acids with oxygen, involves a series of complex
chemical reactions that ultimately lead to the deterioration of the oil's quality and nutritional value
(Shahidi & Zhong, 2010).
Stages of Autoxidation
1. Initiation The initiation stage involves the formation of free radicals, highly reactive species that can
start chain reactions. In Omega-3 oils, this can occur due to exposure to light, heat, or metal catalysts
(Frankel, 2005). The presence of these factors causes the homolytic cleavage of C-H bonds in the
fatty acid molecules, leading to the formation of lipid radicals (R•).
RH→R•+H•
2. Propagation During the propagation stage, the lipid radicals react with oxygen to form peroxyl
radicals (ROO•). These peroxyl radicals are highly reactive and can further react with other lipid
molecules, continuing the chain reaction and producing lipid hydroperoxides (ROOH) (McClements
& Decker, 2000).
R•+O
2
→ROO•
ROO•+RH→ROOH+R•
The lipid hydroperoxides can decompose into secondary products such as aldehydes, ketones, and
alcohols, which contribute to off-flavors and odors in the oil (Choe & Min, 2006).
3. Termination The termination stage involves the reaction of free radicals with each other, forming
non-radical products that do not propagate the chain reaction. This can occur through various
pathways, such as the combination of two peroxyl radicals or the reaction of a peroxyl radical with
a lipid radical (Frankel, 2005).
pág. 8312
R•+R•→R-R
ROO•+R•→ROOR
The termination stage effectively halts the autoxidation process, but by this point, significant oxidative
damage may have already occurred (McClements & Decker, 2000).
Factors Influencing Autoxidation
Degree of Unsaturation Omega-3 fatty acids, being polyunsaturated, have multiple double bonds,
making them more susceptible to oxidation compared to monounsaturated or saturated fats. The higher
the degree of unsaturation, the greater the susceptibility to autoxidation (Shahidi & Zhong, 2010).
Presence of Antioxidants Natural antioxidants, such as vitamin E (tocopherols), can inhibit the
autoxidation process by donating hydrogen atoms to free radicals, stabilizing them and preventing
further propagation. Synthetic antioxidants are also used in the food industry to extend the shelf life of
oils (McClements & Decker, 2000).
Environmental Conditions Exposure to light, heat, and oxygen accelerates the autoxidation process.
Proper storage conditions, such as keeping oils in dark, cool, and airtight containers, can significantly
reduce the rate of oxidation (Choe & Min, 2006).
Measurement of Oxidative Stability
The oxidative stability of Omega-3 oils can be measured using various methods, including: Peroxide
Value (PV) This method quantifies the primary oxidation products (hydroperoxides) formed during the
initial stages of autoxidation. The peroxide value is commonly used as an indicator of the oil's freshness
and the extent of oxidation (Shahidi & Zhong, 2010) y Electrochemical Impedance Spectroscopy (EIS)
EIS is an advanced technique that measures the dielectric properties of oils. By analyzing the imaginary
dielectric permittivity, researchers can infer the extent of oxidation and the presence of oxidative
degradation products (McClements & Decker, 2000).
The standard technique used in the pharmaceutical industry for oxidative characterization of omega-3
oil is the chemical bromatological for peroxide index. This technique consists of the determination of
the peroxide value of the sample measuring the volume needed to titrate a solution of the omega-3 oil
(Rodríguez, Escobar, & Ortiz, 2010 & Spellman, 2009). The titration procedure consists of mixing 5g
of the omega-3 oil with 30mL of a solution of acetic acid – chloroform (3:2). After swirl the solution
pág. 8313
until is completely dissolved 0.5mL of saturated potassium iodide must be added and then swirl for one
minute. Immediately 30mL of distilled water must be added and shacked vigorously. Now the solution
is titrated with 0.1N sodium thiosulfate and mixed until obtain a light yellow color. Then 5mL of starch
is added as indicator, the solution will take a blue gray color. Finally titrate until the blue color
disappears. It is necessary to make a blank determination of the reagents. Then the peroxide value is
given by:
Were V
s
is the titration volume of the sample, V
b
is the titration volume of the blank and W is the
weight of the sample in grams. The bromatological peroxide index technique requires a wide use of
reactives and longtime of measurement making it inefficient for a high number of samples. Rodriguez
et al. (2010) has shown the use of the differential scanning calorimetry as a substitute for the chemical
bromatological technique to measure the oxidative stability of omega-3 oil. Herby this research aims to
find an alternative more efficient method of peroxide characterization using the imaginary dielectric
spectrum to measure the electric properties of omega-3 oil (Barsoukov & Macdonald, 2005).
