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Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples
Accepted Manuscript Title: Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples Author: Fatemeh Rezaei Yadollah Yamini Hamid Aseiabi Shahram Seidi Maryam Rezazadeh PII: DOI: Reference:
S0896-8446(15)30150-9 http://dx.doi.org/doi:10.1016/j.supflu.2015.10.005 SUPFLU 3474
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
2-8-2015 6-10-2015 6-10-2015
Please cite this article as: F. Rezaei, Y. Yamini, H. Aseiabi, S. Seidi, M. Rezazadeh, Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples, The Journal of Supercritical Fluids (2015), http://dx.doi.org/10.1016/j.supflu.2015.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples
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Fatemeh Rezaeia, Yadollah Yaminia*[email protected], Hamid Aseiabia, Shahram Seidib, Maryam Rezazadeha
Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
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Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran. Iran
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* Corresponding author. Tel: +98-21-82883417, fax: +98-21-88006544.
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Abstract
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Supercritical fluid extraction (SFE) followed by supramolecular solvents microextraction (SUPRAS) has been developed for extraction and determination of levonorgestrel (LeV) and megestrol acetate (MA) in blood samples. LeV and MA were employed as model compounds to assess the extraction procedure and were determined by high performance liquid chromatography coupled with ultraviolet detection. SUPRAS is a nano-structured liquid, generated from the amphiphiles through a sequential self-assembly process occurring on two scales; molecular and nano. SUPRAS tests were generated from solutions of reverse micelles of decanoic acid (DeA) in tetrahydrofuran (THF) by addition of water, which acted as the coacervating agent. In SFE-SUPRAS procedure, the blood sample were mixed with anhydrous sodium sulfate and loaded into SFE extraction vessel and extraction was performed in a predetermined time. The DeA solution and SFE (THF) collecting solvent were immediately injected into water for SUPRAS formation. The effective parameters on the SUPRAS efficiency were studied and optimized utilizing rotatable central composite design (RCCD). The Taguchi orthogonal array (OAD) experimental design with an OA16 (45) matrix was employed to optimize the SFE conditions. The calibration plots were linear in the range of 0.5–7.0 mg kg−1 and the limits of detection (LODs) were 0.1 and 0.2 mg kg−1 for MA and LeV, respectively. Analysis of drugs in different blood samples showed that the improved technique has great potential for extraction and determination of LeV and MA in blood samples.
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Keywords
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Supercritical fluid extraction; Nanostructured solvents; Levonorgestrel; Megestrol acetate; Blood samples
1. Introduction
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Todays, emergence of new diseases and so new pharmaceutical compounds and environmental pollutants is a serious threat to the living beings. Therefore, timely and precise determination of trace amounts of different analytes has become an important issue and received major attention from the research community around the world.
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Levonorgestrel is a synthetic female contraceptive hormone used in pregnancy prevention in humans. Although it has been widely used in the world, some deleterious reproductive effects including the turbulence of menstruation and increasing rate of galactophore cancer have been reported as the side effects [1-3]. Megestrol acetate is a type of progestin, which is used in treatment of advanced endometrial cancer and breast cancer. It is also used as an appetite enhancer and to treat weight loss in pediatric and patients with malignancies, cystic fibrosis, and HIV/AIDS [4, 5]. Determination of concentration of these drugs in whole blood may be useful to adjust the expected effective dose and so clinical side effects.
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Since analytes of environmental or biological origin are usually found in complex matrices that disturb the separation and data analysis, a series of steps are required to remove the interfering substances, preconcentrate the analytes, and increase the sensitivity. As a consequence, the research trends have been directed to development of new sample preparation techniques that are faster, simpler, inexpensive, miniaturized, and more environmental friendly. Whole blood is one of the most complicated biological fluids and extraction and clean-up of trace amounts of different analytes in it are the bottle-neck in many analytical procedures. Matrix solid phased dispersion (MSPD) is based on dispersion of sample onto an adsorbent followed by preliminary purification and elution of analytes with a relatively small solvent volume. From the first introduction of MSPD in 1989 by Barker, it has found increasing acceptance in trace analysis of organic compounds using chromatographic techniques due to providing sample homogenization, extraction, and clean up in one step [6-8]. Until now, MSPD in combination with chromatography methods has been successfully applied for determination of different analytes in various biological samples [9-20].
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However, MSPD procedure generally needs longer analytical time and its limit of detection (LOD) is limited [21]. Also, owing to the fact that sorbents are nonselective, further purification of the extracts is often still required to remove coextractants before further analysis. Shen et al. reported combination of MSPD with accelerated solvent extraction (ASE) from soil samples and fish muscle for determination of OCP residues [9, 10]. However, the consumption of hazardous organic solvents in ASE is relatively high, the final extract needs solvent evaporation; and extraction of other matrix interferences is probable, which reduces higher purification of extract.
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In the case of non-polar compounds and complicated matrices such as whole blood, simultaneous application of MSPD with supercritical fluid extraction (SFE) is an intelligent combination of separate MSPD and SFE providing the advantages of both techniques and creating considerable clean extracts. The considerable cleanup of extracts may be attributed to the non-polar nature of carbon dioxide in SFE that only provides the extraction ability of non-polar compounds. In offline SFE, extracts are often collected into one milliliter volumes of a proper solvent, which dilutes the analytes. So, application of a fast and simple preconcentration step instead of solvent evaporation reduces extraction time and loss of the extracts during evaporation step, and increases safety and sensitivity of further analysis. SFE has been combined with different preconcentration techniques such as dispersive liquid-liquid extraction (DLLME) and vesicular based-supramolecular solvent [22-24].
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Supramolecular solvents (SUPRASs) constitute environment friendly alternatives to molecular organic ones for analytical extractions [25, 26]. In 2007, Ruiz et al. [27] developed a supramolecular solvent (SUPRAS) microextraction technique based on analyte partitioning between an alkyl carboxylic acid-based nanostructured solvent and a bulk aqueous sample. The SUPRASs, which are water-immiscible liquids consisting of reverse micelle aggregates of nanoscale dimensions dispersed in a continuous phase (including tetrahydrofuran (THF) and water), have properties exceptionally useful for extraction processes, derived from the particular structure of their supramolecular assemblies [28-30]. A two-step process is needed for SUPRAS formation. First, amphiphilic molecules spontaneously form three-dimensional aggregates above a critical aggregation concentration. Then, the generated nanostructures self-assemble in larger aggregates by the action of an external stimulus and separate from the bulk solution as an immiscible liquid by a phenomenon named coacervation [31]. The objective of this work is optimization and validation of a method based on combination of MSPD with SFE followed by a preconcentration step using a nanostructured supramolecular solvent, which is made up of reverse micelles of decanoic acid (DeA), dispersed in a continuous phase of THF/water for determination of levonorgestrel and megestrol in whole blood samples. Response surface methodology (RSM) was used for optimization of different parameters 3 Page 3 of 22
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affecting the extraction efficiency. To the best of our knowledge, there is no report about the combination of MSPD with SFE. 2. Experimental
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2.1. Chemicals and reagents
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Standards of levonorgestrel (LeV) and megestrol acetate (MA) were kindly donated from the Department of Medical Sciences of Tehran University (Tehran, Iran). Structure and properties of drugs are provided in Table 1. THF was supplied by Merck (Darmstadt, Germany). Decanoic acid was obtained from Fluka (Buchs, Switzerland). Sodium sulfate and other reagents were of analytical grade and obtained from Merck. The ultra-pure water was prepared by a model Aqua Max-Ultra Youngling ultra-pure water purification system (Dongan-gu, South Korea). HPLCgrade methanol and acetonitrile were purchased from Duksan (Gyeonggi-do, South Korea). Microliter syringes (25-500 µL) were purchased from Hamilton (Bonaduz, Switzerland).
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2.2. Apparatus
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Stock standard solutions (1000 µg mL−1) of the analytes were prepared by dissolving a proper amount of each drug in methanol. Mixtures of standard working solutions were prepared by dilution of the stock solutions with ultra-pure water.
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Droplet size and morphology of the prepared SUPRAS were determined by a transmission electron microscope (TEM) model EM10C (Zeiss, Germany) operating at 80 kV accelerated voltage. A Suprex (Pittsburgh, PA, USA) SFE/SFC instrument model MPS/225 in SFE mode was utilized for all extractions. Extractions were accomplished using a 1-mL volume stainless steel extraction vessel. A manual adjustable restrictor from ISCO (USA) was used in the SFE instrument to collect the extracted analytes in a suitable solvent. To prevent sample plugging, the temperatures of body and restrictor point was adjusted at 90 and 95 ºC, respectively. Chromatographic separations were performed with a HPLC instrument including a Varian 9012 HPLC pump (Walnut Creek, CA, USA), a six-port Cheminert HPLC valve from Valco (Houston, TX, USA) with a 20-µL sample loop and equipped with a Varian 9050 UV–Vis detector. Chromatographic data were recorded and analyzed using ChromanaCH software, version 3.6.4 (Tehran, Iran). An ODS-3 column (150 mm × 4.6 mm, with 5-µm particle size) from MZ-Analysentechnik (Mainz, Germany) was applied to separate MA and LeV under isocratic elution conditions. A mixture of ultra-pure water and acetonitrile (30:70) for 10 min and
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100% acetonitrile for 5 min at a flow rate of 1 mL min−1 were used as the mobile phase and the analytes were detected at 257 nm. 2.3. Sample preparation
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To evaluate applicability of the developed extraction method, two blood samples (blood group B+ and O+) were obtained from the Iranian Blood Transfusion Organization (Tehran, Iran). The blood samples were poured in a porcelain mortar containing anhydrous sodium sulfate, and the mixture was blended until an apparently dry material was obtained. Afterwards, the samples were extracted using SFE–SUPRAS procedure. The extracted analytes were analyzed by HPLCUV.
