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A Fiber-in-Needle Reduced-Pressure Microextraction Strategy for Dynamic Headspace Sampling of Aldehydes and Room Temperature Assessment of Edible Oils’ Oxidative Stability
Running Title: Assessment of oxidative stability of edible oils using RP-FIN-GC-FID
A thin film of polypyrrole/graphene oxide nanocomposite was coated on a stainless steel fiber by electrochemical polymerization and used to prepare a fiber-in-needle (FIN) dynamic sampling setup. The FIN was employed for extraction of aldehydes in edible oils, followed by GC-FID determination. To improve release of the analytes from the sample tissue and more effective trapping of them on the nanosorbent, sampling was performed under the reduced-pressure conditions. The influential experimental variables were evaluated. Under the optimal conditions, limits of detection were in the range of 0.1-0.8 µg L-1. Linear dynamic ranges were found over the range of 0.7-50000 µg L-1. The relative standard deviations were obtained 5.9-9%. The proposed reduced-pressure fiber-in-needle dynamic microextraction (RP-FIN) method, coupled to GC-FID was successfully applied for the extraction, preconcentration and quantification of hexanal, heptanal, nonanal, decanal and undecanal in several edible oil real samples and the samples’ oxidative resistances were compared.
KEYWORDS: room temperature microextraction; reduced-pressure fiber-in-needle dynamic sampling; edible oil; oxidative stability; aldehydes
Oxidation is the main factor of edible oils’ instability which leads to change their chemical, physical and nutritional properties (Choe & Min, 2006). Oil’s unsaturated fatty acids in the vicinity of oxygen get involved in non-enzymatic self-oxidation, resulted from chain reactions of released radicals, and form peroxide (Taghvaei & Jafari, 2015). Peroxides as the initial products of oxidation are very instable. They enter the secondary oxidation process and convert to volatile and non-volatile products. The most important class of these compounds are aldehydes (Guillen & Goicoechea, 2008). The unpleasant smell of rancid oil is because of the presence of aldehydes, so that the low level of hexanal (about 5 ng mL-1), as a main member, is easily distinguishable by olfactory (Kalua, Allen, Bedgood, Bishop, Prenzler, & Robards, 2007). Nowadays, the level of putrefaction and durability of oils for food quality control is measured by traditional classic methods such as acidic value, peroxide value, anisidine value and thiobarbituric acid value. These methods are generally time-consuming and costly, require tedious sample preparation and use poisonous materials and solvents (Lee, Chung, Chang, & Lee, 2007). Therefore, sensitive and precise tracing of aldehydes, as the biomarkers of oil quality, is an important issue for food quality control purposes. On the other hand, heat is a reason for enhancing oil oxidation and also increases aldehydes contents of oil sample . Therefore, analysis of aldehydes should be done at low temperatures. It means that the effective release of analytes from the sample matrix must be performed using another strategy, instead of sample matrix heating (Ghiasvand & Pirdadeh-Beiranvand, 2015). A proper alternative is extraction under reduced-pressure condition, which has been developed in recent years (Ghiasvand, Nouriasl, & Yazdankhah, 2018; Psillakis, Yiantzi, Sanchez-Prado, & Kalogerakis, 2012). In this sampling strategy, volatile analytes are easily released from the sample matrix (without need to heating), rapidly diffuse into the headspace and adsorb on the extraction phase by discharge air from the sampling chamber. On the other hand, evacuation of oxygen from the sample headspace prevents further oxidation of fatty acids and production of aldehydes, during extraction process.