Electrochemical impedance spectroscopy is a method widely used to characterize the dielectric
properties of a system (Barsoukov & Macdonald, 2005). This technique requires no sample preparation
and the relatively simple electrical measurements makes it ideal for automated characterization
evaluating a high volume of samples in a short time. The permittivity spectra is obtained from the
measurement of an AC current from the sample as result of an AC voltage input. The ratio between the
voltage and current as function of the applied frequency is given by the impedance (z(ω)).
In general the impedance is a complex number and ω is the angular frequency of the applied potential
(ω=2πf). Experimentally impedance measurements are taken using a capacitance cell in which the
sample is placed and its relative permittivity can be calculated by:
pág. 8314
Where
0
is the vacuum permittivity, C
x
is the capacitance of the cell with the sample and C
0
is the
capacitance of the empty cell (A= cell area, d= cell separation). Relative permittivity is a complex
number
and the complex impedance as function of the applied frequency is represented
in a Blode plot where the frequency is in logarithmic scale. A system can be classified according to the
ratio of the imaginary part of the permittivity to the real part.
In general the higher this ratio more conductive is the sample. A perfect conductor will have a ratio of
while a perfect dielectric (has no conductivity) will have a ratio of
Understanding the autoxidation process is crucial for both the food and pharmaceutical industries. In
the food industry, oxidative stability determines the shelf life and sensory qualities of Omega-3-rich
products. In pharmaceuticals, maintaining the integrity of Omega-3 oils in supplements is essential for
ensuring their therapeutic efficacy and safety (Shahidi & Zhong, 2010; McClements & Decker, 2000).
METHODOLOGY
The study utilized Omega-3 oil samples from the same lot, provided by PROCAPS S.A. The samples
were subjected to autoxidation at room temperature with exposure times of 1, 3, and 5 days to simulate
different levels of oxidative degradation. The specifications of the oil samples, including their initial
peroxide levels and fatty acid composition (samples extracted on May 14, 2009, and analyzed on May
24, 2009), are detailed in Table 1.
Table 1. Omega-3 oil analysis performed
Specifications
Method
Results
Free fatty acid
Iso 660 1996
0.42%
Moisture
Iso662b 1998
0.02%
Iodine index
Aoac 920.158 2005
193 hanus
Peroxide
Aocs cd 18-90 1998
3 meq/kg
EPA
DHA
Aocs ce1b-89 1998
20.0%
12.4%
pág. 8315
The bromatological technique used for peroxide index determination is the one established by United
States Pharmacopeial Convention and Food Ingredient Expert Committee (2009) for fats and oils.
Samples mass were weighed to 5±0.0001g using a Mettler Toledo model XP205DR electronic scale.
High purity reactives and solvents were used. This procedure was performed by PROCAPS S.A.
(Barranquilla- Colombia).
The primary variables considered in this study were peroxide levels and imaginary dielectric
permittivity. Peroxide levels were determined using the chemical bromatological method, providing a
measure of the oxidative degradation of the Omega-3 oil samples. Imaginary dielectric permittivity was
measured using electrochemical impedance spectroscopy (EIS), which offered insights into the
electrical properties of the oil and their correlation with oxidative stability.
To analyze the relationship between these variables, a linear correlation analysis was performed. This
statistical approach helped to establish the connection between peroxide levels and imaginary dielectric
permittivity. The strength and reliability of this correlation were assessed using the RMS value of R²,
which quantified the degree of linearity between the variables and confirmed the potential of EIS as an
effective method for evaluating the oxidative stability of Omega-3 oils.
Experimental Procedure
The bromatological technique for peroxide index determination followed the United States
Pharmacopoeia Convention (USPC, 2009) standards for fats and oils. Five grams of each sample were
weighed with a precision of ±0.0001 g using a Mettler Toledo model XP205DR electronic scale. High
purity reagents and solvents were used, and the procedure was carried out by PROCAPS S.A. in
Barranquilla, Colombia. The results were recorded and analyzed to establish baseline peroxide levels
for each sample.
Imaginary impedance spectra were obtained using a YSI Inc. electrochemical cell model 3402 with a
platinum electrode configuration (M/sample/M). A Solartron signal analyzer model 1260 was used,
covering a frequency range from 3 kHz to 100 kHz. The measurements were performed at a constant
room temperature of 24°C. The impedance data were collected and processed to obtain the imaginary
component of the dielectric permittivity for each sample.
pág. 8316
RESULTS AND DISCUSSION
The results of the bromatological peroxide index analysis for the Omega-3 oil samples show a clear
proportional increase in the peroxide index with the duration of the oil's exposure. This progressive
increase, as shown in Table 2, indicates a continuous oxidation process occurring in the oil samples
over time.