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2.4. MSPD-SFE – SUPRAS procedure
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Half milliliter of blood samples was poured in a porcelain mortar containing 1 g of anhydrous sodium sulfate, and the mixture was blended with the pestle for a few minutes until an apparently dry material was obtained. The homogeneous mixture was placed in the SFE extraction vessel and spiked with 50 µL of standard solution (100 mg L-1), then another filter was placed on the top of the vessel and the vessel was closed. Finally, SFE was carried out using a combination of static extractions to enhance the sample-solvent contact, and thus a better penetration of the fluid in the matrix, followed by the dynamic extraction step, in which the supercritical fluid passed continuously through the extraction chamber. Extractions were conducted under the following conditions: 15 min static extraction, 40 min dynamic extraction at 240 bar and 55ºC, a CO2 flowrate of 0.5 mL min-1 and temperatures of restrictor body and tip equal to 90 and 95ºC, respectively. The extracted analytes were collected in THF (3.0 mL), which was located in a 5.0mL volumetric flask. To increase the collection efficiency and decrease the solvent evaporation, the volumetric flask was placed in an ice bath during the dynamic extraction time. Afterwards, 40 mg of DeA was added to 3.0 mL of SFE collecting solvent (THF). Then, an aliquot of 40.0 mL ultra-pure water with pH value adjusted at 2.5 (by dropwise addition of HCl (0.1 mol L-1)) was poured into a 50-mL homemade centrifuge tube, which is designed for collection of low density organic solvents. The solution that was obtained from SFE step was quickly injected into the aqueous solution using a 5-mL gastight syringe from Hamilton. The SUPRAS, made up of reverse micelles of DeA dispersed in THF:water, spontaneously formed and separated from the THF:water solution as an immiscible liquid. The mixture was centrifuged at 5000 rpm for 5 min to accelerate complete separation of the two immiscible liquids. The coacervate, located at the top of the glass tube, was withdrawn using a microsyringe and 20 µL of it was injected into the HPLC instrument for subsequent analysis. 3. Results and Discussion 5 Page 5 of 22
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In this paper, the SFE was used to extract drugs directly from the MSPD. The MSPD-SFE method integrates the relative merits of MSPD and SFE to achieve selective determination of analytes.
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The blood is disrupted and dispersed thoroughly with the shearing force and hydrophobic– hydrophilic characteristic of adsorbents during MSPD process. This is while after the SFE process, the matrix components are retained by adsorbent while the analytes are extracted by SFE.
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The most serious problem of off-line SFE methods is evaporation of the collecting solvent at the end of extraction to acquire high preconcentration factor. However, this procedure is a timeconsuming step and contaminates the environment and collected analytes may be lost or degraded in this step. Supramolecular solvent microextraction is an extraction method that consumes less organic solvent and very high enrichment factors can be obtained using this method. Combination of MSPD-SFE and SUPRAS was applied for extraction and determination of LeV and MA in blood samples.
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3.1.1. Solvent composition
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3.1. Supramolecular solvent microextraction
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The reverse micelle-based supramolecular solvent applied in this research is spontaneously formed in ternary mixtures of DeA, water, and THF at well-defined proportions. Its formation occurs through two sequential self-assembly processes. First, DeA molecules aggregate as reverse micelles in THF and then, upon the addition of water, they rearrange into larger reverse micelles that separate from the bulk solution as an immiscible liquid via a mechanism that remains elusive. The immiscible liquid is made up of reverse micelles, THF, and a little amount of water. The excellent dissolution properties of reverse micelles and the low volume of the coacervates obtained make them very attractive to be used in analytical extractions. In order to set up efficient extraction schemes, it is important to understand the intermolecular forces driving the extraction process. Hydrocarbon chains of DeA molecules forming aggregates in these SUPRASs extend into and are surrounded by the THF, while their carboxylic groups are solvated by water in the interior of the aggregates. DeA reverse micelles provide a two-fold mechanism for substrate solubilization, namely hydrophobic interactions in the surfactant tails at the micellar surface and hydrogen bonds in the polar head groups at the micellar core [27, 32]. Consequently, the expected driving forces for the extraction were van der Waals interactions between the hydrocarbon chains of the DeA and the drugs aromatic framework, and hydrogen bonds on account of the acceptor and donor groups of the analytes molecules.
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3.1.2. Characterization of the nano-structured solvent
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3.1.3. Optimization of supramolecular solvent microextraction
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The shape and size of the aggregates of DeA were investigated by TEM (Fig. 1). The target SUPRASs were formed by injecting a mixture containing 200 mg DeA and 10 mL THF into 30 mL ultra-pure water (pH = 2), then sonication of the tube for 5 min. As can be seen, the DeA aggregates were nearly spherical in shape with an average diameter of about 10–20 nm, and they tended to aggregate to larger particles.
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The optimized response value for one factor is often influenced by other factors, which may impose substantial impacts on the determination signal. These factor interactions can only be measured by multivariate optimization strategies such as factorial designs. Response surface methodology (RSM) is effective for responses that are influenced by many factors as well as by their interactions and was originally described by Box and Wilson [33]. In this work, rotatable central composite design (RCCD) was utilized to achieve the optimal conditions. The software package Design-Expert 8.0.6 trial version (Stat-Ease Inc., MN, USA) was used for experimental design, data analysis, and response surfaces.
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CCD is a second-order model correlating the response function with the independent factors; so that the amount of response can be predicted at any point within the factor domain even if the point has not been included in the design. The model takes the following general form for independent variables [34]:
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where Y is the process response or output (dependent variable), k is the number of the patterns, i and j are the index numbers for pattern, β0 is the offset term, βi is the linear effect, βii is the squared effect, βij is the interaction effect, and Xi and Xj are the levels of the independent variables. With four factors and six center points, totally 30 experiments had to be run for five-level investigation of variables in rotatable CCD. The total number of experimental runs (N), suggested by RCCD, is obtained by the following equation:
wherein f is the number of variables and
(2) is the number of center points. The independent
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variables were pH (A), DeA weight (B, mg), THF volume (C, mL), and ionic strength (D, % w/v NaCl). The sum of peak areas for each run was selected as the response objective for the study. The model was described as follows:
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Equation 3 relates the response to the coded amount of the variables in the range of −2 to 2. To this end, the high and low levels of each variable were considered 2 and −2, respectively. Therefore, the response could be predicted in each level of variables.
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Analysis of variance (ANOVA) was performed, which showed that the model was significant (Table 4). Also, the “lack of fit” was not significant (p = 0.05), which implied that the model fitted the data.
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The ANOVA result showed that the F value of 6.69 is significant and there is only 0.01% likelihood that large model F value could occur by chance. The response equation fitted the experimental data with R2-value of 0.9260, indicating 92.60% variability in the response. The goodness-of-fit of the model to the experimental data shown in Fig. 2 has an adjusted R2 = 0.8569.
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For graphical interpretation of the interactions, the use of three-dimensional plots (3D) of the model is highly recommended. This is helpful for visualizing the relationship between the responses and the experimental levels of each factor. These plots were obtained for a given pair of factors, while maintaining the other factors fixed at their optimal values (Fig. 2). The pH value of the sample is a significant factor that may affect the extraction recovery of the analytes as well as the state of DeA, which plays an essential role in the stability of reverse micelle in aqueous solutions. Acidification of the sample is usually required to have the neutral forms of these compounds and thus increasing recoveries. Also, the coacervation phenomenon occurs from protonated alkyl carboxylic acids; so, extractions must be carried out at pH values below 4 [26]. At higher pH values, solubilization of deprotonated DeA molecules in the waterTHF phase in equilibrium with the SUPRAS occurred and that resulted in reduction of formed SUPRAS volume. Therefore, the effect of pH on microextraction of analytes was studied in the pH range of 1–5. As can be seen in Fig. 2A, the best extraction efficiency of the analytes was obtained at pH 2.5. Addition of salt is widely used in microextraction techniques to improve the partitioning of analytes into the organic through the salting out effect. This is while it was reported that addition 8 Page 8 of 22
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of NaCl decreases the efficiency of dispersion of extraction phase and mass transfer in aqueous medium, leading to a decrease in extraction efficiency. To investigate the influence of salt addition on performance of SUPRASF extraction, various experiments were performed by adding different amounts of NaCl (0–20% w/v) to the sample solution. It was found that the peak area increased with the increase of ionic strength in the range of 0% -5.0% and then decreased at higher values (Fig. 2B).
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SUPRAS-to-sample phase volume ratios were a function of the concentrations of DeA and THF, which are both major components of this solvent. According to Fig. 2C, the peak area increases first and then decreases by increasing the volume of THF for both analytes. Since at a low volume of THF, a reverse micelle phase could not be well formed; therefore, it resulted in a low recovery rate. When the THF volume increased from 2 to 3 mL, the extraction efficiency increased due to the improved dispersion procedure and when the THF volume increased from 3 to 6 mL, the extraction efficiency decreased. This type of dependence indicated that progressively more THF was incorporated into the solvent as the percentage of THF in the solution increased and consequently, the reverse micelles became more and more diluted.
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DeA weight affects producing reverse micelle and volume of extraction phase, thus influencing the extraction efficiency. The effect of DeA weight on the extraction efficiency was studied in the range of 30-70 mg. As the obtained results show, the peak areas of the analyte were increased by increasing DeA amount to 40 mg and then decreased because of increasing DeA weight, increase in the coacerva volume, and decrease in the concentration of analytes. Accordingly, weight of DeA was adjusted at 40 mg for further studies. According to the overall results of the optimization study, the following experimental conditions were chosen: THF volume, 3.0 mL; pH, 2.5; DeA weight, 40.0 mg, and concentration of sodium chloride, 5.0% (w/v). 3.2. Optimization of matrix solid phased dispersion-supercritical fluid extraction SFE, a sustainable green technology, has led to a wide range of applications in the past decades. Like many other processes, SFE is sometimes criticized for its large number of factors that need to be properly adjusted before every single run. Experimental design and proper statistical analysis with small number of trials in adjusting the SFE parameters has become popular in this regard. Taguchi methods have been widely used to optimize the reaction variables by formulating the minimum number of experiments. This approach helps to identify the influence of individual factors and establish the relationship between variables and operational conditions [35, 36]. The Taguchi experimental method, an orthogonal array design, was applied to study the possible influence on the performance of the MSPD-SFE method of five factors (each factor at four levels); namely temperature, pressure, static time, dynamic time, and the modifier (methanol) volume. Sixteen experiments were performed to estimate the best conditions for the SFE of MA and LeV (Table 3). ANOVA was performed for the experimental data obtained. 9 Page 9 of 22
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Table 4 presents the ANOVA results for the effects of investigated parameters. The ANOVA results showed that all factors were statistically significant at P< 0.05. Furthermore, from the percentage contribution (Table 4), it can be deduced that the most important factor contributing to the extraction efficiency was the modifier volume (methanol, 40.276%). Further experiments were performed under the optimal conditions and the results showed that under the optimal conditions obtained from the OA16 (45) matrix, the recoveries were similar to the optimal performance calculated using the following expression:
where T is the grand total of all results, N is the total number of the results, Aopt is the
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performance under optimal conditions; and , , , , and are the average performances of the dynamic time, static time, temperature, pressure, and modifier volume at those optimal levels, respectively. Based on the above equation, under the optimal conditions, the performance is estimated using only the significant factors (all the factors in this study) [37].
