- Email: [email protected]
In situ decomposition of deep eutectic solvent as a novel approach in liquid-liquid microextraction
Analytica Chimica Acta 1065 (2019) 49e55
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
In situ decomposition of deep eutectic solvent as a novel approach in liquid-liquid microextraction b, Christina Vakh a, Juraj Kucha r b, Andra s Simon c, Andrey Shishov a, *, Renata Chroma Vasil Andruch b, Andrey Bulatov a a b c
Institute of Chemistry, Saint Petersburg State University, RU-198504, Saint Petersburg, Russia rik, SK-04154, Kosice, Slovakia Institute of Chemistry, University of P.J. Safa Department of General and Analytical Chemistry, Budapest University of Technology and Economics, H-1111, Budapest, Hungary
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Simple and efficient approach for sample pretreatment. Liquid-liquid microextraction based on deep eutectic solvent decomposition. Deep eutectic solvent decomposition investigation. In situ dispersion of organic phase and extraction of hydrophobic analyte(s). Preconcentration of 17b-estradiol using proposed approach.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2018 Received in revised form 11 March 2019 Accepted 17 March 2019 Available online 20 March 2019
In this study, a novel approach for effective liquid-liquid microextraction based on deep eutectic solvent (DES) decomposition was suggested for the first time. It was established that DESs synthesized from tetrabutylammonium bromide and long-chain alcohols decomposed in aqueous phase resulting in in situ dispersion of organic phase and extraction of hydrophobic analyte(s). It this process long-chain alcohol acted as an extraction solvent and tetrabutylammonium bromide acted as a dispersive agent and promoted mass transfer between aqueous and organic phases as a salting out agent. Phenomenon of DES decomposition was studied in detail and applied for separation and preconcentration in chemical analysis for the first time. The developed approach was applied for 17b-estradiol microextraction from transdermal gel samples as a proof-of-concept example. The results showed that the in situ dispersed organic phase obtained can provide efficient extraction of 17b-estradiol with good extraction recovery (95 ± 5%) and excellent reproducibility (6%). The reported approach proves to be fast, simple, and inexpensive. © 2019 Elsevier B.V. All rights reserved.
Keywords: Liquid-liquid microextraction Deep eutectic solvent In situ organic phase formation 17b-estradiol
1. Introduction
* Corresponding author. E-mail address: [email protected] (A. Shishov). https://doi.org/10.1016/j.aca.2019.03.038 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Deep eutectic solvents (DESs) [1] can be considered as a green alternative to conventional extraction solvents for separation and preconcentration of analytes from variety of matrices. The eutectic mixture is generally composed of hydrogen bond acceptor and
50
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
hydrogen bond donor. Therefore, DESs are a class of solvents composed of a mixture that forms a eutectic with a much lower melting point than either of the individual components [2]. They are characterized by new favorable features. Among them, we can mention chemical stability, a wide range of viscosity, negligible volatility at room temperature and simple preparation. Due to these characteristics they have attracted interest in analytical chemistry as effective extraction solvents, where research of their application is still at the beginning [3e7]. Application of water-miscible, hydrophilic DESs as extraction solvents in analytical chemistry is associated with certain difficulties since the addition of DESs to the aqueous samples results in a homogeneous mixture. Therefore, addition of an emulsifier solvent (usually tetrahydrofuran) is necessary to obtain a cloudy solution, followed by centrifugation to separate the DES phase containing analytes [8]. Another way to overcome this limitation, is an application of hydrophobic, water-insoluble DESs [9] based on fatty acids or long-chain alcohols as a hydrogen bond donor. In a number of works it has been shown that such DESs are stable in aqueous solutions and can be used to extract various analytes from aqueous media [10,11]. At the same time, DESs with at least one water-soluble component are not stable in water and their decomposition is observed [12]. Therefore, the question of the usability of hydrophobic DESs for the analysis of aqueous media remains open. In this study we established that DESs synthesized from tetrabutylammonium bromide (TBABr) and long-chain alcohols are decomposed in aqueous phase resulting in in situ dispersed organic phase formation. Reagents formed during this process, long-chain alcohol can act as an extraction solvent and on the other hand TBABr acts as a dispersive agent and can promote mass transfer between aqueous and organic phases as a salting out agent. Obviously, this feature can open new opportunities for separation and preconcentration of target hydrophobic analytes from various matrixes. To the best of our knowledge, this phenomenon has not been presented in literature. To demonstrate the efficiency of the suggested approach, the strategy was applied to 17b-estradiol (E2) (Fig. S1) microextraction from transdermal gel samples followed by its determination based on the high-performance liquid chromatography with UV detection (HPLC-UV) as a proof-of-concept example. E2 is a type of steroidal estrogen that is widely used in gynecology for hormone therapy as well in hormone replacement therapy [13,14]. 2. Experimental 2.1. Reagents and solutions All chemicals and reagents were of analytical grade. The steroid hormone E2 was purchased from Sigma-Aldrich (Germany). Other chemicals were purchased from various suppliers: heptanol, toluene-4-sulfonic acid (Sigma-Aldrich, Germany), TBABr, decanol, amylalcohol and isopropyl alcohol (Vekton, Russia), octanol, dodecanol (Reachim, Russia), acetone (Ekos 1, Russia), acetonitrile (Biochem Chemopharma, France) and methanol (J.T. Baker, Netherlands). The stock solution of E2 at a concentration level of 1 g L1 was prepared by dissolving the reagent in methanol and the obtained solution was stored in a dark place at 5 C and used within 1 month. The working solution was prepared just before the experiment by dilution of the stock solution with deionized water. Ultra-pure water from Millipore Milli-Q RG system (Millipore, USA) was used throughout the work. The DESs were prepared by mixing of various long-chain alcohols and TBABr in various molar ratios under heating and stirring at 80 C until a clear liquid was formed.
