Reversed Phase Dispersive Liquid–Liquid Microextraction for Sensitive Ion Chromatographic Determination of Metal Cations in Sesame Oil

Reversed-phase dispersive liquid–liquid microextraction (RP-DLLME) was proposed in the present work to separate and preconcentrate metal cations in sesame oil before their detection through ion chromatography-conductivity detection (IC-CDD). This method facilitates the direct extraction of cations like Na+, Pb2+, K+, Mg2+, and Ca2+ from oil specimens into an aqueous micro-drop to inject into the chromatography column. Four parameters were included in the process as the pH of water, volume of water and THF, and centrifugation time through a response surface technique. Five replicated analyses were performed under optimized circumstances (1.5 mL THF as a disperser and 60 μL water at pH 9 as an extraction solvent). Thus, recoveries of 110%, 108%, 107.2%, 104.2%, and 106.8% were displayed with respective standard deviations of 10.3, 9.8, 7.3, 9.7, and 6.7 for K+, Na+, Mg2+, Pb2+, and Ca2+. The detection limits (3σ) for the method were 0.007 μg mL−1 for K+, 0.001 μg mL−1 for Na+, 0.011 μg mL−1 for Mg2+, 0.008 μg mL−1 for Pb2+, and 0.009 μg mL−1 for Ca2+. The method could successfully determine the existence of metal cations in four sesame oil specimens.


Introduction
One of the earliest oilseed crops used by humans is sesame (Sesamum indicum L.).It is a member of the Pedaliaceae family with one of the greatest oil contents of any seed.This product is grown in different areas of the world (Zech-Matterne et al. 2015).Sesame possesses many therapeutic anti-inflammatory and antioxidant properties along with reducing anxiety and nerve damage.Sesame seeds are rich in fat, protein, calcium and vitamins, iron, potassium, magnesium, sodium, and other minerals (Wei et al. 2022).
Oils are highly consumed food sources containing natural cations.Minor components, for instance, in olive oil, which are present in very low amounts (about 2% of oil weight), include more than 300 different chemical compounds (Lechhab et al. 2022a(Lechhab et al. , 2022b;;Lozano-Sánchez et al. 2010).Trace levels of Cu, Fe, Zn, Ni, Cr, Mn, and Co are known to increase the rate of oxidation reaction and might produce peroxides, aldehydes, and ketones (de Souza et al. 2022;Tokay and Bağdat 2016).Considering the important role of cations in nutritional and human health problems, efficient and simple pretreatment stages are generally needed for extracting and measuring these cations before analyzing the natural specimens (Afkhami et al. 2009).
Extraction is the most basic technique for preparation of sample where a small quantity of sample tissue is separated.Metal cations are measured in vegetable oils using different analytical approaches such as atomic absorption spectrometer, ICP-MS, flame atomic absorption spectrometer, and graphite furnace (Baran and Yaşar 2012;Pehlivan et al. 2008;Tüzen 2003;Zeiner et al. 2005).RP-DLLME method was used in the present work for measuring several cations in sesame oil.Assadi et al. introduced DLLME as an auspicious alternative technique for the classic liquid-liquid extraction (LLE) (Rezaee et al. 2006).DLLME is an effective preconcentration technique to extract and preconcentrate analytes from complex specimens.
The DLLME applications are mostly concentrated on simple water samples (Leong et al. 2014;Rezaee et al. 1 3 2010;Rykowska et al. 2018;Saraji and Boroujeni 2014;Zgoła-Grześkowiak and Grześkowiak 2011).We established a new type of DLLME in 2010 called reversed-phase DLLME (RP-DLLME) (Hashemi et al. 2010) to determine hydrophilic compounds from aqueous solutions.In the RP-DLLME, the target analytes were extracted from oil specimens into the aqueous extractant unlike conventional DLLME.In other words, an aqueous solvent is a good preference as an extractant when the sample is immiscible with water.The solvent polarity is overturned by the suggested RP-DLLME technique in the ordinary (normal-phase) DLLME and the toxic solvents are replaced with water, which can be injected directly into ion chromatography (IC) or reversed-phase liquid chromatographic columns.This method can properly extract polar species from nonpolar specimens like oils (Hashemi et al. 2011;Rezaeinejad and Hashemi 2021).
As ordinary DLLME is not appropriate for immiscible with water non-polar samples, using RP-DLLME for these samples, the technique will be broadened (Godoy-Caballero et al. 2013;Hosseini et al. 2015;Kalschne et al. 2020;Mohebbi et al. 2018;Rashidipour et al. 2019).In a recent study, an RP-DLLME method was developed by our research group (Rezaeinejad and Hashemi 2021) for the extraction of inorganic anions from virgin olive oil before their determination by IC.In the current work, we assessed the possibility of using RP-DLLME to extract metal cations from low-concentration samples like virgin sesame oil to determine them directly by an IC method.A multivariate optimization technique known as central composite design (response surface) was used to extract conditions.By this method, the effects of the variables influencing the extraction process and their possible interactions can be simultaneously calculated.Hence, a detailed quantitative model is produced for the response (s).