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Under the optimal conditions, the confidence interval (CI) of the performance is calculated using the following expression:
(5)
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where F (1, n2) is the F-value from the F-table at a required confidence level at the degrees of freedom (DOFs) of 1 and of error, n2; Ne is the effective number of replications, and Ve is the variance of error term from ANOVA. The effect of pressure on the results obtained from MSPD-SFE – SUPRAS of drugs was examined in the range of 180-360 bar. These results indicated that increasing pressure from 180 to 240 bar enhanced the extraction recovery, which was due to increased supercritical carbon dioxide (SC-CO2) density at higher pressures. However, above 240 bar, an increase in pressure leads to a reduction in the extraction efficiency, which can probably be attributed to reduced diffusion rates of the extracted materials from the sample matrix to the supercritical fluid environment. Therefore, 240 bar was selected as the optimum pressure in the subsequent studies. Temperature is an important variable for the SFE extraction. The effect of temperature on the results achieved from MSPD-SFE–SUPRAS was scrutinized in the range of 318-348 K. The extraction depends on a complex balance between the supercritical carbon dioxide density and vapor pressure of drugs. As the temperature increases, the extraction efficiency increases drastically up to 328 K. This arises from enhancement of vapor pressure of the compounds at high temperature. However, above 328 K, the extraction efficiency decreases, due to the density 10 Page 10 of 22
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diminution of supercritical fluid at high temperatures. Therefore, 328 K was selected as the optimum temperature in the following studies. The similar temperature and pressure behavior has been reported previously [23]. The effect of temperature on the extraction efficiency of the drugs may be discussed by its effect on the solute solubility around crossover region too [38].
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Time is one of the main factors for exhausted extraction and is an important index for evaluation of extraction efficiency. Shorter extraction time could cause incomplete extraction and longer extraction time could be time and solvent wasting. In order to obtain high yield of analytes, a primary extraction step in the static mode was performed. This step was followed by a dynamic extraction to enhance solubility of analytes in the supercritical fluid. In this study, the effects of static extraction period on the results achieved from SFE-SUPRAS of drugs were scrutinized at four levels; 5, 10, 15, and 20 min. The results exhibited that by increasing the static time from 5 to 15 min, the extraction efficiency can be increased effectively. For this reason, a static time of 15 min was applied. This step would lead to better penetration of the fluid into the matrix compared with the only dynamic extraction mode. The effect of dynamic extraction time was studied at four levels; 20, 30, 40, and 50 min. By increasing the dynamic time from 10 to 40 min, the extraction efficiency increased. Nevertheless, above 40 min, an increase in the dynamic time led to a reduction in the collection efficiency of the analytes. Long dynamic extraction times may cause unwanted physical movement of matrix components to the trapping device as well as loss of analytes from the trap due to being blown off from the trap and volatilization of the trapping solvent, which was not beneficial to increase the extraction recovery. Hence, dynamic extraction time of 40 min was selected for subsequent experiments. The extraction efficiency of compounds of interest can be enhanced significantly by addition of a polar modifier to supercritical CO2. Moreover, modifier content was demonstrated to be a crucial factor that affects the recovery of polar compounds. Effect of modifier volume (methanol) was studied at four levels in the range of 0-75 µL. The results showed that by increasing the modifier volume from 0 to 50 µL, extraction efficiencies for the analytes increased. In the presence of methanol, solubility of the analytes in CO2 can be increased. However, by increasing the methanol volume from 50 to 75 µL, a reduction in the extraction efficiency of the drugs can be obtained. Hence, 50 µL of the modifier was added into the samples in subsequent experiments. 3.3. Quantitative analysis The calibration curves obtained under optimized conditions are summarized in Table 5. Linear dynamic ranges varied in the range of 0.5-7.0 mg kg-1 for LeV and MA. Coefficients of determinations (R2) ranged from 0.9983 to 0.9989. Intraday (n = 3) and interday standard deviations were calculated by extracting the drugs from blood samples at 1.0 mg kg-1 level. Intraand interday RSDs% lower than 7.1% and 10.2%, were obtained respectively. The LODs, based 11 Page 11 of 22
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on S/N of 3, ranged between 0.1 to 0.2 mg kg-1 for two drugs and the ER% obtained were in the range of 39-47% for LeV and MA. 3.4. Analysis of blood samples
4. Conclusions
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In order to evaluate applicability of the developed extraction method for analysis of LeV and MA in real samples, two blood samples were selected and the drugs were extracted using the proposed method under the optimal conditions. Sample preparation for real samples was performed according to Section 2.4. The blood samples were analyzed by HPLC-UV after SFE– SUPRAS procedure. A recovery study was carried out by spiking the samples at concentration levels of 1.0 and 2.0 mg kg-1. Each treatment was done in triplicates, and the mean results are shown in Table 6. The relative recoveries of the two drugs from the spiked samples ranged from 90% to 98% with a precision (RSDs %) of 6.3–7.2%. The results revealed that the blood matrices had little effect on the MSPD-SFE–SUPRAS procedure. Fig. 3 illustrates the typical chromatograms of the extracted drugs from blood sample (group B+) before and after spiking with the drugs at 1.0 mg kg-1 level. The Lev and MA peaks were eluted around 8 and 10 min from the column respectively.
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Supramolecular solvents have outstanding properties for microextraction. They combine the capability of solubilizing solutes in a wide polarity range with the ability to achieve high enrichment factors, mainly arising from the mixed-mode mechanisms and multiple binding sites they can provide. In this research, combination of MSPD with SFE followed by supramolecular solvent extraction was proposed as valuable tools for extraction of LeV and MA from blood samples. The proposed method is environmental friendly, precise, reproducible, and linear over a broad concentration range. Besides, very small matrix effects were observed when the proposed MSPD-SFE-SUPRAS technique was applied to blood samples spiked with the analytes. Also, after completion of SFE procedure, the extra steps (vaporization of large volume of toxic organic solvent, which is time-consuming and has inappropriate environmental behavior) needed before final analysis were eliminated. The proposed method possesses great potential in analysis of trace organic compounds in real solid samples.
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[8] M. García-López, P. Canosa, I. Rodríguez, Trends and recent applications of matrix solidphase dispersion, Analytical and Bioanalytical Chemistry 391 (2008) 963–974.
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[9] Z.L. Shen, D. Yuan, H. Zhang, M. Hu, J. H. Zhu, X.Q. Zhang, Q. D. Suc, Matrix solid phase dispersion-accelerated solvent extraction for determination of OCP residues in fish muscles, J. Chinese Chemical Society 58 (2011) 494-502. [10] Z.L. Shen, J.B. Cai, Y. Gao, X.L. Zhu, Q.D. Su, Matrix solid phase dispersion-accelerated solvent extraction for determination of OCP residues in fish muscles, Chinese Journal of Analytical Chemistry 33 (2005) 1318-1320. [11] L. Guo, M. Guan, C. Zhao, H. Zhang, Molecularly imprinted matrix solid-phase dispersion for extraction of chloramphenicol in fish tissues coupled with high-performance liquid chromatography determination, Analytical and Bioanalytical Chemistry 392 (2008) 1431-1438. [12] Y. Zhang, X. Xu, H. Liu, Y. Zhai, Y. Sun, S. Sun, H. Zhang, A. Yu, Y. Wang, Matrix solidphase dispersion extraction of sulfonamides from blood, J. Chromatographic Science 50 (2012) 131-136. [13] S. Bogialli, R. Curini, A. Di Corcia, M. Nazzari, M. L. Polci, Rapid confirmatory assay for determining 12 sulfonamide antimicrobials in milk and eggs by matrix solid-phase dispersion 13 Page 13 of 22
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and liquid chromatography-mass spectrometry, J. Agriculture and Food Chemistry 51 (2003) 4225-4232.
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[14] E.M. Kristenson, E.G.J. Haverkate, C.J. Slooten, L. Ramos, R.J.J. Vreuls, U.A.T. Brinkman, Suitability of several carbon sorbents for the fractionation of various sub-groups of toxic polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, J. Chromatography A 917 (2001) 277-286.
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[15] S.A. Barker, Matrix solid phase dispersion, J. Biochemical and Biophysical Methods 70 (2007) 151-162.
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[16] S.A. Barker, Matrix solid-phase dispersion, J. Chromatography A 885 (2000) 115-127.
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[17] J.J. Ramos, R. Rial-Otero, L. Ramos, J.L. Capelo, Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits, J. Chromatography A 1212 (2008) 145-149.
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[18] E. Sobhanzadeh, N.K. Abu Bakar, M.R. Bin Abas, K. Nemati, Low temperature followed by matrix solid-phase dispersion-sonication procedure for the determination of multiclass pesticides in palm oil using LC-TOF-MS, J. Hazardous Materials186 (2011) 1308-1313.
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[19] Y. Moliner-Martinez, P. Campíns-Falcó, C. Molins-Legua, L. Segovia-Martínez, A. SecoTorrecillas, Miniaturized matrix solid phase dispersion procedure and solid phase microextraction for the analysis of organochlorinated pesticides and polybrominated diphenylethers in biota samples by gas chromatography electron capture detection, J. Chromatography A 1216 (2009) 6741-6745. [20] S.A. Rodrigues, S. S. Caldas, E. G. Primel, A simple; efficient and environmentally friendly method for the extraction of pesticides from onion by matrix solid-phase dispersion with liquid chromatography-tandem mass spectrometric detection Analytica Chimica Acta 678 (2010) 8289. [21] N. Furusawa, A toxic reagent-free method for normal-phase matrix solid-phase dispersion extraction and reversed-phase liquid chromatographic determination of aldrin, dieldrin, and DDTs in animal fats, Analytical and Bioanalytical Chemistry 378 (2004) 2004-2007. [22] M.H. Naeeni, Y.Yamini, M. Rezaee, Combination of supercritical fluid extraction with dispersive liquid-liquid microextraction for extraction of organophosphorus pesticides from soil and marine sediment samples, J. Supercritical Fluids 57 (2011) 219-226.
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[23] M. Rezaee, Y. Yamini, M. Moradi, A. Saleh, M. Faraji, M.H. Naeeni, Supercritical fluid extraction combined with dispersive liquid–liquid microextraction as a sensitive and efficient sample preparation method for determination of organic compounds in solid samples, J. Supercritical Fluids 55 (2010) 161-168.