2.2. Apparatus For characterization of DESs and understanding of the extraction mechanism, various techniques were applied including, Fouriertransform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), gas chromatography with mass spectrometric detection (GC-MS), HPLC-UV and Karl Fischer titration. FTIR spectra were recorded by an IR Affinity-1 spectrometer with Shimadzu own software (Shimadzu, Japan). 1H NMR spectra were measured using a Bruker Avance 400 spectrometer in deuterated dimethyl sulfoxide (DMSO‑d6) at 25.0 C with Bruker Fusion-SV for Windows. A Simultaneous Thermal Analyzer STA 449 F3 Jupiter (Netzsch, Germany) equipped with aluminum DSC sample pans (TA Instruments, Germany) with Proteus® software was used for the DSC study. An 831 KF Coulometer (Metrohm, Switzerland) was used for the determination of water in DES and organic phase obtained after extraction. A GC-MS-QP2010 Ultra gas chromatograph mass spectrometer (Shimadzu, Japan) with GCMSsolution Ver. 2.6 software was used for the determination of heptanol in DES and organic phase obtained after extraction. The GC-MS system was fitted with a SPB624 MS capillary column (6% cyanopropyl phenyl and 94% dimethyl polysiloxane, 30 m, 0.25 mm i.d., 0.25 mm coating). A HPLC system LC-20 Prominence (Shimadzu, Japan) with Clarity Chromatography Software equipped with UV-Vis detector was used for the TBABr and E2 determination. Separations were carried out in a reversed-phase mode using Luna C18 column (150 4 mm, 5 mm particles size) (Phenomenex). 2.3. Sample pretreatment Two different transdermal gels Divigel (containing E2, carbopol 974P, triethanolamine, propylene glycol, ethanol, water) and Oestrogel (containing E2, carbopol 980, triethanolamine, ethanol, water) were purchased from a local pharmacy (St. Petersburg, Russia). A 50 mg of the gel sample was accurately weighted in a 2 mL conical tube and diluted with 1.5 mL of water. The tube was manually shaken for 1 min and the obtained solution was subsequently used for the microextraction procedure. 2.4. Microextraction procedure 1.5 mL of sample solution was rapidly injected by a syringe into a 5 mL Eppendorf polypropylene tube containing 250 mL of DES (TBABr and heptanol at a 1:2 M ratio) (Fig. 1). During the aqueous phase injection, the DES was decomposed and obtained organic phase was dispersed into aqueous phase resulting extraction of analyte (Please see video file in electronic Supplementary materials for details). There after the tube was centrifuged for 3 min at 5000 rpm. Afterwards, 75 mL of top organic phase was easily withdrawn using an automatic pipette, transferred to another clean and dry test tube and diluted with 50 mL of methanol to reduce the viscosity. The resulting solution was directly injected into the HPLC-UV system. Supplementary video related to this article can be found at https://doi.org/10.1016/j.aca.2019.03.038. 2.5. GC-MS procedure For the determination of heptanol the injector temperature was kept at 220 C while the interface temperature and the ion source were 200 C. Helium was used as a carrier gas at a flow rate of 2 mL min1. The sample volume of 1 mL was injected in splitless mode with 1 min of purge time. The electron ionization source was run at 70 eV. A library search using NIST, NBS and Wiley GC-MS
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
51
Fig. 1. Schematic of liquid-liquid microextraction based on in situ decomposition of deep eutectic solvent.
libraries was carried out for the identification. 2.6. HPLC-UV procedure for TBABr determination For the determination of TBABr, a mixture of acetonitrile and 5$103 mol L1 of toluene-4-sulfonic acid in a ratio of 65:35 (v/v) at flow rate of 1 mL min1 was used as mobile phase [15]. The absorbance measurement was carried out at 254 nm. The temperature of column was set at 40 C. The volume of injected sample was 20 mL. 2.7. HPLC-UV procedure for E2 determination For the determination of E2, the mobile phase consisted of water and acetonitrile at a 55:45 (v/v) ratio. The total flow rate of the isocratic gradient module was maintained at 1 mL min1. The temperature of column was set at 40 C; the injection volume was 20 mL, and the detection wavelength was 280 nm. The total run time was 8 min and the retention time of E2 was 6.1 min. 2.8. Reference procedure The method reported earlier by Du et al. [16] with several minor modifications was used as the reference method. Briefly, 0.5 mL of methanol containing of 100 mL of C2H2Cl4 was rapidly injected into a sample solution prepared according to the procedure described in the Sample pretreatment section. The mixture was gently shaken for 1 min and then centrifuged for 2 min at 4000 rpm. The sedimented phase was evaporated to near-dryness under reduced pressure and dissolved in 100 mL of methanol, and obtained solution was taken for HPLC-UV analysis. 3. Results and discussion 3.1. Preliminary considerations The microextraction procedure based on DES decomposition