Reagents and Materials
Tartaric acid and oxalic acid were gained from Merck with the maximum available purity.HPLC-grade acetonitrile, cyclohexane, ethylacetate, tetrahydrofuran (THF), and other chemicals were bought from Merck (Darmstadt, Germany) and utilized as received.Other solvents and salts of magnesium nitrate, sodium nitrate, potassium nitrate, calcium nitrate, and lead nitrate were prepared with the purity degree from Merck company.A stoke sample was Dane-Talai sesame oil.

Apparatus
The samples were analyzed by IC using a Shimadzu (Model L-10AD) instrument that had two reciprocating pumps, a DGU-14A in-line degasser, a CT10-10AC type oven, a manual high-pressure injection valve (20 μL injection loop) and a CDD detector (Model CDD-10 AVP).The data was collected and processed with Class-vp v.R 6.1 software.The analytical column was a Shim-pack IC-A3 column with 4.6 mm i.d.× 150 mm dimensions and 5-μm polyacrylate resin particles.A 25-μL HPLC microsyringe (F-LC, SGE, Australia) was used to withdraw and inject the samples.A 2-mL PE syringe was used for the RP-DLLME procedure.The extracts were centrifuged with a model 5810, Hamburg, centrifuge.A glass test tube with an elongated conical bottom was used in the RP-DLLME procedure to help withdraw the sedimented phase with a syringe as reported elsewhere (Hashemi et al. 2011).The glass tube was about 10 cm long and 12 mm wide.

RP-DLLME Method
The RP-DLLME procedure involved the following steps: A diluted sample was prepared by mixing 2.0 mL of virgin sesame oil and 2.0 mL of cyclohexane (diluter) in a glass test tube with a lengthened conical bottom.An extraction solvent consisting of 150 μL of water (pH 7) and 1.2 mL of THF (disperser) was prepared in a microvial and rapidly injected into the diluted sample using a 2-mL PE syringe.This resulted in the formation of a cloudy solution as the water droplets were dispersed into the sample.The glass test tube was placed inside a centrifuge tube and centrifuged for 10 min at 2500 rpm.The sedimented aqueous phase was completely transferred to a microvial using a 250-μL microsyringe and a portion of it was directly injected into the 20-μL injection loop of the IC column for analysis.
The IC separation of metal cations was performed using 4 mmol L −1 oxalic acid as the eluent in isocratic mode at a flow rate of 1 mL min -1 .A half-fraction CCD method using Minitab statistical software was employed to optimize the parameters affecting the extraction efficiency by the RP-DLLME method.The parameters investigated and optimized were the type and volume of extraction and dispersive solvents, the extraction time and the pH of the extraction solvent.Table 1 shows the abbreviations and levels of each factor used in the design.

Diluent Solvent
The present study aimed to extract potasium, sodium, lead, calcium, and magnesium ions from sesame oil into an aqueous micro-drop using the RP-DLLME process.For this purpose, the sample may be injected directly into an HPLC column with no pretreatment more.The sample viscosity was too higher for dispersing the solvent according to primary experiments, and used directly for the extraction.Thus, using a diluter solvent, the viscosity of sesame oil was reduced, thus increasing the cloud formation efficiency.Cyclohexane is a relatively nonexpensive and non-toxic organic solvent used commonly to remove heavy lipids from sesame oil extracts before HPLC analysis.Thus, various combinations of this solvent were examined along with the oil sample.In line with the studies on olive oil (Rezaeinejad et al. 2021), using a 1:1 mixture resulted in the most proper viscosity and cloud formation condition.