In recent years, a variety of analytical procedures have been employed for quantification of volatile and non-volatile aldehydes in different food samples. Magnetic solid-phase extraction coupled (Liu, Yuan, & Feng, 2015), headspace solid-phase microextraction (HS-SPME) (Yang & Peppard, 1994), automated dynamic headspace sampling (Jaeho, Dong-Won, Xi, Hwang, & You-Shin, 2011), liquid-phase microextraction (Sghaier, Vial, Sassiat, Thiebaut, Watiez, Breton, et al., 2016) and IR spectroscopy (Wójcicki, Khmelinskii, Sikorski, & Sikorska, 2015) have been used for this purpose. Microextraction techniques are one of the hottest issues in a multidisciplinary area of science including analytical chemistry, biology, medicine, pharmacy and environmental chemistry (Ghiasvand, Hajipour, & Heidari, 2016). A simple overview of the recent scientific publications show that SPME is the most used technique among the microextraction sample preparation strategies, due to its practical and analytical features (Kaykhaii & Linford, 2017). It is performed in direct-immersion, headspace, and membrane protected sampling modes (Zhang & Pawliszyn, 1993). Additionally, many researchers have tried to improve the analytical performances and application aspects of this flourishing technique by development its new needle-based and syringe-based configurations including needle-trap device (M. Heidari, Bahrami, Ghiasvand, Emam, Shahna, & Soltanian, 2015), in-tube solid-phase microextraction (Moliner-Martinez, Herráez-Hernández, Verdú-Andrés, Molins-Legua, & Campíns-Falcó, 2015), inside needle capillary adsorption trap (Ghiasvand & Yazdankhah, 2017), fiber-packed needle solid-phase microextraction (Saito, Ueta, Ogawa, Abe, Yogo, Shirai, et al., 2009) and fiber-in-needle solid-phase microextraction (Behfar, Ghiasvand, & Yazdankhah, 2017) , dynamic solid-phase extraction (Jackson, Borba, Meija, Mills, Haverstick, Olson, et al., 2016), and microextraction by packed sorbent (Rahimi, Hashemi, Badiei, & Safdarian, 2016). Needle-trap device (NTD) was developed to compensate the limitations of fibre-SPME, while it has also suffers from drawbacks like high memory effect and uncontrollable packing and compressing of the sorbent (Chen, Yang, Hu, Cheng, Chen, & Ouyang, 2018; N. Heidari, Ghiasvand, & Abdolhosseini, 2017). Fibre-in-needle solid-phase microextraction (FIN-SPME) is a newly developed sampling strategy that has merged the features of fire-SPME and NTD . It uses a coated fiber (similar to conventional SPME) that is fixed inside a needle (similar to NTD), while is free from the problems such as sorbent packing, clogging and high memory effect. The most outstanding advantage of FIN-SPME is feasibility of dynamic extraction, which remarkably increases the sensitivity of the method.
In this research, polypyrrole/graphene oxide nanocomposite (PPy/GO) was synthesized and coated simultaneously on the surface of a stainless steel fiber, using an in-situ electropolymerization process. A FIN device was prepared using the coated fibre. The FIN setup was coupled with GC-FID and employed for dynamic extraction and preconcentration of aldehydes in edible oils, through a reduced-pressure microextraction procedure. The reduced-pressure condition causes the analytes to be released from the sample tissue without heating. The RP-FIN-GC method guarantees low temperature and rapid extraction of aldehydes, while eliminates oxygen and its related problems. It does not need organic solvents; no derivatization step is required and is performed directly on the oil sample, without any manipulation and sample preparation.
Reagents and Supplies. Extra pure pyrrole (99.5%), sodium dodecyl sulfate (SDS) and potassium hexachloroplatinate (IV) (K2PtCl6) were purchased from Merck company (Darmstadt, Germany). All inorganic acids, bases and organic solvents were of analytical reagent grade and provided by Merck. Graphene oxide was synthesized according to the modified Hummers’ method (Kumar, Singh, Verma, & Bhatti, 2014). Hexanal (Hex), heptanal (Hep), nonanal (Non), decanal (Dec), and undecanal (Undec), all of purity > 99.0%, were purchased from Sigma Aldrich (Germany). A mixture stock solution of the aldehydes (1000 μg mL-1) was prepared by dissolving appropriate amounts of them in methanol. The working standard solutions were prepared weekly by diluting the standard stock solution. All aldehydes stock and working standard solutions were kept at 4°C.