involved several steps. The first step included injection of the aqueous sample phase into the DES phase using a syringe (Fig. 1). This reverse injection sequence was chosen because the DESs are viscous liquids and their aspiration into the syringe is difficult. During aqueous sample injection instantaneous and spontaneous decomposition of the DES was observed resulting in in situ dispersion of organic phase and extraction of target hydrophobic analyte into extraction solvent droplets. Finally, after centrifugation the upper phase was collected. Undoubtedly, that the composition of the DES may affect its stability in aqueous phase. Usually, DESs are obtained by the formation of hydrogen bond between the hydrogen bond acceptor agent and the functional group of hydrogen bond donor agent. Several hydrophobic DESs synthesized from TBABr as the hydrogen bond acceptor and long-chain alcohols (amyl alcohol, heptanol, octanol, decanol and dodecanol) as the hydrogen bond donor in molar ratio of 1:2 were studied for E2 microextraction. It was established, that all DESs studied were decomposed into the aqueous phase resulting long-chain alcohols phase dispersion. However, the obtained extraction efficiency depended on the hydrogen bond donor type. The higher extraction efficiency and better repeatability (n ¼ 3) was obtained for the DES based on TBABr and heptanol at a 1:2 M ratio (Fig. 2). This fact can be explained by decreasing of the long-chain alcohols solubility in water. On the one hand amyl alcohol has higher solubility and as result analyte solubility in aqueous phase is increased. On the other hand, dispersion efficiency of organic phase is reduced with decreasing solubility of the alcohol resulting poor extraction efficiency. 3.2. Investigation of the DES structure Based on preliminary studies the DESs synthesized from TBABr and heptanol at various molar ratio was chosen for future experiments. FTIR spectra were recorded and DSC investigations were carried out for confirmation of the DESs formation. The FTIR spectra revealed several absorption bands according to the composition of the DESs at various molar ratio of TBABr and
52
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
Fig. 2. Effect of DES composition (volume of aqueous phase, 1.5 mL; concentration of 17b-estradiol, 10 mg L1; volume of DES phase, 250 mL).
heptanol (1:1, 1:2, and 1:3, respectively). Several absorption bands connected to the methyl and methylene groups of both compounds were identified. The dominant peaks in the region 2960e2858 cm1 arising from (CeH) stretching vibration, existence of the above mentioned groups corroborates the broader band with the minimum at 1467 cm1 dedicated to the eCH2e deformation vibration and also bands of the eCH3 group's deformation vibration that were found at 1458 cm1 and 1380 cm1, respectively. Skeletal vibrations of e(CH2)4e group in heptanol was visible by bands about 740 cm1. As it was evident from Fig. S2 the intensity varied according to the ratio between TBABr and heptanol. The peaks about 3350 cm1 indicated stretching vibrations of the OeH group of the primary alcohol. However, broader character hid the shift due to interaction of the OeH$$$Br type as it was found in mixture of water and TBABr [17]. Absorption bands of n(CeN) asymmetrical and symmetrical stretching vibrations in the region 1059e882 cm1 confirmed the TBABr. Unfortunately, the IR spectroscopy cannot clearly confirm the association between heptanol and TBABr as the differences between the IR spectra of each components and mixtures were insignificant. As it is mentioned above the difference was found mainly in intensity of the absorption bands. Differential scanning calorimetry investigations of the DES (TBABr and heptanol at a 1:2 M ratio) synthesized and initial components were also performed in the range from 30 C to 110 C at a rate of 5 C min1. In the thermograms (Fig. S3), the peaks corresponding to TBABr and heptanol melting were observed, yielding melting temperatures of 105 C and 34 C, respectively. The position of the peak in the graph of the heattemperature dependence confirmed a significant decrease in the melting point value of the mixture as compared with its precursors. The lowering of melting temperatures 10 C was observed for the DESs. From the thermograms obtained, it can be concluded that the thermal behavior of DES was different from the behavior of the individual components.
GC-MS. It was established that 85 ± 2% (m/m) of the original TBABr was dissolved into the aqueous phase. In the aqueous phase dissociation of TBABr was observed and a peak of TBAþ was presented on the chromatogram (Fig. S4). The concentrations of heptanol in organic phase was found to be 78 ± 2% (m/m) (Fig. S5). Moreover, the obtained organic phase was analyzed by the KarlFisher method, and it was established that it contains 7.01 ± 0.07% of water. Thus, the extraction mechanism was associated with the DES decomposition as a consequence of TBABr dissolution into aqueous sample phase and heptanol hydration. From this point of view TBABr can be considered as dispersive agent as in conventional dispersive liquid-liquid microextraction (DLMME), where dispersive solvent is used for organic phase dispersion. However, TBABr as quaternary ammonium salt containing bromide ion can also act as salting out agent and promote mass transfer between the two phases. Both DES before extraction (TAQ1) and top phase after the extraction (TAQ2) were also studied by 1H NMR (Please see electronic Supplementary materials for details). The results obtained are in good agreement with the results obtained by the GC-MS method. To demonstrate above mentioned features, the developed approach was compared with the DLMME procedure using the mixture of heptanol and various polar water-miscible solvents. For this purpose, 250 mL of extraction mixture containing of heptanol and various dispersive solvents (isopropyl alcohol, methanol, acetone or acetonitrile) in ratio 2:1 v/v was injected into 1.5 mL of aqueous analyte solution (10 mg L1) by the syringe. After centrifugation organic phase was analyzed by HPLC-UV. It was shown (Fig. 3), that extraction efficiency obtained for DES was higher than extraction efficiency for the mixtures of heptanol with dispersive solvents studied. The result obtained confirmed fact, that polar organic dispersive solvents increased solubility of E2 in an aqueous phase.