Choosing Disperser Solvent
In microextraction approaches, the dispersant solvent volume is severely important, because of altering the extraction solvent solubility into the sample, thus influencing the extraction efficacy.For a moderately polar disperser, the extraction solvent (water) is dispersed in RP-DLLME, as very finer droplets into the organic phase (in this case, the diluted oil).The main prerequisite is miscibility of the disperser solvent with the organic phase and the extraction solvent.Therefore, three organic solvents were investigated as dispersers in this study including THF, ethanol, and methanol.Though, reasonable results were found only by THF.The cloudy solution was not stable or very lower recoveries were achieved for the other solvents.Thus, THF was chosen as the disperser in following experiments.

Optimization by the Central Composite Design
Some initial tests were done and then a half-fraction central composite design was used to find the best extraction conditions with the RP-DLLME technique we proposed.
The factorial designs are used to screen the important factors.Then, more advanced second-order models (response surface designs) were performed to find the optimal experimental settings.For this purpose, a central composite design (CCD) model was used to optimize the extraction conditions.CCD combines a two-level factorial design with extra points (the stars) and at least one point in the center of the experimental region.This helps us fit quadratic polynomials with properties like orthogonality or rotatability.We usually repeat the center points to estimate the experimental errors (pure errors) better.The factors we considered were disperser volume (Vdis), extraction solvent volume (Vw), centrifugation time, with 2500 rpm (Tc), and pH of the water phase.Selection of the upper and lower levels of each factor was based on some preliminary experiments and our previous studies (Table 1).
The peak areas corresponding to the extracted quantities of K + , Na + , Pb 2+ , Ca 2+ , and Mg 2+ were considered as the CCD model's response functions for optimizing the stated variables.Table 2 displays the levels of the respective response values and experimental variables of the central composite design.
The Minitab software response optimizer function was used to optimize the studied factors simultaneously as follows: Vw = 60 μL, Vdis = 1.5 mL, pH = 9, and Tc = 16 min.The optimization plot for the central composite design is represented in Fig. 1.
The 3D surfaces can represent the association between the levels of these factors and the response.The plot of each response level was drown for a dependent variable based on two independent variables at the actual variables' confidence level.The combined effect of the two variables is displayed by the curvature of the response (Fig. 2).
Table 3 shows the regression coefficients, as well as T and P values for each mode in the model.As seen, for all cations, the most significant factor is water pH with the lowest value of p.

RP-DLLME of Sesame Oil Samples
Using the RP-DLLME technique, the analytes in four different sesame oil products were examined.Table 5 shows the concentrations of Mg 2+ , K + , Na + , Pb 2+ , and Ca 2+ in the specimens.
The samples nos.1-3 were prepared from local markets, but sample no. 4 was manually obtained by a especial mincer in a traditional shop in Khorramabad, Iran (Sonati sesame oil).According to the results, the much lower level of Na + , K + , Pb 2+ , Ca 2+ , and Mg 2+ was found in the Sonati sesame oil.

Conclusion
Sesame oil is among the oldest human food sources, as one of the sesame products.Naturally, it contains different metal cations; some of which may be also added to the oil as contamination while producing.These metal cations can be harmful or beneficial to human health based on their concentration.Hence, using RP-DLLME method in this research as an efficient and suitable condensation, K + , Na + , Mg 2+ , Pb 2+ , and Ca 2+ metal cations were extracted from sesame oil.It is a fast simple, and inexpensive method, which may be coupled with IC directly.It was indicated that RP-DLLME technique can be utilized for extracting the cations of samples with a non-aqueous environment as a fast and easy method.
The proposed process was compared with some reported methods used to extract and determine inorganic cations from various oils (Table 6).The RP-DLLME-IC-CDD had the detection limits much lower with higher recovery.

Fig. 1
Fig.1The optimization plot for the central composite design, comprising two dependent and five independent variables

Fig. 2
Fig.2The response surface plots for the mg of extracted a Na + , b K + , c Pb 2+ , d Mg 2+ , e Ca 2+

Table 3
The regression coefficients estimated by the CCD model and statistical t student (T) and probability (P) values for the studied factors and their interactions + , Na + , Mg 2+ , Pb 2+ , and Ca 2+ under the optimized conditions for Etka virgin sesame oil from Khoramabad

Table 5
The quantity of Na + , Pb 2+ , K + , Mg 2+ , and Ca 2+ in four sesame oil samples defined by the RP-DLLME-HPLC technique

Table 6
Comparing the proposed RP-DLLME-IC-CDD technique and some other reported methods to extract and determine inorganic cations in various oils a The limit of detection, in μg kg −1 b The limit of detection, in μg L −1