The imaginary component of the complex relative permittivity of Omega-3 oil as a function of the
applied voltage frequency at a constant temperature of 24°C. The spectra for each oxidation time begin
to converge at frequencies above 50 kHz, eventually overlapping at approximately 100 kHz, as shown
in Figure 3. This convergence suggests that EIS is sensitive to changes in the oxidative state of the oil,
with the imaginary permittivity serving as a reliable indicator of peroxide levels.
Figure 3. Imaginary component of the complex relative permittivity of Omega-3 oil as function of the
applied voltage frequency at a constant temperature of 24°C
It was observed that for frequencies above 50 kHz, the imaginary permittivity spectra for each oxidation
time begin to converge until they overlap around 100 kHz. In this frequency region, the electric response
of the Omega-3 oil is independent of the peroxide index. However, between 1 kHz and 50 kHz, a
considerable difference among the spectra is observed. The imaginary permittivity of Omega-3 oil
decreases as the peroxide index increases. The minimum values of the imaginary component of the
complex permittivity obtained were 38 x 10⁻³, 36 x 10⁻³, and 34 x 10⁻³ for the first, third, and fifth days,
respectively.
pág. 8317
The DC conductivity is directly proportional to the imaginary permittivity, as expressed by the equation:
Therefore, the DC conductivity of Omega-3 oil decreases with higher peroxide index values.
Correlation
The analysis evaluated the linear correlation between peroxide levels and imaginary dielectric
permittivity, revealing a strong linear relationship with a correlation coefficient of R² = 0.99. As shown
in Figure 4, this high correlation confirms the potential of EIS as a sensitive and reliable alternative
method for assessing oxidative stability in Omega-3 oils.
Figure 4. Peroxide index as function of the Omega-3 oil imaginary permittivity for different exposure
days.
CONCLUSIONS
This study has demonstrated that electrochemical impedance spectroscopy (EIS) can effectively
characterize the oxidative stability of Omega-3 oils. The strong linear correlation between peroxide
levels and the imaginary dielectric permittivity highlights the sensitivity and reliability of EIS in
detecting oxidative changes. This technique offers a faster, non-destructive alternative to traditional
chemical methods, providing significant advantages for quality control in both the food and
pharmaceutical industries.
In the food industry, the ability to monitor oxidative stability in real-time can enhance product quality
pág. 8318
and extend shelf life. EIS can facilitate more precise interventions to prevent rancidity and maintain
sensory attributes, ultimately improving consumer satisfaction and reducing waste (Choe & Min, 2006).
The work of Rodríguez et al. (2015) on the oxidative stability of sacha inchi oil underscores the
importance of efficient monitoring methods. While traditional methods were used in their study, the
integration of EIS could provide a more efficient and precise approach.
For the pharmaceutical industry, maintaining the integrity of Omega-3 supplements is crucial for
ensuring their therapeutic efficacy and safety. EIS offers a reliable method for rapid quality assessment,
helping manufacturers comply with stringent regulatory standards and deliver high-quality products to
the market (Shahidi & Zhong, 2010). Studies such as those by Rodríguez, Escobar, and Ortiz (2010),
which explored the oxidative stability of fish oil using bromatological techniques and differential
scanning calorimetry, highlight the potential for EIS to provide similar insights more efficiently.
Future research should focus on further validating the EIS technique for various types of Omega-3 oils
and under different storage and processing conditions. Additionally, exploring the integration of EIS
with other analytical methods, as demonstrated by Sanaeifar and Jafari (2019) with dielectric
spectroscopy and computer vision for olive oil, could enhance the comprehensiveness of oxidative
stability assessments. Investigating the potential application of EIS in monitoring other types of lipids
and nutritional compounds could also broaden its utility.
Despite its advantages, the implementation of EIS in routine quality control faces challenges, including
the initial setup costs and the need for specialized knowledge to interpret the data. Addressing these
challenges through the development of user-friendly software and cost-effective solutions will be
essential for widespread adoption (Frankel, 2005). The advancements made by Flynn et al. (2018) in
applying EIS to lipid bilayers and by Bocian et al. (2021) in creating a chemical stability index for
gasoline demonstrate the ongoing evolution and potential for EIS across various applications,
emphasizing the need for continued innovation and adaptation in this field.
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