3.3. Extraction mechanism investigation
3.4.1. Effect of DES volume The volume of the DES phase may result in preconcentration or dilution of the extracted analyte. On the one hand the volume of the DES phase should be as small as possible to achieve high enrichment factor. On the other hand, the use small volume of the DES phase can give rise to poor reproducibility. The effect of the DES volume was investigated in the range from 200 to 600 mL. Each measurement was performed three times. By increasing the DES volume, the dilution effect predominated and the analytical signal was decreased (Fig. S6). On the other hand, it is too difficult to withdraw an extraction phase obtained if the applied DES volume
The TBABr is water-soluble component of the DES and it was assumed that DES decomposition was associated with TBABr dissolution into aqueous phase while heptanol was responsible for formation of dispersed organic phase. To determine TBABr and heptanol concentration after extraction 1.5 mL of aqueous phase was rapidly injected by the syringe into the 5 mL polypropylene tube containing 250 mL of DES (TBABr and heptanol at a 1:2 M ratio). After centrifugation the aqueous phase was analyzed by HPLC-UV and the organic phase was analyzed by
3.4. Optimization of extraction procedure
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
53
Fig. 3. Effect of dispersive solvent type (volume of aqueous phase, 1.5 mL; concentration of 17b-estradiol, 10 mg L1).
was less than 250 mL. Therefore, a DES volume of 250 mL was selected for further experiments. In this case 100 ± 5 mL of organic phase was separated. 3.4.2. Effect of sample volume The sample volume can influence on the extraction efficiency and enrichment factor (EF). The sample volume was varied from 0.5 to 2.0 mL, where by a volume of 1.5 mL was found to provide the best results in both analytical signal and reproducibility. Therefore, a 1.5 mL sample volume was chosen. The estimated EF was 15. The EF value was estimated as the ratio between the analyte concentration in the separated organic phase (Csop) and the initial concentration of analyte (C0) in the sample phase: EF ¼ Csop/C0. Extraction recovery (ER) value was also calculated as ER ¼ (Csop∙Vsop/C0∙V0) 100%, were Csop and C0 are analyte concentrations in the separated organic phase and in the initial sample phase, respectively; Vsop and V0 are volumes of separated organic phase and sample, respectively. The estimated ER was (95 ± 5) %. 3.5. Quality assurance/quality control To ensure an adequate level of quality assurance and a quality control of measurements, the developed procedure was utilized for the determination of E2 in transdermal gel samples. To obtain calibration curve, eight analyte standard solutions were prepared via the proposed microextraction procedure and the final analysis was performed by HPLC-UV. The results obtained showed that the curve obtained was linear in the range of 0.5e100 mg L1. The regression equation was Y ¼ 53113 C (where Y corresponds to the peak area and C means concentration of E2 expressed in mg L1), the coefficient of determination was 0.9954.
To determine the limit of detection (LOD) of the proposed procedure, initially, a blank sample was analyzed. The LOD was determined based on a signal-to-noise ratio (S/N) of 3. Also, the limit of quantification (LOQ) was determined based on S/N ¼ 10. On this basis, the LOD level was 0.15 mg L1, and the LOQ value was found to be 0.5 mg L1. To evaluate the precision of the developed procedure, the repeatability of the peak areas obtained was investigated for five replicate microextractions and the deionized water sample spiked at a 10 mg L1 level. This parameter was expressed as the relative standard deviation (RSD) and was provided for both the intra- and inter-day precisions. As it can be seen in Table 1S, the relative standard deviations obtained were below 8.07%. To assess the extraction recovery and enrichment factor, the equations presented in section 3.4.2 were applied. Based on the results obtained, the extraction recovery was (95 ± 5) %, and enrichment factor in the value 15 was achieved. To investigate the accuracy of the procedure, the relative recoveries obtained from analysis of real samples spiked with known amounts of E2 under investigation at different concentration levels were calculated. Spiking levels were chosen regarding real content levels and method quantification limits of 15, 30, and 45 mg L1 for transdermal gel 1 (Divigel) and 10, 20, and 30 mg L1 for transdermal gel 2 (Oestrogel). The obtained results are summarized in Table 1. The results obtained showed that the different matrices used for samples had no significant effect on the microextraction efficiency and obtaining high relative recoveries (from 89 to 105%) approved this fact. The mentioned samples were analyzed using reference procedure described in Section 2.8. As it can be seen from Table 1, the analytical results agreed well with the results obtained with reference method [16]. F-values 19.2 indicated insignificant
Table 1 The results of determination of 17b-estradiol (mg L1) in transdermal gel samples (n ¼ 3, P ¼ 0.95, Fk ¼ 19.2, tk ¼ 2.78). Sample
Added
Developed procedure
Reference procedure
F-test
t-test
Recovery, %
1
0 15 30 45 0 10 20 30
36.3 ± 5.8 49.7 ± 6.6 63.4 ± 3.4 77.8 ± 8.5 20.0 ± 2.8 30.6 ± 1.3 39.8 ± 2.5 48.6 ± 4.9
35.0 ± 6.5 48.7 ± 9.1 62.9 ± 10.1 75.1 ± 11.4 21.9 ± 3.3 32.4 ± 8.4 46.6 ± 7.9 52.8 ± 3.4
0.79 0.53 0.11 0.55 0.73 0.03 0.14 2.10
0.29 0.18 0.11 0.38 0.83 0.59 1.94 1.16
89 90 92 105 99 95
2
54
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
Table 2 Comparison of the proposed procedure with those described in the literature. Sample
Detection
Sample pretreatment
LOD, mg L1
RSD, %
Recovery,%
Total analysis time, min
Reference
Gel Tablets Tablets Gel
HPLC-UV HPLC-FLD DPV HPLC-UV
Dissolution Dissolution Dissolution Dissolution
0.01 0.01 5.5 0.15
1.94 1.89 0.99 6
99.6 101.6 100.5 90e101
26 22 15 (Dissolution) 12
19 20 21 This work
in in in in
20 mL of acetonitrile 75 mL of methanol supporting electrolyte 1.5 mL of water and LLME (250 mL of DES)
HPLC-UV e high-performance liquid chromatography with ultraviolet detection, HPLC-FLD e high-performance liquid chromatography with fluorescence detection, DPV e differential pulse voltammetry, LLME e liquideliquid microextraction.