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[24] F. Rezaei, Y. Yamini, M. Moradi, H. Asiabi, Determination of diphenylamine residue in fruit samples by supercritical fluid extraction followed by vesicular based-supramolecular solvent microextraction, J. Supercritical Fluids 100 (2015) 79-85. [25] F. Rezaei, Y. Yamini, M. Moradi, B. Daraei, Supramolecular solvent-based hollow fiber liquid phase microextraction of benzodiazepines, Analytica Chimica Acta 804 (2013) 135-142.
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[26] F. Rezaei, Y. Yamini, M. Moradi, A comparison between emulsification of reverse micellebased supramolecular solvent and solidification of vesicle-based supramolecular solvent for the microextraction of triazines, J. Chromatography A 1327 (2014) 155-159.
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[27] F.J. Ruiz, S. Rubio, D. Perez-Bendito, Water-induced coacervation of alkyl carboxylic acid reverse micelles: phenomenon description and potential for the extraction of organic compounds Analytical Chemistry 79 (2007) 7473-7485.
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[28] A. Ballesteros-Go´mez, M.D. Sicilia, S. Rubio, Supramolecular solvents in the extraction of organic compounds, Analytica Chimica Acta 677 (2010) 108-130.
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[29] A. García-Prieto, L. Lunar, S. Rubio, D. Pérez-Bendito, Decanoic acid reverse micellebased coacervates for the microextraction of bisphenol A from canned vegetables and fruits, Analytica Chimica Acta 617 (2008) 51-58. [30] E.M. Costi, M.D. Sicilia, S. Rubio, Supramolecular solvents in solid sample microextractions: application to the determination of residues of oxolinic acid and flumequine in fish and shellfish, J. Chromatography A 1217 (2010) 1447-1454. [31] M. Moradi, Y. Yamini, F. Rezaei, E. Tahmasebi, A. Esrafili, Development of a new and environment friendly hollow fiber-supported liquid phase microextraction using vesicular aggregate-based supramolecular solvent, Analyst 137 (2012) 3549-3557. [32] F. Rezaei, Y. Yamini, M. Moradi, B. Ebrahimpour, Solid phase extraction as a cleanup step before microextraction of diclofenac and mefenamic acid using nanostructured solvent, Talanta 105 (2013) 173-178. [33] G.E.P. Box, K.B. Wilson, On the experimental attainment of optimum conditions, J. the Royal Statistical Society: Series B 13 (1951) 1-45. 15 Page 15 of 22
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[34] B. Muir, S. Quick, B.J. Slater, D.B. Cooper, M.C. Moran, C.M. Timperley, W.A. Carrick, C.K. Burnell, Analysis of chemical warfare agents: II. Use of thiols and statistical experimental design for the trace level determination of vesicant compounds in air samples, J. Chromatography A 1068 (2005) 315-326.
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[35] P. Billot, B. Pitard, Taguchi design experiments for optimizing the gas chromatographic analysis of residual solvents in bulk pharmaceuticals, J. Chromatography 623 (1992) 305-313.
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[36] Y. Yamini, A. Saleh, M. Khajeh, Orthogonal array design for the optimization of supercritical carbon dioxide extraction of platinum (IV) and rhenium (VII) from a solid matrix using cyanex 301, Separation and Purification Technology 61 (2008) 109-114.
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[37] E. Tahmasebi, Y. Yamini, A. Saleh, Extraction of trace amounts of pioglitazone as an antidiabetic drug with hollow fiber liquid phase microextraction and determination by highperformance liquid chromatography-ultraviolet detection in biological fluids, J. Chromatography B 877 (2009) 1923-1929.
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[38] Y. Yamini, M. Tayyebi, M. Moradi, A. Vatanarac, Solubility of megestrol acetate and levonorgestrel in supercritical carbon dioxide, Thermochimica Acta 569 (2013) 48–54.
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Fig. 1 TEM micrographs of supramolecular solvent produced from water-induced coacervation of DeA reverse micelle. Fig. 2 Three-dimensional representation of the response surfaces where (A) pH and DeA amount (mg), (B) concentration of sodium chloride (%w/v) and DeA amount (mg), (C) pH and THF volume (mL), and (D) THF volume (mL) and DeA amount (mg). Fig. 3 HPLC-UV chromatograms of blood sample after MSPD-SFE-SUPRAS: (a) non-spiked, (b) 1 mg kg−1 spiked with the target analytes. Table 1
Structure of the drugs used and their physicochemical properties
16 Page 16 of 22
Structure
MW (g/mol)
LeV
C21H22O2
312.5
MA
C24H32O4
384.5
b
Tm (K)
Log P
479 ± 1
3.5
4.0
Molecular weight. Melting temperature.
M
b
488 ± 1
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a
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Formula
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a
Name
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Table 2 Experimental factors, levels, and analysis of variance (ANOVA) table for response surface quadratic model
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Symbol
Levels (-
)
(-1)
(0)
(+1)
(+
4
5
60
70
5
6
10
15
20
F -value
p-value
A
1
2
3
DeA amount (mg)
B
30
40
50
Volume of THF (mL)
C
2
3
4
Ionic strength (% w/v NaCl)
D
0
5
Source
df
Mean squares
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4.93E+06
14
3.19E+05
13.4
< 0.0001
A-pH
31554.38
1
31554.38
1.2
0.2906
B-DeA
2.02E+05
1
4.81E+05
7.66
0.0144
C-THF
3.87E+05
1
3.87E+05
14.71
0.0016
D-NaCl
1.53E+06
1
1.53E+06
58.01
< 0.0001
AB
1.95E+05
1
1.95E+05
7.4
0.0158
91870.65
1
91870.65
3.49
0.0812
2.30E+05
1
2.30E+05
8.76
0.0097
1.14E+05
1
1.14E+05
4.34
0.0548
59441.01
1
59441.01
2.26
0.1534
1.86E+05
1
1.86E+05
7.08
0.0178
A2
6.57E+05
1
5.55E+05
24.98
0.0002
B2
5.81E+05
1
1.89E+05
22.09
0.0003
C2
3.47E+05
1
2.74E+05
13.2
0.0025
D2
1.61E+05
1
1.13E+05
6.13
0.0257
Residual
3.94E+05
15
47720.09
Lack of fit
3.21E+05
10
18 64213.13
2.18
0.2021
Pure error
73670
5
14734
13.4
AD BC BD CD
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AC
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Model
M
Sum of squares
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pH
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Factors
)
Significant
Not significant Page 18 of 22
Temperature (K)
Pressure (bar)
Modifier volume (µL)
1
318
180
0
2
318
240
25
3
318
300
50
4
318
330
5
328
180
6
328
240
7
328
300
8
328
9
338
10
338
12 13 14 15 16
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5
Dynamic time (min) 20
30
15
40
75
20
50
75
10
40
50
5
50
25
20
20
330
0
15
30
180
25
15
50
240
0
20
40
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M
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10
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11
Static time (min)
cr
No.
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Table 3 OA16 (45) experimental design for optimization of MSPD-SFE of LeV and MA.
338
300
75
5
30
338
330
50
10
20
348
1800
50
20
30
348
240
75
15
20
348
300
0
10
50
348
330
25
5
40
Table 4. ANOVA results for optimization of MSPD-SUPRAS of LeV and MA.
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DOFa
Sum of squares
Variance
F-ratiob
Pure sum of squares
PC%c
Temperature (K)
3
10185621.57
3395207.19
213.466
10137906.36
21.854
Pressure (bar)
3
5792680.56
1930893.52
121.401
5744965.35
12.384
Methanol volume (µL)
3
279785.313
93261.771
3467.876
279704.634
40.276
Static time (min)
3
142232.8
47410.933
1762.943
142152.121
20.469
Dynamic time (min)
3
25722.073
8574.024
318.819
25641.394
3.692
Error
32
860.576
15905.069
Total
47
694454.001
Fcritical (3, 32; 0.05) = 3.24.
c
Percent contribution.
Analyte
cr
Precisiona (RSD%, n=3)
Linearity R2
LOD (mg kg-1)
LeV
0.5-7.0
0.9983
0.2
MA
0.5-7.0
0.9989
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d
LDR (mg kg-1)
0.1
Inter-day
Intra-day
ER (%)
9.8
7.1
39
10.2
6.7
47
Data were calculated based on the extraction of 1.0 mg kg-1 of LeV and MA.
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a
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b
100
an
Degrees of freedom.
0.184
M
a
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Factor
Table 5 Figures of merit for the MSPD-SFE-SUPRAS of LeV and MA. Table 6 Results obtained from analysis of LeV and MA in blood samples.
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Blood B+
Initial concentration (mg kg-1)
ndc
nd
RRa %
91
94
RSD (n = 3)
6.9
7.1
RRb %
94
RSD (n = 3)
6.3
Initial concentration (mg kg-1)
nd
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MA
90
cr
Blood O+
LeV
us
Sample
RRa %
RRb % RSD (n = 3) 1 mg kg-1 of LeV and MA were spiked to blood samples.
b
2 mg kg-1 of LeV and MA were spiked to blood samples.
95
6.4
6.8
90
93
7.2
6.8
te
Highlight
d
M
a
nd
98
an
RSD (n = 3)
6.7
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Matrix solid phased dispersion combined with supercritical fluid extraction (MSPD-SFE). MSPD-SFE followed by supramolecular solvents microextraction (SUPRAS). MSPD-SFE-SUPRAS was applied for extraction of levonorgestrel and megestrol acetate. The extracted analytes were determined by HPLC-UV. Developed technique has great potential for determination of LeV and MA in blood samples.
Supercritical fluid extraction followed by supramolecular solvents microextraction has been developed for extraction and determination of levonorgestrel and megestrol acetate in blood samples.
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S0896-8446(15)30150-9 http://dx.doi.org/doi:10.1016/j.supflu.2015.10.005 SUPFLU 3474
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
2-8-2015 6-10-2015 6-10-2015
Please cite this article as: F. Rezaei, Y. Yamini, H. Aseiabi, S. Seidi, M. Rezazadeh, Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples, The Journal of Supercritical Fluids (2015), http://dx.doi.org/10.1016/j.supflu.2015.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Supercritical fluid extraction followed by nanostructured supramolecular solvent extraction for extraction of levonorgestrel and megestrol from whole blood samples
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Fatemeh Rezaeia, Yadollah Yaminia*[email protected], Hamid Aseiabia, Shahram Seidib, Maryam Rezazadeha
Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
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Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran. Iran
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* Corresponding author. Tel: +98-21-82883417, fax: +98-21-88006544.