Instrumentation. Gas chromatographic separations and determinations were carried out using a Shimadzu GC-2010 Plus AF gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID-2010 Plus) and a split/splitless injector (SPL-2010 Plus) system. It was run with GC solution (version 2.4) software. Separations were accomplished using a HT-5 fused-silica capillary column (15 m × 0.32 mm × 0.5 µm). Heating the samples was done using a Heidolph MR 3001-K magnetic heater-stirrer (Kelheim, Germany). The RP-FIN extractions were performed using 40 mL sample vials with plastic crimp caps and PTFE-coated silicone septa (Supelco, Bellefonte, PA, USA). Fourier transform infrared spectra were recorded by an FT-IR 8400 spectrometer (Shimadzu, Kyoto, Japan) in the transmittance mode, and employed to characterize the functional groups of the nanomaterials. A VEGA\TESCAN CM120 (Brno, Czech Republic) field-emission scanning electron microscope (FE-SEM) was used to study the morphology of the PPy/GO nanocomposite coating. A handmade reduced-pressure setup was fabricated and used for CP-FIN sampling of the aldehydes. A Flexiflo Enternal peristaltic pump (Ross Products, Columbus, Ohio, USA) with 1.2 mbar ultimate vacuum, was used to evacuate the vacuum chamber. The sample matrices was used for circulating the sample headspace through the RP-FIN system. A stainless steel needle (12 cm L, 0.7 mm I.D., 0.9 mm O.D) and a 140 mm-long stainless steel fiber (Vita Needle Co., Needham, MA, USA) were used to fabricate the FIN device. Commercial SPME fibers with polydimethylsiloxane (PDMS), polydimethylsiloxane/carboxen (PDMS/CAR) and polyacrylate (PA) coatings were purchased from Supelco (Supelco, Bellefonte, PA, USA).
GC-FID Separation and Determination of Aldehydes.To acquire the best separation and determination conditions, GC temperature program was optimized by performing different exploratory experiments. The best oven temperature program was started from 60 ºC and raised to 100 ºC with a rate of 8 ºC min-1. Then, the temperature was raised to 280 ºC by a rate of 25 ºC min-1 and held constant for 2 min. Consequently, the total run time was 13.2 min. Both injector and detector were set at 280 ºC and runs were conducted in a split mode (split ratio: 1/10). Ultrapure nitrogen (99.999%) was used as carrier gas at a flow rate of 1 mL min-1. The flow rates of makeup gas nitrogen) and FID gases (hydrogen and air) were adjusted at 30, 30, and 300 mL min-1, respectively. Several standard solutions of the analytes with different concentrations were prepared and injected directly into GC-FID system, in order to find the retention times and obtain the instrumental linear dynamic ranges.
Modification of the Sorbent Substrate and Preparation of the FIN Device.To obtain a porous and adhesive substrate, the surface of the stainless steel wire was platinized using an emended electrophoretic procedure (Ghiasvand, Dowlatshah, Nouraei, Heidari, & Yazdankhah, 2015). It has been demonstrated that, this approach leads to tight attachment of the coating to the substrate and results in durable, mechanically strong and chemically resistant fiber coating. Then, PPy-GO electrosynthesis and its simultaneous deposition onto the surface of substrate was done. For this purpose, 1 mL redistilled pyrrole was added to 60 mL of 0.05M SDS electrolyte solution in a Pyrex flask and sonicated in an ultrasonic bath for 3 min in order to be homogeneous. Then, 0.2 g of synthesized graphene oxide was added and the mixture sonicated for complete dispersion (30 min at 45°C). In the next step, the mixture was transferred into a 40-mL electrochemical cell and the fiber and normal connected to the anode and cathode of a DC power supply, respectively. A 1-V potential was applied between the electrodes (distance 1 cm, I ≈ 30 µA), along with stirring of the mixture for 30 min. Consequently, a thin‐layer of the PPy/GO nanocomposite was deposited on the surface of the fibre. The GO nanosheets are embedded in PPy as pyrrole monomers are migrating, polymerizing and attaching on the surface of fibre. Then, the PPy/GO fiber was removed, washed with water and dried. It was initially checked using a stereomicroscope for adequate thickness and uniformity of the coating. Finally, the prepared fibre was conditioned in the GC injector at 260°C for 30 min, under the nitrogen atmosphere. The morphology and structure of the nanocomposite film were studied by SEM and FT‐IR instruments, as described previously. The structure of PPy-GO nanocomposite as well as the bonding between PPy and GO were demonstrated in the FT-IR spectra. On the other hand, the SEM images revealed that the GO sheets properly embedded in PPy matrix. In other words, the interactions between PPy and GO resulted in homogeneous dispersion of PPy throughout GO nanosheets. Additionally, the porous structure of the prepared nanosorbent was clearly observable. Finally, after the characterization, the fibre was fixed inside a stainless-steel needle to fabricate the FIN device.