difference in precision between both methods at the 95% confidence level. t-values 2.78 indicated insignificant difference between the results obtained using these methods (n ¼ 3). The chromatogram of transdermal gel sample (Divigel) is shown in Fig. S7.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.03.038. References
3.6. Comparison of the developed procedure with other procedures Table 2 shows figures of merit for the proposed procedure and those of other procedures reported for the determination of E2 in pharmaceutical samples. As it can be seen, LOD of the proposed procedure is comparable with those reported for the other procedures. The proposed procedure is characterized by lower consumption of organic solvents and therefore offers low waste production. Moreover, the total analysis time is less in comparison with the developed procedures based on HPLC-UV [19], HPLC-FLD [20] and DPV [21]. 4. Conclusion A really effective liquid-liquid microextraction approach based on deep eutectic solvent decomposition was suggested for the first time. Above all, it was revealed and confirmed that extraction mechanism was associated with the DES decomposition as a consequence of TBABr dissolution into aqueous sample phase and heptanol hydration. This methodology can be considered as a mode of dispersive liquid-liquid microextraction. It is known, that the addition of dispersive solvents in conventional DLLME can decrease the partition coefficients of some analytes into the extraction solvents [18]. Unlike conventional DLLME, in the developed procedure the hydrogen bond acceptor (TBABr) during DES dissolution and decomposition on the one hand acted as the dispersive agent, on the other hand it promoted mass transfer between aqueous and organic phases as a salting out agent. As a result, two simultaneously occurring effects (organic phase dispersion and salting out effect) provided efficient extraction of E2 with good extraction recovery (95 ± 5%), excellent reproducibility (6%) and short time (5 min). Comparative studies have indicated that the developed approach is a reliable analytical tool with wide potential applications in analysis of various sample matrixes and it has the opportunity to be coupled with other instrumental methods. Acknowledgements This work was supported by the Russian Foundation for Basic Research (project no. 18-33-20004) and by Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic (VEGA 1/0010/15). Scientific research was performed using the equipment of the Research Park of St. Petersburg State University (Chemical Analysis and Materials Research Centre, Centre for Magnetic Resonance, Centre of Thermal Analysis and Calorimetry and the Chemistry Educational Centre of Research Park of St. Petersburg State University).
[1] A.P. Abbott, D. Boothby, G. Capper, D.L. Davies, R.K. Rasheed, Deep Eutectic Solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids, J. Am. Chem. Soc. 126 (2004) 9142e9147, https:// doi.org/10.1021/ja048266j. [2] B. Tang, K.H. Row, Recent developments in deep eutectic solvents in chemical sciences, Monatshefte Fur Chemie 144 (2013) 1427e1454, https://doi.org/ 10.1007/s00706-013-1050-3. [3] A. Shishov, A. Bulatov, M. Locatelli, S. Carradori, V. Andruch, Application of deep eutectic solvents in analytical chemistry, in: A review, Microchemical Journal, vol. 135, 2017, pp. 33e38, https://doi.org/10.1016/j.microc.2017.07. 015. [4] L. Nugbienyo, A. Shishov, S. Garmonov, L. Moskvin, V. Andruch, A. Bulatov, Flow method based on liquid-liquid extraction using deep eutectic solvent for the spectrofluorimetric determination of procainamide in human saliva, Talanta 168 (2017) 307e312. https://doi.org/10.1039/c3ay40284c. [5] S.C. Cunha, J.O. Fernandes, Extraction techniques with deep eutectic solvents, Trac. Trends Anal. Chem. 105 (2018) 225e239, https://doi.org/10.1016/ j.trac.2018.05.001. [6] A. Shishov, N. Volodina, D. Nechaeva, S. Gagarinova, A. Bulatov, Deep eutectic solvents as a new kind of dispersive solvent for dispersive liquid-liquid microextraction, RSC Adv. 8 (2018) 38146e38149, https://doi.org/10.1039/ c8ra07300g. [7] A.Y. Shishov, M.V. Chislov, D.V. Nechaeva, L.N. Moskvin, A.V. Bulatov, A new approach for microextraction of non-steroidal anti-inflammatory drugs from human urine samples based on in-situ deep eutectic mixture formation, J. Mol. Liq. 272 (2018) 738e745, https://doi.org/10.1016/j.molliq.2018.10.006. [8] A. Shishov, N. Volodina, D. Nechaeva, S. Gagarinova, A. Bulatov, An automated homogeneous liquid-liquid microextraction based on deep eutectic solvent for the HPLC-UV determination of caffeine in beverages, Microchem. J. 144 (2019) 469e473. [9] D.J.G.P. Van Osch, L.F. Zubeir, A. Van Den Bruinhorst, M.A.A. Rocha, M.C. Kroon, Hydrophobic deep eutectic solvents as water-immiscible extractants, Green Chem. 17 (2015) 4518e4521. http://doi.org/10.1039/c5gc01451d. [10] S.M. Yousefi, F. Shemirani, S.A. Ghorbanian, Hydrophobic deep eutectic solvents in developing microextraction methods based on solidification of floating drop: application to the trace HPLC/FLD determination of PAHs, Chromatographia 81 (2018) 1201e1211. http://doi.org/10.1007/s10337-0183548-7. [11] S. Zhu, J. Zhou, H. Jia, H. Zhang, Liquideliquid microextraction of synthetic pigments in beverages using a hydrophobic deep eutectic solvent, Food Chem. 243 (2018) 351e356. http://doi.org/10.1016/j.foodchem.2017.09.141. [12] A. Florindo, L.C. Branco, I.M. Marrucho, Development of hydrophobic deep eutectic solvents for extraction of pesticides from aqueous environments, Fluid Phase Equilib. 