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Abstract
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Supercritical fluid extraction (SFE) followed by supramolecular solvents microextraction (SUPRAS) has been developed for extraction and determination of levonorgestrel (LeV) and megestrol acetate (MA) in blood samples. LeV and MA were employed as model compounds to assess the extraction procedure and were determined by high performance liquid chromatography coupled with ultraviolet detection. SUPRAS is a nano-structured liquid, generated from the amphiphiles through a sequential self-assembly process occurring on two scales; molecular and nano. SUPRAS tests were generated from solutions of reverse micelles of decanoic acid (DeA) in tetrahydrofuran (THF) by addition of water, which acted as the coacervating agent. In SFE-SUPRAS procedure, the blood sample were mixed with anhydrous sodium sulfate and loaded into SFE extraction vessel and extraction was performed in a predetermined time. The DeA solution and SFE (THF) collecting solvent were immediately injected into water for SUPRAS formation. The effective parameters on the SUPRAS efficiency were studied and optimized utilizing rotatable central composite design (RCCD). The Taguchi orthogonal array (OAD) experimental design with an OA16 (45) matrix was employed to optimize the SFE conditions. The calibration plots were linear in the range of 0.5–7.0 mg kg−1 and the limits of detection (LODs) were 0.1 and 0.2 mg kg−1 for MA and LeV, respectively. Analysis of drugs in different blood samples showed that the improved technique has great potential for extraction and determination of LeV and MA in blood samples.
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Keywords
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Supercritical fluid extraction; Nanostructured solvents; Levonorgestrel; Megestrol acetate; Blood samples
1. Introduction
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Todays, emergence of new diseases and so new pharmaceutical compounds and environmental pollutants is a serious threat to the living beings. Therefore, timely and precise determination of trace amounts of different analytes has become an important issue and received major attention from the research community around the world.
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Levonorgestrel is a synthetic female contraceptive hormone used in pregnancy prevention in humans. Although it has been widely used in the world, some deleterious reproductive effects including the turbulence of menstruation and increasing rate of galactophore cancer have been reported as the side effects [1-3]. Megestrol acetate is a type of progestin, which is used in treatment of advanced endometrial cancer and breast cancer. It is also used as an appetite enhancer and to treat weight loss in pediatric and patients with malignancies, cystic fibrosis, and HIV/AIDS [4, 5]. Determination of concentration of these drugs in whole blood may be useful to adjust the expected effective dose and so clinical side effects.
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Since analytes of environmental or biological origin are usually found in complex matrices that disturb the separation and data analysis, a series of steps are required to remove the interfering substances, preconcentrate the analytes, and increase the sensitivity. As a consequence, the research trends have been directed to development of new sample preparation techniques that are faster, simpler, inexpensive, miniaturized, and more environmental friendly. Whole blood is one of the most complicated biological fluids and extraction and clean-up of trace amounts of different analytes in it are the bottle-neck in many analytical procedures. Matrix solid phased dispersion (MSPD) is based on dispersion of sample onto an adsorbent followed by preliminary purification and elution of analytes with a relatively small solvent volume. From the first introduction of MSPD in 1989 by Barker, it has found increasing acceptance in trace analysis of organic compounds using chromatographic techniques due to providing sample homogenization, extraction, and clean up in one step [6-8]. Until now, MSPD in combination with chromatography methods has been successfully applied for determination of different analytes in various biological samples [9-20].
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However, MSPD procedure generally needs longer analytical time and its limit of detection (LOD) is limited [21]. Also, owing to the fact that sorbents are nonselective, further purification of the extracts is often still required to remove coextractants before further analysis. Shen et al. reported combination of MSPD with accelerated solvent extraction (ASE) from soil samples and fish muscle for determination of OCP residues [9, 10]. However, the consumption of hazardous organic solvents in ASE is relatively high, the final extract needs solvent evaporation; and extraction of other matrix interferences is probable, which reduces higher purification of extract.
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In the case of non-polar compounds and complicated matrices such as whole blood, simultaneous application of MSPD with supercritical fluid extraction (SFE) is an intelligent combination of separate MSPD and SFE providing the advantages of both techniques and creating considerable clean extracts. The considerable cleanup of extracts may be attributed to the non-polar nature of carbon dioxide in SFE that only provides the extraction ability of non-polar compounds. In offline SFE, extracts are often collected into one milliliter volumes of a proper solvent, which dilutes the analytes. So, application of a fast and simple preconcentration step instead of solvent evaporation reduces extraction time and loss of the extracts during evaporation step, and increases safety and sensitivity of further analysis. SFE has been combined with different preconcentration techniques such as dispersive liquid-liquid extraction (DLLME) and vesicular based-supramolecular solvent [22-24].
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Supramolecular solvents (SUPRASs) constitute environment friendly alternatives to molecular organic ones for analytical extractions [25, 26]. In 2007, Ruiz et al. [27] developed a supramolecular solvent (SUPRAS) microextraction technique based on analyte partitioning between an alkyl carboxylic acid-based nanostructured solvent and a bulk aqueous sample. The SUPRASs, which are water-immiscible liquids consisting of reverse micelle aggregates of nanoscale dimensions dispersed in a continuous phase (including tetrahydrofuran (THF) and water), have properties exceptionally useful for extraction processes, derived from the particular structure of their supramolecular assemblies [28-30]. A two-step process is needed for SUPRAS formation. First, amphiphilic molecules spontaneously form three-dimensional aggregates above a critical aggregation concentration. Then, the generated nanostructures self-assemble in larger aggregates by the action of an external stimulus and separate from the bulk solution as an immiscible liquid by a phenomenon named coacervation [31]. The objective of this work is optimization and validation of a method based on combination of MSPD with SFE followed by a preconcentration step using a nanostructured supramolecular solvent, which is made up of reverse micelles of decanoic acid (DeA), dispersed in a continuous phase of THF/water for determination of levonorgestrel and megestrol in whole blood samples. Response surface methodology (RSM) was used for optimization of different parameters 3 Page 3 of 22
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affecting the extraction efficiency. To the best of our knowledge, there is no report about the combination of MSPD with SFE. 2. Experimental
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2.1. Chemicals and reagents
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Standards of levonorgestrel (LeV) and megestrol acetate (MA) were kindly donated from the Department of Medical Sciences of Tehran University (Tehran, Iran). Structure and properties of drugs are provided in Table 1. THF was supplied by Merck (Darmstadt, Germany). Decanoic acid was obtained from Fluka (Buchs, Switzerland). Sodium sulfate and other reagents were of analytical grade and obtained from Merck. The ultra-pure water was prepared by a model Aqua Max-Ultra Youngling ultra-pure water purification system (Dongan-gu, South Korea). HPLCgrade methanol and acetonitrile were purchased from Duksan (Gyeonggi-do, South Korea). Microliter syringes (25-500 µL) were purchased from Hamilton (Bonaduz, Switzerland).
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2.2. Apparatus
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Stock standard solutions (1000 µg mL−1) of the analytes were prepared by dissolving a proper amount of each drug in methanol. Mixtures of standard working solutions were prepared by dilution of the stock solutions with ultra-pure water.
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Droplet size and morphology of the prepared SUPRAS were determined by a transmission electron microscope (TEM) model EM10C (Zeiss, Germany) operating at 80 kV accelerated voltage. A Suprex (Pittsburgh, PA, USA) SFE/SFC instrument model MPS/225 in SFE mode was utilized for all extractions. Extractions were accomplished using a 1-mL volume stainless steel extraction vessel. A manual adjustable restrictor from ISCO (USA) was used in the SFE instrument to collect the extracted analytes in a suitable solvent. To prevent sample plugging, the temperatures of body and restrictor point was adjusted at 90 and 95 ºC, respectively. Chromatographic separations were performed with a HPLC instrument including a Varian 9012 HPLC pump (Walnut Creek, CA, USA), a six-port Cheminert HPLC valve from Valco (Houston, TX, USA) with a 20-µL sample loop and equipped with a Varian 9050 UV–Vis detector. Chromatographic data were recorded and analyzed using ChromanaCH software, version 3.6.4 (Tehran, Iran). An ODS-3 column (150 mm × 4.6 mm, with 5-µm particle size) from MZ-Analysentechnik (Mainz, Germany) was applied to separate MA and LeV under isocratic elution conditions. A mixture of ultra-pure water and acetonitrile (30:70) for 10 min and
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100% acetonitrile for 5 min at a flow rate of 1 mL min−1 were used as the mobile phase and the analytes were detected at 257 nm. 2.3. Sample preparation
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To evaluate applicability of the developed extraction method, two blood samples (blood group B+ and O+) were obtained from the Iranian Blood Transfusion Organization (Tehran, Iran). The blood samples were poured in a porcelain mortar containing anhydrous sodium sulfate, and the mixture was blended until an apparently dry material was obtained. Afterwards, the samples were extracted using SFE–SUPRAS procedure. The extracted analytes were analyzed by HPLCUV.
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2.4. MSPD-SFE – SUPRAS procedure
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Half milliliter of blood samples was poured in a porcelain mortar containing 1 g of anhydrous sodium sulfate, and the mixture was blended with the pestle for a few minutes until an apparently dry material was obtained. The homogeneous mixture was placed in the SFE extraction vessel and spiked with 50 µL of standard solution (100 mg L-1), then another filter was placed on the top of the vessel and the vessel was closed. Finally, SFE was carried out using a combination of static extractions to enhance the sample-solvent contact, and thus a better penetration of the fluid in the matrix, followed by the dynamic extraction step, in which the supercritical fluid passed continuously through the extraction chamber. Extractions were conducted under the following conditions: 15 min static extraction, 40 min dynamic extraction at 240 bar and 55ºC, a CO2 flowrate of 0.5 mL min-1 and temperatures of restrictor body and tip equal to 90 and 95ºC, respectively. The extracted analytes were collected in THF (3.0 mL), which was located in a 5.0mL volumetric flask. To increase the collection efficiency and decrease the solvent evaporation, the volumetric flask was placed in an ice bath during the dynamic extraction time. Afterwards, 40 mg of DeA was added to 3.0 mL of SFE collecting solvent (THF). Then, an aliquot of 40.0 mL ultra-pure water with pH value adjusted at 2.5 (by dropwise addition of HCl (0.1 mol L-1)) was poured into a 50-mL homemade centrifuge tube, which is designed for collection of low density organic solvents. The solution that was obtained from SFE step was quickly injected into the aqueous solution using a 5-mL gastight syringe from Hamilton. The SUPRAS, made up of reverse micelles of DeA dispersed in THF:water, spontaneously formed and separated from the THF:water solution as an immiscible liquid. The mixture was centrifuged at 5000 rpm for 5 min to accelerate complete separation of the two immiscible liquids. The coacervate, located at the top of the glass tube, was withdrawn using a microsyringe and 20 µL of it was injected into the HPLC instrument for subsequent analysis. 3. Results and Discussion 5 Page 5 of 22
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In this paper, the SFE was used to extract drugs directly from the MSPD. The MSPD-SFE method integrates the relative merits of MSPD and SFE to achieve selective determination of analytes.