Fabrication of the RP-FIN Setup. The reduced-pressure setup was fabricated by modification of a previously described system (Beiranvand & Ghiasvand, 2017). Its main part, vacuum chamber, has been consisted a 100-mL vacuum flask (Fig. 1), which was sealed using a silicone stopper. There was two suitable holes in the stopper to accommodate two septa for injection of the FIN device and another normal needle. A third septum was also accommodated for the injection of oil sample. The FIN device and second needle were connected to the peristaltic pump to recirculate the headspace of the sample. The lateral exit valve was used for evacuation of the chamber by the vacuum pump. Swagelok USA qualitative standard was applied to check possible leaks in the system.
Preparation of the Model Matrix. Using a real sample matrix or a model matrix is obligatory to obtain reliable results for optimization of extraction procedures (Ghiasvand & Hajipour, 2016). This necessity is more serious for analysis of complex samples such as edible oils. A model matrix is a real sample without analyte or a blank matrix, which its physicochemical properties are similar to the sample matrix (Ghiasvand & Hajipour, 2016). To follow this rule, a sesame oil sample was used as the model matrix, after an especial processing to remove its aldehydes content. Sesame oil contains the lowest content of polyunsaturated fatty acids, which are responsible for oxidation and aldehyde production, in comparison with other edible oils (Chung, Lee, & Choe, 2004). Additionally, sesame oil contains lignan that has strong antioxidative properties (Fukuda, Nagata, Osawa, & Namiki, 1986). For this purpose, 500 mL of same oil was poured into a 1000 mL vacuum container and was subjected to vacuum for 20 min at 25 ºC along with continuous stirring. Because of evacuating, the air and reducing the pressure all aldehydes were removed from the oil matrix. Subsequently, a model matrix, without aldehydes was obtained. It was stored at 4 ºC in a fridge for subsequent uses. The storage vial was kept under vacuum to avoid oxidation of the sample. Absence of aldehydes in this model matrix was checked using GC analysis.
RP-FIN Sampling of Aldehydes in Oil Samples.Based on the depiction in Fig. 1, the valve was opened and the vacuum pump turned on. After complete evacuation (45 s), the valve was closed and the pump turned off. Then, 5 mL of oil sample was injected into the chamber using a gastight syringe. In this way, the analytes are easily released from the sample matrix and quickly diffuse into the headspace. In the trapping step, the peristaltic pump was turned on to circulate the headspace through the FIN device at a flow rate of 12 mL min-1. After completion of the extraction (15 min), the vacuum valve was opened to break down vacuum and FIN retracted from the septum, its end was sealed using a septum and injected into the GC-FID instrument for desorption and quantification of the aldehydes, at 280 ºC for 3 min. The exposing of the fiber to heat and carrier gas in the GC injector was made feasible by its specific configuration, which is represented schematically in Fig. 2.
RESULT AND DISCUSSION
In order to optimize the procedure and to achieve a reliable and applicable strategy, the influential experimental variables including desorption conditions, temperature and time of extraction, and flowrate of circulation of the headspace were studied. As an important prerequisite, the synthesized nanocomposite was characterized. The functional groups and morphology of the prepared PPy/GO nanocomposite was studied by FT-IR and SEM instruments. The details can be found in our previous report (Behfar, Ghiasvand, & Yazdankhah, 2017). The SEM images of the PPy/GO nanocomposite (Fig. 3) showed that the sorbent has been covered uniformly on the surface of platinized stainless steel fibre. The exploratory pphysical tests demonstrated that the fiber coating was resistant to impact and abrasion.
Desorption Conditions.In the SPME-GC methods, an important priority is optimization of desorption conditions for obtaining reliable analysis. High temperatures can cause faster desorption and vaporization of the analytes in the injector with less carryover effect, but temperature is limited by the thermal stability of the sorbent and the analytes. On the other hand, longer times can lead to complete desorption of analytes and their transport to the GC column, but may cause peak broadening or reduce the lifetime of the sorbent. Therefore, optimum desorption conditions must be carefully optimized. For this purpose, different times (0.5-5 min) and temperatures (250-300 ̊C) were evaluated for desorption of the extracted aldehydes from the PPy/GO coated fiber. Consequently, 3 min at 280 ̊C was selected as the best condition for complete desorption of the analytes with the least carryover (< 3%).