448 (2017) 135e142. https://doi.org/10.1016/j.fluid.2017. 04.002. [13] S. Pozdniakova, M. Guitart-Mampel, G. Garrabou, G. Di Benedetto, Y. Ladilov, V. Regitz-Zagrosek, 17b-Estradiol reduces mitochondrial cAMP content and cytochrome oxidase activity in a phosphodiesterase 2-dependent manner, Br. J. Pharmacol. 175 (2018) 3876e3890, https://doi.org/10.1111/bph.14455. [14] H. Kuhl, Pharmacology of estrogens and progestogens: influence of different routes of administration, Climacteric 8 (2005) 3e63, https://doi.org/10.1080/ 13697130500148875. [15] J. Poon, A. Katsifis, M. Fulham, Alternate HPLC method for the analysis of tetrabutylammonium hydroxide in [18F]fluorodeoxythymidine (FLT), J. Liq. Chromatogr. Relat. Technol. 40 (2017) 667e670. [16] X. Du, X. Wang, Y. Li, F. Ye, Q. Dong, Ch Huang, Determination of estrone and 17b-estradiol in water samples using dispersive liquid-liquid microextraction followed by LC, Chromatographia 71 (2009) 405e410. https://doi.org/10. 1365/s10337-009-1455-7. [17] H. Kleeberg, IR-Spectroscopic investigation of the hydration of tetrabutylammonium bromide in methylene chloride, J. Solut. Chem. (1986) 169e176. https://doi.org/10.1007/BF00646288.
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55 [18] J. Regueiro, M. Llompart, C. Garcia-Jares, J.C. Garcia-Monteagudo, R. Cela, Ultrasound-assisted emulsification-microextraction of emergent contaminants and pesticides in environmental waters, J. Chromatogr. A 1190 (2008) 27e38. https://doi.org/10.1016/j.chroma.2008.02.091. , L. Matysova , J. Sícha, [19] L. Havlíkov a, L. Nov akova P. Solich, Determination of estradiol and its degradation products by liquid chromatography, J. Chromatogr. A 1119 (2006) 216e223, https://doi.org/10.1016/j.chroma.
55
2006.01.085. [20] B. Yilmaz, Y. Kadioglu, Determination of 17 b-estradiol in pharmaceutical preparation by UV spectrophotometry and high performance liquid chromatography methods, Arab. J. Chem. 10 (2017) S1422eS1428, https://doi.org/ 10.1016/j.arabjc.2013.04.018. [21] B. Salcı, I. Biryol, Voltammetric investigation of b - estradiol, J. Pharm. Biomed. Anal. 28 (2002) 753e759, https://doi.org/10.1016/S0731-7085(01)00676-8.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
In situ decomposition of deep eutectic solvent as a novel approach in liquid-liquid microextraction b, Christina Vakh a, Juraj Kucha r b, Andra s Simon c, Andrey Shishov a, *, Renata Chroma Vasil Andruch b, Andrey Bulatov a a b c
Institute of Chemistry, Saint Petersburg State University, RU-198504, Saint Petersburg, Russia rik, SK-04154, Kosice, Slovakia Institute of Chemistry, University of P.J. Safa Department of General and Analytical Chemistry, Budapest University of Technology and Economics, H-1111, Budapest, Hungary
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Simple and efficient approach for sample pretreatment. Liquid-liquid microextraction based on deep eutectic solvent decomposition. Deep eutectic solvent decomposition investigation. In situ dispersion of organic phase and extraction of hydrophobic analyte(s). Preconcentration of 17b-estradiol using proposed approach.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 November 2018 Received in revised form 11 March 2019 Accepted 17 March 2019 Available online 20 March 2019
In this study, a novel approach for effective liquid-liquid microextraction based on deep eutectic solvent (DES) decomposition was suggested for the first time. It was established that DESs synthesized from tetrabutylammonium bromide and long-chain alcohols decomposed in aqueous phase resulting in in situ dispersion of organic phase and extraction of hydrophobic analyte(s). It this process long-chain alcohol acted as an extraction solvent and tetrabutylammonium bromide acted as a dispersive agent and promoted mass transfer between aqueous and organic phases as a salting out agent. Phenomenon of DES decomposition was studied in detail and applied for separation and preconcentration in chemical analysis for the first time. The developed approach was applied for 17b-estradiol microextraction from transdermal gel samples as a proof-of-concept example. The results showed that the in situ dispersed organic phase obtained can provide efficient extraction of 17b-estradiol with good extraction recovery (95 ± 5%) and excellent reproducibility (6%). The reported approach proves to be fast, simple, and inexpensive. © 2019 Elsevier B.V. All rights reserved.