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The blood is disrupted and dispersed thoroughly with the shearing force and hydrophobic– hydrophilic characteristic of adsorbents during MSPD process. This is while after the SFE process, the matrix components are retained by adsorbent while the analytes are extracted by SFE.
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The most serious problem of off-line SFE methods is evaporation of the collecting solvent at the end of extraction to acquire high preconcentration factor. However, this procedure is a timeconsuming step and contaminates the environment and collected analytes may be lost or degraded in this step. Supramolecular solvent microextraction is an extraction method that consumes less organic solvent and very high enrichment factors can be obtained using this method. Combination of MSPD-SFE and SUPRAS was applied for extraction and determination of LeV and MA in blood samples.
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3.1.1. Solvent composition
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3.1. Supramolecular solvent microextraction
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The reverse micelle-based supramolecular solvent applied in this research is spontaneously formed in ternary mixtures of DeA, water, and THF at well-defined proportions. Its formation occurs through two sequential self-assembly processes. First, DeA molecules aggregate as reverse micelles in THF and then, upon the addition of water, they rearrange into larger reverse micelles that separate from the bulk solution as an immiscible liquid via a mechanism that remains elusive. The immiscible liquid is made up of reverse micelles, THF, and a little amount of water. The excellent dissolution properties of reverse micelles and the low volume of the coacervates obtained make them very attractive to be used in analytical extractions. In order to set up efficient extraction schemes, it is important to understand the intermolecular forces driving the extraction process. Hydrocarbon chains of DeA molecules forming aggregates in these SUPRASs extend into and are surrounded by the THF, while their carboxylic groups are solvated by water in the interior of the aggregates. DeA reverse micelles provide a two-fold mechanism for substrate solubilization, namely hydrophobic interactions in the surfactant tails at the micellar surface and hydrogen bonds in the polar head groups at the micellar core [27, 32]. Consequently, the expected driving forces for the extraction were van der Waals interactions between the hydrocarbon chains of the DeA and the drugs aromatic framework, and hydrogen bonds on account of the acceptor and donor groups of the analytes molecules.
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3.1.2. Characterization of the nano-structured solvent
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3.1.3. Optimization of supramolecular solvent microextraction
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The shape and size of the aggregates of DeA were investigated by TEM (Fig. 1). The target SUPRASs were formed by injecting a mixture containing 200 mg DeA and 10 mL THF into 30 mL ultra-pure water (pH = 2), then sonication of the tube for 5 min. As can be seen, the DeA aggregates were nearly spherical in shape with an average diameter of about 10–20 nm, and they tended to aggregate to larger particles.
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The optimized response value for one factor is often influenced by other factors, which may impose substantial impacts on the determination signal. These factor interactions can only be measured by multivariate optimization strategies such as factorial designs. Response surface methodology (RSM) is effective for responses that are influenced by many factors as well as by their interactions and was originally described by Box and Wilson [33]. In this work, rotatable central composite design (RCCD) was utilized to achieve the optimal conditions. The software package Design-Expert 8.0.6 trial version (Stat-Ease Inc., MN, USA) was used for experimental design, data analysis, and response surfaces.
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CCD is a second-order model correlating the response function with the independent factors; so that the amount of response can be predicted at any point within the factor domain even if the point has not been included in the design. The model takes the following general form for independent variables [34]:
(1)
where Y is the process response or output (dependent variable), k is the number of the patterns, i and j are the index numbers for pattern, β0 is the offset term, βi is the linear effect, βii is the squared effect, βij is the interaction effect, and Xi and Xj are the levels of the independent variables. With four factors and six center points, totally 30 experiments had to be run for five-level investigation of variables in rotatable CCD. The total number of experimental runs (N), suggested by RCCD, is obtained by the following equation:
wherein f is the number of variables and
(2) is the number of center points. The independent
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variables were pH (A), DeA weight (B, mg), THF volume (C, mL), and ionic strength (D, % w/v NaCl). The sum of peak areas for each run was selected as the response objective for the study. The model was described as follows:
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(3)
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Equation 3 relates the response to the coded amount of the variables in the range of −2 to 2. To this end, the high and low levels of each variable were considered 2 and −2, respectively. Therefore, the response could be predicted in each level of variables.
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Analysis of variance (ANOVA) was performed, which showed that the model was significant (Table 4). Also, the “lack of fit” was not significant (p = 0.05), which implied that the model fitted the data.
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The ANOVA result showed that the F value of 6.69 is significant and there is only 0.01% likelihood that large model F value could occur by chance. The response equation fitted the experimental data with R2-value of 0.9260, indicating 92.60% variability in the response. The goodness-of-fit of the model to the experimental data shown in Fig. 2 has an adjusted R2 = 0.8569.
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For graphical interpretation of the interactions, the use of three-dimensional plots (3D) of the model is highly recommended. This is helpful for visualizing the relationship between the responses and the experimental levels of each factor. These plots were obtained for a given pair of factors, while maintaining the other factors fixed at their optimal values (Fig. 2). The pH value of the sample is a significant factor that may affect the extraction recovery of the analytes as well as the state of DeA, which plays an essential role in the stability of reverse micelle in aqueous solutions. Acidification of the sample is usually required to have the neutral forms of these compounds and thus increasing recoveries. Also, the coacervation phenomenon occurs from protonated alkyl carboxylic acids; so, extractions must be carried out at pH values below 4 [26]. At higher pH values, solubilization of deprotonated DeA molecules in the waterTHF phase in equilibrium with the SUPRAS occurred and that resulted in reduction of formed SUPRAS volume. Therefore, the effect of pH on microextraction of analytes was studied in the pH range of 1–5. As can be seen in Fig. 2A, the best extraction efficiency of the analytes was obtained at pH 2.5. Addition of salt is widely used in microextraction techniques to improve the partitioning of analytes into the organic through the salting out effect. This is while it was reported that addition 8 Page 8 of 22
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of NaCl decreases the efficiency of dispersion of extraction phase and mass transfer in aqueous medium, leading to a decrease in extraction efficiency. To investigate the influence of salt addition on performance of SUPRASF extraction, various experiments were performed by adding different amounts of NaCl (0–20% w/v) to the sample solution. It was found that the peak area increased with the increase of ionic strength in the range of 0% -5.0% and then decreased at higher values (Fig. 2B).
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SUPRAS-to-sample phase volume ratios were a function of the concentrations of DeA and THF, which are both major components of this solvent. According to Fig. 2C, the peak area increases first and then decreases by increasing the volume of THF for both analytes. Since at a low volume of THF, a reverse micelle phase could not be well formed; therefore, it resulted in a low recovery rate. When the THF volume increased from 2 to 3 mL, the extraction efficiency increased due to the improved dispersion procedure and when the THF volume increased from 3 to 6 mL, the extraction efficiency decreased. This type of dependence indicated that progressively more THF was incorporated into the solvent as the percentage of THF in the solution increased and consequently, the reverse micelles became more and more diluted.
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DeA weight affects producing reverse micelle and volume of extraction phase, thus influencing the extraction efficiency. The effect of DeA weight on the extraction efficiency was studied in the range of 30-70 mg. As the obtained results show, the peak areas of the analyte were increased by increasing DeA amount to 40 mg and then decreased because of increasing DeA weight, increase in the coacerva volume, and decrease in the concentration of analytes. Accordingly, weight of DeA was adjusted at 40 mg for further studies. According to the overall results of the optimization study, the following experimental conditions were chosen: THF volume, 3.0 mL; pH, 2.5; DeA weight, 40.0 mg, and concentration of sodium chloride, 5.0% (w/v). 3.2. Optimization of matrix solid phased dispersion-supercritical fluid extraction SFE, a sustainable green technology, has led to a wide range of applications in the past decades. Like many other processes, SFE is sometimes criticized for its large number of factors that need to be properly adjusted before every single run. Experimental design and proper statistical analysis with small number of trials in adjusting the SFE parameters has become popular in this regard. Taguchi methods have been widely used to optimize the reaction variables by formulating the minimum number of experiments. This approach helps to identify the influence of individual factors and establish the relationship between variables and operational conditions [35, 36]. The Taguchi experimental method, an orthogonal array design, was applied to study the possible influence on the performance of the MSPD-SFE method of five factors (each factor at four levels); namely temperature, pressure, static time, dynamic time, and the modifier (methanol) volume. Sixteen experiments were performed to estimate the best conditions for the SFE of MA and LeV (Table 3). ANOVA was performed for the experimental data obtained. 9 Page 9 of 22
(4)
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Table 4 presents the ANOVA results for the effects of investigated parameters. The ANOVA results showed that all factors were statistically significant at P< 0.05. Furthermore, from the percentage contribution (Table 4), it can be deduced that the most important factor contributing to the extraction efficiency was the modifier volume (methanol, 40.276%). Further experiments were performed under the optimal conditions and the results showed that under the optimal conditions obtained from the OA16 (45) matrix, the recoveries were similar to the optimal performance calculated using the following expression:
where T is the grand total of all results, N is the total number of the results, Aopt is the
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performance under optimal conditions; and , , , , and are the average performances of the dynamic time, static time, temperature, pressure, and modifier volume at those optimal levels, respectively. Based on the above equation, under the optimal conditions, the performance is estimated using only the significant factors (all the factors in this study) [37].