Extraction Temperature and Time. Generally, extraction temperature imposes a bilateral effect in the conventional HS-SPME analysis, due to the exothermic nature of the sorption process (Ghiasvand, Hajipour, & Heidari, 2016). Higher extraction temperatures result in higher headspace concentrations thermodynamically, because of increase in partial vapor pressure and Henry’s constant. Contrariwisely, higher sample temperature reduces the tendency of fiber to adsorbed analytes. Therefore, HS-SPME typically has an optimum extraction temperature (Moradi, Kaykhaii, Ghiasvand, Shadabi, & Salehinia, 2012). In order to study the effect of temperature on the extraction efficiency of aldehydes, the RP-FIN-GC method was conducted in different temperatures over the range of 25-100 °C. The findings revealed that increase in sample temperature results in a significant rise in the extracted amounts (Fig. S-1). However, it should be considered that high temperatures can cause more oxidation and produce more aldehyde contents in edible oils, during the extraction process. This phenomenon will make significant errors in the aldehydes measurement. Consequently, 25 °C was chosen as the optimal extraction temperature for further researches.
To evaluate the effect of extraction time on the efficiency of the developed method, 5-25 min were examined as the extraction time. The results showed that peak areas of the analytes increased by increasing extraction time up to 15 minutes and then remained constant. Therefore, 15 min was considered as the optimal extraction time.
Influence of Sampling Flow Rate.In dynamic SPME, sampling flow rate is a compromise between extraction time and the sorbent characteristics. The sorbent amount and its efficiency are basically constant and cannot be changed during the extraction, while time can be varied. Higher flowrate leads to shorter extraction time, but is limited by the sorbent capacity and sorption kinetics. For this reason, some exploratory tests were done and an estimated flowrate was chosen (10 mL-1), before optimization of extraction time. For final evaluation and optimization, 3, 6, 12, 18 and 24 mL min-1 were examined as sampling flowrates (Fig. S-2). The results indicated that by increasing the flowrate up to 12 mL min-1 peak areas of the analytes increased and after that started to decline slightly. At higher flowrates the analyte molecules pass through the sorbent bed faster than the speed needed to reach the equilibrium. Consequently, 12 mL min-1 was considered as the optimum flow rate of sampling.
Comparison of the PPy/GO Nanocomposite with Other Sorbents.To ensure the reliability and superiority of the prepared sorbent in comparison with other widely used sorbents, it was compared with PA, PDMS and PDMS/CAR commercial fibers as well as a handmade nanostructured PPy coated fiber. The PPy fiber was prepared as described previously (Abdolhosseini, Ghiasvand, & Heidari, 2017). To this end, all the fibers were applied under the optimized experimental conditions. The results (Fig. S-3) show that the PPy/GO sorbent possesses higher extraction efficiency than other examined fibers. On the other hand, the prepared fiber has not the problems corresponded to the commercial fibers such as low capacity, high-cost, swelling in organic solvents and fragility.
Comparison of RP-FIN with Atmospheric-Pressure FIN and Conventional HS-SPME Methods. To prove the preference of RP-FIN sampling strategy, it was compared with atmospheric-pressure FIN (AP-FIN) and conventional HS-SPME under the optimized conditions. For conducting conventional HS-SPME, the PPy/GO coated fiber was mounted in a handmade SPME fiber holder and expose to the headspace of the extraction chamber (based on Fig. 1) of the sample at atmospheric pressure. The AP-FIN sampling was performed exactly same as described in section 2.6., except for applying vacuum. The results are presented in Fig. S-4. It is obvious that the efficiency of the RP-FIN procedure is remarkably higher than the AP-FIN and HS-SPME techniques. It can be rationalized by considering the effect of reduced-pressure on the extraction efficiency. Evacuation of the extraction chamber enhances significantly the release of analytes from the sample tissue and accelerates their diffusion into the headspace, which has been diluted by vacuum. On the other hand, the air molecules are evacuated and any possible interferences or competition with the analytes is avoided.
Analytical Performance. The analytical figures of merit of the RP-FIN-GC-FID method for analysis if the aldehydes in edible oils, including linear dynamic ranges (LDRs), limits of detection (LODs) and relative standard deviations (RSDs) were obtained under the optimized experimental conditions. The results are summarized in Table S-1. It was revealed that all analytes showed good linearity’s in the range of 0.7-50000 µg L-1 with acceptable correlation coefficients (R2 > 0.99).The LODs, based on signal-to-noise ratio of three, were found to be 0.1-0.8 µg L-1. The repeatability values (RSDs) were obtained in range of 5.9-9.0%, for six repetitive analysis of an oil sample containing 1 µg mL-1 of hexanal, heptanal, nonanal, decanal and undecanal.