Keywords: Liquid-liquid microextraction Deep eutectic solvent In situ organic phase formation 17b-estradiol
1. Introduction
* Corresponding author. E-mail address: [email protected] (A. Shishov). https://doi.org/10.1016/j.aca.2019.03.038 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Deep eutectic solvents (DESs) [1] can be considered as a green alternative to conventional extraction solvents for separation and preconcentration of analytes from variety of matrices. The eutectic mixture is generally composed of hydrogen bond acceptor and
50
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
hydrogen bond donor. Therefore, DESs are a class of solvents composed of a mixture that forms a eutectic with a much lower melting point than either of the individual components [2]. They are characterized by new favorable features. Among them, we can mention chemical stability, a wide range of viscosity, negligible volatility at room temperature and simple preparation. Due to these characteristics they have attracted interest in analytical chemistry as effective extraction solvents, where research of their application is still at the beginning [3e7]. Application of water-miscible, hydrophilic DESs as extraction solvents in analytical chemistry is associated with certain difficulties since the addition of DESs to the aqueous samples results in a homogeneous mixture. Therefore, addition of an emulsifier solvent (usually tetrahydrofuran) is necessary to obtain a cloudy solution, followed by centrifugation to separate the DES phase containing analytes [8]. Another way to overcome this limitation, is an application of hydrophobic, water-insoluble DESs [9] based on fatty acids or long-chain alcohols as a hydrogen bond donor. In a number of works it has been shown that such DESs are stable in aqueous solutions and can be used to extract various analytes from aqueous media [10,11]. At the same time, DESs with at least one water-soluble component are not stable in water and their decomposition is observed [12]. Therefore, the question of the usability of hydrophobic DESs for the analysis of aqueous media remains open. In this study we established that DESs synthesized from tetrabutylammonium bromide (TBABr) and long-chain alcohols are decomposed in aqueous phase resulting in in situ dispersed organic phase formation. Reagents formed during this process, long-chain alcohol can act as an extraction solvent and on the other hand TBABr acts as a dispersive agent and can promote mass transfer between aqueous and organic phases as a salting out agent. Obviously, this feature can open new opportunities for separation and preconcentration of target hydrophobic analytes from various matrixes. To the best of our knowledge, this phenomenon has not been presented in literature. To demonstrate the efficiency of the suggested approach, the strategy was applied to 17b-estradiol (E2) (Fig. S1) microextraction from transdermal gel samples followed by its determination based on the high-performance liquid chromatography with UV detection (HPLC-UV) as a proof-of-concept example. E2 is a type of steroidal estrogen that is widely used in gynecology for hormone therapy as well in hormone replacement therapy [13,14]. 2. Experimental 2.1. Reagents and solutions All chemicals and reagents were of analytical grade. The steroid hormone E2 was purchased from Sigma-Aldrich (Germany). Other chemicals were purchased from various suppliers: heptanol, toluene-4-sulfonic acid (Sigma-Aldrich, Germany), TBABr, decanol, amylalcohol and isopropyl alcohol (Vekton, Russia), octanol, dodecanol (Reachim, Russia), acetone (Ekos 1, Russia), acetonitrile (Biochem Chemopharma, France) and methanol (J.T. Baker, Netherlands). The stock solution of E2 at a concentration level of 1 g L1 was prepared by dissolving the reagent in methanol and the obtained solution was stored in a dark place at 5 C and used within 1 month. The working solution was prepared just before the experiment by dilution of the stock solution with deionized water. Ultra-pure water from Millipore Milli-Q RG system (Millipore, USA) was used throughout the work. The DESs were prepared by mixing of various long-chain alcohols and TBABr in various molar ratios under heating and stirring at 80 C until a clear liquid was formed.