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Under the optimal conditions, the confidence interval (CI) of the performance is calculated using the following expression:
(5)
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where F (1, n2) is the F-value from the F-table at a required confidence level at the degrees of freedom (DOFs) of 1 and of error, n2; Ne is the effective number of replications, and Ve is the variance of error term from ANOVA. The effect of pressure on the results obtained from MSPD-SFE – SUPRAS of drugs was examined in the range of 180-360 bar. These results indicated that increasing pressure from 180 to 240 bar enhanced the extraction recovery, which was due to increased supercritical carbon dioxide (SC-CO2) density at higher pressures. However, above 240 bar, an increase in pressure leads to a reduction in the extraction efficiency, which can probably be attributed to reduced diffusion rates of the extracted materials from the sample matrix to the supercritical fluid environment. Therefore, 240 bar was selected as the optimum pressure in the subsequent studies. Temperature is an important variable for the SFE extraction. The effect of temperature on the results achieved from MSPD-SFE–SUPRAS was scrutinized in the range of 318-348 K. The extraction depends on a complex balance between the supercritical carbon dioxide density and vapor pressure of drugs. As the temperature increases, the extraction efficiency increases drastically up to 328 K. This arises from enhancement of vapor pressure of the compounds at high temperature. However, above 328 K, the extraction efficiency decreases, due to the density 10 Page 10 of 22
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diminution of supercritical fluid at high temperatures. Therefore, 328 K was selected as the optimum temperature in the following studies. The similar temperature and pressure behavior has been reported previously [23]. The effect of temperature on the extraction efficiency of the drugs may be discussed by its effect on the solute solubility around crossover region too [38].
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Time is one of the main factors for exhausted extraction and is an important index for evaluation of extraction efficiency. Shorter extraction time could cause incomplete extraction and longer extraction time could be time and solvent wasting. In order to obtain high yield of analytes, a primary extraction step in the static mode was performed. This step was followed by a dynamic extraction to enhance solubility of analytes in the supercritical fluid. In this study, the effects of static extraction period on the results achieved from SFE-SUPRAS of drugs were scrutinized at four levels; 5, 10, 15, and 20 min. The results exhibited that by increasing the static time from 5 to 15 min, the extraction efficiency can be increased effectively. For this reason, a static time of 15 min was applied. This step would lead to better penetration of the fluid into the matrix compared with the only dynamic extraction mode. The effect of dynamic extraction time was studied at four levels; 20, 30, 40, and 50 min. By increasing the dynamic time from 10 to 40 min, the extraction efficiency increased. Nevertheless, above 40 min, an increase in the dynamic time led to a reduction in the collection efficiency of the analytes. Long dynamic extraction times may cause unwanted physical movement of matrix components to the trapping device as well as loss of analytes from the trap due to being blown off from the trap and volatilization of the trapping solvent, which was not beneficial to increase the extraction recovery. Hence, dynamic extraction time of 40 min was selected for subsequent experiments. The extraction efficiency of compounds of interest can be enhanced significantly by addition of a polar modifier to supercritical CO2. Moreover, modifier content was demonstrated to be a crucial factor that affects the recovery of polar compounds. Effect of modifier volume (methanol) was studied at four levels in the range of 0-75 µL. The results showed that by increasing the modifier volume from 0 to 50 µL, extraction efficiencies for the analytes increased. In the presence of methanol, solubility of the analytes in CO2 can be increased. However, by increasing the methanol volume from 50 to 75 µL, a reduction in the extraction efficiency of the drugs can be obtained. Hence, 50 µL of the modifier was added into the samples in subsequent experiments. 3.3. Quantitative analysis The calibration curves obtained under optimized conditions are summarized in Table 5. Linear dynamic ranges varied in the range of 0.5-7.0 mg kg-1 for LeV and MA. Coefficients of determinations (R2) ranged from 0.9983 to 0.9989. Intraday (n = 3) and interday standard deviations were calculated by extracting the drugs from blood samples at 1.0 mg kg-1 level. Intraand interday RSDs% lower than 7.1% and 10.2%, were obtained respectively. The LODs, based 11 Page 11 of 22
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on S/N of 3, ranged between 0.1 to 0.2 mg kg-1 for two drugs and the ER% obtained were in the range of 39-47% for LeV and MA. 3.4. Analysis of blood samples
4. Conclusions
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In order to evaluate applicability of the developed extraction method for analysis of LeV and MA in real samples, two blood samples were selected and the drugs were extracted using the proposed method under the optimal conditions. Sample preparation for real samples was performed according to Section 2.4. The blood samples were analyzed by HPLC-UV after SFE– SUPRAS procedure. A recovery study was carried out by spiking the samples at concentration levels of 1.0 and 2.0 mg kg-1. Each treatment was done in triplicates, and the mean results are shown in Table 6. The relative recoveries of the two drugs from the spiked samples ranged from 90% to 98% with a precision (RSDs %) of 6.3–7.2%. The results revealed that the blood matrices had little effect on the MSPD-SFE–SUPRAS procedure. Fig. 3 illustrates the typical chromatograms of the extracted drugs from blood sample (group B+) before and after spiking with the drugs at 1.0 mg kg-1 level. The Lev and MA peaks were eluted around 8 and 10 min from the column respectively.
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te
Supramolecular solvents have outstanding properties for microextraction. They combine the capability of solubilizing solutes in a wide polarity range with the ability to achieve high enrichment factors, mainly arising from the mixed-mode mechanisms and multiple binding sites they can provide. In this research, combination of MSPD with SFE followed by supramolecular solvent extraction was proposed as valuable tools for extraction of LeV and MA from blood samples. The proposed method is environmental friendly, precise, reproducible, and linear over a broad concentration range. Besides, very small matrix effects were observed when the proposed MSPD-SFE-SUPRAS technique was applied to blood samples spiked with the analytes. Also, after completion of SFE procedure, the extra steps (vaporization of large volume of toxic organic solvent, which is time-consuming and has inappropriate environmental behavior) needed before final analysis were eliminated. The proposed method possesses great potential in analysis of trace organic compounds in real solid samples.
References [1] S.L. Camp, D.S. Wilkerson, T.R. Raine, The benefits and risks of over-the-counter availability of levonorgestrel emergency contraception, Contraception 68 (2003) 309-317.
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[2] T. Backman, Benefit-risk assessment of the levonorgestrel intrauterine system in contraception, Drug Safety 27 (2004) 1185-1204. [3] T.G. Watson, B.J. Stewart, A sensitive direct radioimmunoassay for assessing D-norgestrel levels in human plasma, Annals of Clinical Biochemistry 25 (1988) 280-287.
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[4] H. Adlercreutz, P.B. Eriksen, M.S. Christensen, Plasma concentrations of megestrol acetate and medroxyprogesterone acetate after single oral administration to healthy subjects. J. Pharmaceutical and Biomedical Analysis 1 (1983) 153-162.
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[5] M. Samei, A. Vatanara, Sh. Fatemi, A.R. Najafabadi, Process variables in the formation of nanoparticles of megestrol acetate through rapid expansion of supercritical CO2. J. Supercritical Fluids 70 (2012) 1-7.
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[6] S.A. Barker, A.R. Long, C.R. Short, Isolation of drug residues from tissues by solid phase dispersion, J. Chromatography 475 (1989) 353-361.
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[7] Y.K. Li, P. Fan, Y.R. Xu, Y. Huang, Q.F. Hu, Study on determination of triterpenoids in chaenomeles by high performance liquid chromatography and sample preparation with matrix solid phase dispersion, J. Chinese Chemical Society 55 (2008) 1332-1337.
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[8] M. García-López, P. Canosa, I. Rodríguez, Trends and recent applications of matrix solidphase dispersion, Analytical and Bioanalytical Chemistry 391 (2008) 963–974.
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[9] Z.L. Shen, D. Yuan, H. Zhang, M. Hu, J. H. Zhu, X.Q. Zhang, Q. D. Suc, Matrix solid phase dispersion-accelerated solvent extraction for determination of OCP residues in fish muscles, J. Chinese Chemical Society 58 (2011) 494-502. [10] Z.L. Shen, J.B. Cai, Y. Gao, X.L. Zhu, Q.D. Su, Matrix solid phase dispersion-accelerated solvent extraction for determination of OCP residues in fish muscles, Chinese Journal of Analytical Chemistry 33 (2005) 1318-1320. [11] L. Guo, M. Guan, C. Zhao, H. Zhang, Molecularly imprinted matrix solid-phase dispersion for extraction of chloramphenicol in fish tissues coupled with high-performance liquid chromatography determination, Analytical and Bioanalytical Chemistry 392 (2008) 1431-1438. [12] Y. Zhang, X. Xu, H. Liu, Y. Zhai, Y. Sun, S. Sun, H. Zhang, A. Yu, Y. Wang, Matrix solidphase dispersion extraction of sulfonamides from blood, J. Chromatographic Science 50 (2012) 131-136. [13] S. Bogialli, R. Curini, A. Di Corcia, M. Nazzari, M. L. Polci, Rapid confirmatory assay for determining 12 sulfonamide antimicrobials in milk and eggs by matrix solid-phase dispersion 13 Page 13 of 22
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and liquid chromatography-mass spectrometry, J. Agriculture and Food Chemistry 51 (2003) 4225-4232.
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[14] E.M. Kristenson, E.G.J. Haverkate, C.J. Slooten, L. Ramos, R.J.J. Vreuls, U.A.T. Brinkman, Suitability of several carbon sorbents for the fractionation of various sub-groups of toxic polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, J. Chromatography A 917 (2001) 277-286.
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[15] S.A. Barker, Matrix solid phase dispersion, J. Biochemical and Biophysical Methods 70 (2007) 151-162.
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[16] S.A. Barker, Matrix solid-phase dispersion, J. Chromatography A 885 (2000) 115-127.
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[17] J.J. Ramos, R. Rial-Otero, L. Ramos, J.L. Capelo, Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits, J. Chromatography A 1212 (2008) 145-149.
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[18] E. Sobhanzadeh, N.K. Abu Bakar, M.R. Bin Abas, K. Nemati, Low temperature followed by matrix solid-phase dispersion-sonication procedure for the determination of multiclass pesticides in palm oil using LC-TOF-MS, J. Hazardous Materials186 (2011) 1308-1313.
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[19] Y. Moliner-Martinez, P. Campíns-Falcó, C. Molins-Legua, L. Segovia-Martínez, A. SecoTorrecillas, Miniaturized matrix solid phase dispersion procedure and solid phase microextraction for the analysis of organochlorinated pesticides and polybrominated diphenylethers in biota samples by gas chromatography electron capture detection, J. Chromatography A 1216 (2009) 6741-6745. [20] S.A. Rodrigues, S. S. Caldas, E. G. Primel, A simple; efficient and environmentally friendly method for the extraction of pesticides from onion by matrix solid-phase dispersion with liquid chromatography-tandem mass spectrometric detection Analytica Chimica Acta 678 (2010) 8289. [21] N. Furusawa, A toxic reagent-free method for normal-phase matrix solid-phase dispersion extraction and reversed-phase liquid chromatographic determination of aldrin, dieldrin, and DDTs in animal fats, Analytical and Bioanalytical Chemistry 378 (2004) 2004-2007. [22] M.H. Naeeni, Y.Yamini, M. Rezaee, Combination of supercritical fluid extraction with dispersive liquid-liquid microextraction for extraction of organophosphorus pesticides from soil and marine sediment samples, J. Supercritical Fluids 57 (2011) 219-226.