For more assurance of the reliability of the developed strategy, its analytical performances were compared with some similar reported methods for analysis of aldehydes in edible oils (Garrido-Delgado, Mercader-Trejo, Arce, & Valcárcel, 2011; Gromadzka & Wardencki, 2010; Jaeho, Dong-Won, Xi, Hwang, & You-Shin, 2011; Kaykhaii & Linford, 2017; Ma, Ji, Tan, Chen, Luo, Wang, et al., 2014; Ramezani, Mirzajani, & Kardani, 2015). The results are given in Table 1. It is clear that the proposed technique has wider LDRs than all other mentioned methods. Its RSD is also acceptable compared with the reported procedures. However, the LOD of DHS-TD-UV-IMS is slightly lower than the present method. It should be noticed that the present strategy is a microextraction solvent-less method and uses simple and low-cost facilities that are available in many food laboratories.
Determination of Aldehydes in Edible Oil Samples.To evaluate the performance and applicability of the developed method for measurement of aldehydes, several edible oil samples were prepared from local supermarkets in Khoramabad (Lorestan, Iran) and analyzed under the proposed procedure. All samples were also fortified with 100 µg L-1 of the analytes and analyzed as added/found strategy. Additionally, the resulted values were compared with those obtained by validated HS-SPME-GC-FID method (Table 2). The statistical evaluation was applied to check an acceptable agreement between the results of the two applied methods. The findings showed no significant differences between the results.
To assess the applicability of the RP-FIN-GC-FID method for study of oxidative stability, it was employed for analysis of hexanal in six edible oil samples, which have been kept at different temperatures for varying times (Table 3). The results demonstrated that sunflower and soybean oils have the lowest thermal stability, while the highest thermal stability is related to sesame, olive and corn oils. The most favorable thermal and storage oxidative stability was related to sesame oil. Sunflower oil showed the higher rate of hexanal production in comparison with other samples, in case of heating and/or storage. Oil oxidation and aldehyde production are depended to amount of polyunsaturated fatty acids in oil. Unsaturated fatty acid can oxidize in the presence of oxygen and this phenomenon intensified at higher temperatures and longer times. Therefore, the content of polyunsaturated fatty acids in edible oils is the main responsible factor for the oxidation stability (Taghvaei & Jafari, 2015). Among the studied samples, sunflower oil has the highest percentage of polyunsaturated fatty acid (65-70%), whereas sesame oil contains the lowest amount. The presence of lignan and other natural antioxidants in sesame oil are other boosting variables for oxidative resistivity (Chung, Lee, & Choe, 2004). As a general result, the developed RP-FIN-GC-FID method can easily applied for sensitive monitoring of oxidation extent easily, in a short time and without using toxic solvents.
In conclusion, by considering the strength and weakness of the other reported procedures, a novel, low-cost and efficient reduced-pressure fiber-in-needle microextraction setup was introduced for dynamic sampling of aldehydes in edible oils. Using the PPy/GO nanocomposite, as the sorbent, enhanced the trapping efficiency of the analytes of interest. On the other hand, the reduced-pressure condition improved release of the analytes from the sample matrix as well as their trapping onto the sorbent. Additionally, evacuation of air from the extraction vial make the method suitable for oxygen sensitive samples and analytes. The feasibility of room-temperature analysis of aldehydes in edible oils is the main result aroused from the combination of the mentioned features, while the existing methods almost expose the sample to heat during the extraction process. The proposed RP-FIN sampling strategy showed remarkable higher efficiency than conventional HS-SPME and atmospheric-pressure FIN methods. In comparison with most of other published reports, this method has wider LDRs and lower LODs. The RP-FIN-GC-FID setup/procedure was successfully applied for determination of aldehydes in different edible oils. It was also used for comparing the oxidative stability of the oil samples under different storage times and temperatures. The developed setup and method can be used for analysis other heat-and oxygen-sensitive analytes.
The authors sincerely acknowledge Mrs. Zahra Ghasemi, English translator and instructor, for proofreading this article.
Conflict of interest:
The authors have no conflicts of interest.
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