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[23] M. Rezaee, Y. Yamini, M. Moradi, A. Saleh, M. Faraji, M.H. Naeeni, Supercritical fluid extraction combined with dispersive liquid–liquid microextraction as a sensitive and efficient sample preparation method for determination of organic compounds in solid samples, J. Supercritical Fluids 55 (2010) 161-168.
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[24] F. Rezaei, Y. Yamini, M. Moradi, H. Asiabi, Determination of diphenylamine residue in fruit samples by supercritical fluid extraction followed by vesicular based-supramolecular solvent microextraction, J. Supercritical Fluids 100 (2015) 79-85. [25] F. Rezaei, Y. Yamini, M. Moradi, B. Daraei, Supramolecular solvent-based hollow fiber liquid phase microextraction of benzodiazepines, Analytica Chimica Acta 804 (2013) 135-142.
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[26] F. Rezaei, Y. Yamini, M. Moradi, A comparison between emulsification of reverse micellebased supramolecular solvent and solidification of vesicle-based supramolecular solvent for the microextraction of triazines, J. Chromatography A 1327 (2014) 155-159.
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[27] F.J. Ruiz, S. Rubio, D. Perez-Bendito, Water-induced coacervation of alkyl carboxylic acid reverse micelles: phenomenon description and potential for the extraction of organic compounds Analytical Chemistry 79 (2007) 7473-7485.
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[28] A. Ballesteros-Go´mez, M.D. Sicilia, S. Rubio, Supramolecular solvents in the extraction of organic compounds, Analytica Chimica Acta 677 (2010) 108-130.
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[29] A. García-Prieto, L. Lunar, S. Rubio, D. Pérez-Bendito, Decanoic acid reverse micellebased coacervates for the microextraction of bisphenol A from canned vegetables and fruits, Analytica Chimica Acta 617 (2008) 51-58. [30] E.M. Costi, M.D. Sicilia, S. Rubio, Supramolecular solvents in solid sample microextractions: application to the determination of residues of oxolinic acid and flumequine in fish and shellfish, J. Chromatography A 1217 (2010) 1447-1454. [31] M. Moradi, Y. Yamini, F. Rezaei, E. Tahmasebi, A. Esrafili, Development of a new and environment friendly hollow fiber-supported liquid phase microextraction using vesicular aggregate-based supramolecular solvent, Analyst 137 (2012) 3549-3557. [32] F. Rezaei, Y. Yamini, M. Moradi, B. Ebrahimpour, Solid phase extraction as a cleanup step before microextraction of diclofenac and mefenamic acid using nanostructured solvent, Talanta 105 (2013) 173-178. [33] G.E.P. Box, K.B. Wilson, On the experimental attainment of optimum conditions, J. the Royal Statistical Society: Series B 13 (1951) 1-45. 15 Page 15 of 22
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[34] B. Muir, S. Quick, B.J. Slater, D.B. Cooper, M.C. Moran, C.M. Timperley, W.A. Carrick, C.K. Burnell, Analysis of chemical warfare agents: II. Use of thiols and statistical experimental design for the trace level determination of vesicant compounds in air samples, J. Chromatography A 1068 (2005) 315-326.
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[35] P. Billot, B. Pitard, Taguchi design experiments for optimizing the gas chromatographic analysis of residual solvents in bulk pharmaceuticals, J. Chromatography 623 (1992) 305-313.
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[36] Y. Yamini, A. Saleh, M. Khajeh, Orthogonal array design for the optimization of supercritical carbon dioxide extraction of platinum (IV) and rhenium (VII) from a solid matrix using cyanex 301, Separation and Purification Technology 61 (2008) 109-114.
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[37] E. Tahmasebi, Y. Yamini, A. Saleh, Extraction of trace amounts of pioglitazone as an antidiabetic drug with hollow fiber liquid phase microextraction and determination by highperformance liquid chromatography-ultraviolet detection in biological fluids, J. Chromatography B 877 (2009) 1923-1929.
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[38] Y. Yamini, M. Tayyebi, M. Moradi, A. Vatanarac, Solubility of megestrol acetate and levonorgestrel in supercritical carbon dioxide, Thermochimica Acta 569 (2013) 48–54.
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Fig. 1 TEM micrographs of supramolecular solvent produced from water-induced coacervation of DeA reverse micelle. Fig. 2 Three-dimensional representation of the response surfaces where (A) pH and DeA amount (mg), (B) concentration of sodium chloride (%w/v) and DeA amount (mg), (C) pH and THF volume (mL), and (D) THF volume (mL) and DeA amount (mg). Fig. 3 HPLC-UV chromatograms of blood sample after MSPD-SFE-SUPRAS: (a) non-spiked, (b) 1 mg kg−1 spiked with the target analytes. Table 1
Structure of the drugs used and their physicochemical properties
16 Page 16 of 22
Structure
MW (g/mol)
LeV
C21H22O2
312.5
MA
C24H32O4
384.5
b
Tm (K)
Log P
479 ± 1
3.5
4.0
Molecular weight. Melting temperature.
M
b
488 ± 1
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a
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Formula
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a
Name
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Table 2 Experimental factors, levels, and analysis of variance (ANOVA) table for response surface quadratic model
17 Page 17 of 22
Symbol
Levels (-
)
(-1)
(0)
(+1)
(+
4
5
60
70
5
6
10
15
20
F -value
p-value
A
1
2
3
DeA amount (mg)
B
30
40
50
Volume of THF (mL)
C
2
3
4
Ionic strength (% w/v NaCl)
D
0
5
Source
df
Mean squares
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4.93E+06
14
3.19E+05
13.4
< 0.0001
A-pH
31554.38
1
31554.38
1.2
0.2906
B-DeA
2.02E+05
1
4.81E+05
7.66
0.0144
C-THF
3.87E+05
1
3.87E+05
14.71
0.0016
D-NaCl
1.53E+06
1
1.53E+06
58.01
< 0.0001
AB
1.95E+05
1
1.95E+05
7.4
0.0158
91870.65
1
91870.65
3.49
0.0812
2.30E+05
1
2.30E+05
8.76
0.0097
1.14E+05
1
1.14E+05
4.34
0.0548
59441.01
1
59441.01
2.26
0.1534
1.86E+05
1
1.86E+05
7.08
0.0178
A2
6.57E+05
1
5.55E+05
24.98
0.0002
B2
5.81E+05
1
1.89E+05
22.09
0.0003
C2
3.47E+05
1
2.74E+05
13.2
0.0025
D2
1.61E+05
1
1.13E+05
6.13
0.0257
Residual
3.94E+05
15
47720.09
Lack of fit
3.21E+05
10
18 64213.13
2.18
0.2021
Pure error
73670
5
14734
13.4
AD BC BD CD
te
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AC
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Model
M
Sum of squares
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pH
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Factors
)
Significant
Not significant Page 18 of 22
Temperature (K)
Pressure (bar)
Modifier volume (µL)
1
318
180
0
2
318
240
25
3
318
300
50
4
318
330
5
328
180
6
328
240
7
328
300
8
328
9
338
10
338
12 13 14 15 16
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5
Dynamic time (min) 20
30
15
40
75
20
50
75
10
40
50
5
50
25
20
20
330
0
15
30
180
25
15
50
240
0
20
40
te
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an
10
Ac ce p
11
Static time (min)
cr
No.
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Table 3 OA16 (45) experimental design for optimization of MSPD-SFE of LeV and MA.
338
300
75
5
30
338
330
50
10
20
348
1800
50
20
30
348
240
75
15
20
348
300
0
10
50
348
330
25
5
40
Table 4. ANOVA results for optimization of MSPD-SUPRAS of LeV and MA.
19 Page 19 of 22
DOFa
Sum of squares
Variance
F-ratiob
Pure sum of squares
PC%c
Temperature (K)
3
10185621.57
3395207.19
213.466
10137906.36
21.854
Pressure (bar)
3
5792680.56
1930893.52
121.401
5744965.35
12.384
Methanol volume (µL)
3
279785.313
93261.771
3467.876
279704.634
40.276
Static time (min)
3
142232.8
47410.933
1762.943
142152.121
20.469
Dynamic time (min)
3
25722.073
8574.024
318.819
25641.394
3.692
Error
32
860.576
15905.069
Total
47
694454.001
Fcritical (3, 32; 0.05) = 3.24.
c
Percent contribution.
Analyte
cr
Precisiona (RSD%, n=3)
Linearity R2
LOD (mg kg-1)
LeV
0.5-7.0
0.9983
0.2
MA
0.5-7.0
0.9989
te
d
LDR (mg kg-1)
0.1
Inter-day
Intra-day
ER (%)
9.8
7.1
39
10.2
6.7
47
Data were calculated based on the extraction of 1.0 mg kg-1 of LeV and MA.
Ac ce p
a
us
b
100
an
Degrees of freedom.
0.184
M
a
ip t
Factor
Table 5 Figures of merit for the MSPD-SFE-SUPRAS of LeV and MA. Table 6 Results obtained from analysis of LeV and MA in blood samples.
20 Page 20 of 22
Blood B+
Initial concentration (mg kg-1)
ndc
nd
RRa %
91
94
RSD (n = 3)
6.9
7.1
RRb %
94
RSD (n = 3)
6.3
Initial concentration (mg kg-1)
nd
ip t
MA
90
cr
Blood O+
LeV
us
Sample
RRa %
RRb % RSD (n = 3) 1 mg kg-1 of LeV and MA were spiked to blood samples.
b
2 mg kg-1 of LeV and MA were spiked to blood samples.
95
6.4
6.8
90
93
7.2
6.8
te
Highlight
d
M
a
nd
98
an
RSD (n = 3)
6.7
Ac ce p
Matrix solid phased dispersion combined with supercritical fluid extraction (MSPD-SFE). MSPD-SFE followed by supramolecular solvents microextraction (SUPRAS). MSPD-SFE-SUPRAS was applied for extraction of levonorgestrel and megestrol acetate. The extracted analytes were determined by HPLC-UV. Developed technique has great potential for determination of LeV and MA in blood samples.
Supercritical fluid extraction followed by supramolecular solvents microextraction has been developed for extraction and determination of levonorgestrel and megestrol acetate in blood samples.
21 Page 21 of 22
22
Page 22 of 22
d
te
Ac ce p us
an
M
cr
ip t