2.2. Apparatus For characterization of DESs and understanding of the extraction mechanism, various techniques were applied including, Fouriertransform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), gas chromatography with mass spectrometric detection (GC-MS), HPLC-UV and Karl Fischer titration. FTIR spectra were recorded by an IR Affinity-1 spectrometer with Shimadzu own software (Shimadzu, Japan). 1H NMR spectra were measured using a Bruker Avance 400 spectrometer in deuterated dimethyl sulfoxide (DMSO‑d6) at 25.0 C with Bruker Fusion-SV for Windows. A Simultaneous Thermal Analyzer STA 449 F3 Jupiter (Netzsch, Germany) equipped with aluminum DSC sample pans (TA Instruments, Germany) with Proteus® software was used for the DSC study. An 831 KF Coulometer (Metrohm, Switzerland) was used for the determination of water in DES and organic phase obtained after extraction. A GC-MS-QP2010 Ultra gas chromatograph mass spectrometer (Shimadzu, Japan) with GCMSsolution Ver. 2.6 software was used for the determination of heptanol in DES and organic phase obtained after extraction. The GC-MS system was fitted with a SPB624 MS capillary column (6% cyanopropyl phenyl and 94% dimethyl polysiloxane, 30 m, 0.25 mm i.d., 0.25 mm coating). A HPLC system LC-20 Prominence (Shimadzu, Japan) with Clarity Chromatography Software equipped with UV-Vis detector was used for the TBABr and E2 determination. Separations were carried out in a reversed-phase mode using Luna C18 column (150 4 mm, 5 mm particles size) (Phenomenex). 2.3. Sample pretreatment Two different transdermal gels Divigel (containing E2, carbopol 974P, triethanolamine, propylene glycol, ethanol, water) and Oestrogel (containing E2, carbopol 980, triethanolamine, ethanol, water) were purchased from a local pharmacy (St. Petersburg, Russia). A 50 mg of the gel sample was accurately weighted in a 2 mL conical tube and diluted with 1.5 mL of water. The tube was manually shaken for 1 min and the obtained solution was subsequently used for the microextraction procedure. 2.4. Microextraction procedure 1.5 mL of sample solution was rapidly injected by a syringe into a 5 mL Eppendorf polypropylene tube containing 250 mL of DES (TBABr and heptanol at a 1:2 M ratio) (Fig. 1). During the aqueous phase injection, the DES was decomposed and obtained organic phase was dispersed into aqueous phase resulting extraction of analyte (Please see video file in electronic Supplementary materials for details). There after the tube was centrifuged for 3 min at 5000 rpm. Afterwards, 75 mL of top organic phase was easily withdrawn using an automatic pipette, transferred to another clean and dry test tube and diluted with 50 mL of methanol to reduce the viscosity. The resulting solution was directly injected into the HPLC-UV system. Supplementary video related to this article can be found at https://doi.org/10.1016/j.aca.2019.03.038. 2.5. GC-MS procedure For the determination of heptanol the injector temperature was kept at 220 C while the interface temperature and the ion source were 200 C. Helium was used as a carrier gas at a flow rate of 2 mL min1. The sample volume of 1 mL was injected in splitless mode with 1 min of purge time. The electron ionization source was run at 70 eV. A library search using NIST, NBS and Wiley GC-MS
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
51
Fig. 1. Schematic of liquid-liquid microextraction based on in situ decomposition of deep eutectic solvent.
libraries was carried out for the identification. 2.6. HPLC-UV procedure for TBABr determination For the determination of TBABr, a mixture of acetonitrile and 5$103 mol L1 of toluene-4-sulfonic acid in a ratio of 65:35 (v/v) at flow rate of 1 mL min1 was used as mobile phase [15]. The absorbance measurement was carried out at 254 nm. The temperature of column was set at 40 C. The volume of injected sample was 20 mL. 2.7. HPLC-UV procedure for E2 determination For the determination of E2, the mobile phase consisted of water and acetonitrile at a 55:45 (v/v) ratio. The total flow rate of the isocratic gradient module was maintained at 1 mL min1. The temperature of column was set at 40 C; the injection volume was 20 mL, and the detection wavelength was 280 nm. The total run time was 8 min and the retention time of E2 was 6.1 min. 2.8. Reference procedure The method reported earlier by Du et al. [16] with several minor modifications was used as the reference method. Briefly, 0.5 mL of methanol containing of 100 mL of C2H2Cl4 was rapidly injected into a sample solution prepared according to the procedure described in the Sample pretreatment section. The mixture was gently shaken for 1 min and then centrifuged for 2 min at 4000 rpm. The sedimented phase was evaporated to near-dryness under reduced pressure and dissolved in 100 mL of methanol, and obtained solution was taken for HPLC-UV analysis. 3. Results and discussion 3.1. Preliminary considerations The microextraction procedure based on DES decomposition
involved several steps. The first step included injection of the aqueous sample phase into the DES phase using a syringe (Fig. 1). This reverse injection sequence was chosen because the DESs are viscous liquids and their aspiration into the syringe is difficult. During aqueous sample injection instantaneous and spontaneous decomposition of the DES was observed resulting in in situ dispersion of organic phase and extraction of target hydrophobic analyte into extraction solvent droplets. Finally, after centrifugation the upper phase was collected. Undoubtedly, that the composition of the DES may affect its stability in aqueous phase. Usually, DESs are obtained by the formation of hydrogen bond between the hydrogen bond acceptor agent and the functional group of hydrogen bond donor agent. Several hydrophobic DESs synthesized from TBABr as the hydrogen bond acceptor and long-chain alcohols (amyl alcohol, heptanol, octanol, decanol and dodecanol) as the hydrogen bond donor in molar ratio of 1:2 were studied for E2 microextraction. It was established, that all DESs studied were decomposed into the aqueous phase resulting long-chain alcohols phase dispersion. However, the obtained extraction efficiency depended on the hydrogen bond donor type. The higher extraction efficiency and better repeatability (n ¼ 3) was obtained for the DES based on TBABr and heptanol at a 1:2 M ratio (Fig. 2). This fact can be explained by decreasing of the long-chain alcohols solubility in water. On the one hand amyl alcohol has higher solubility and as result analyte solubility in aqueous phase is increased. On the other hand, dispersion efficiency of organic phase is reduced with decreasing solubility of the alcohol resulting poor extraction efficiency. 3.2. Investigation of the DES structure Based on preliminary studies the DESs synthesized from TBABr and heptanol at various molar ratio was chosen for future experiments. FTIR spectra were recorded and DSC investigations were carried out for confirmation of the DESs formation. The FTIR spectra revealed several absorption bands according to the composition of the DESs at various molar ratio of TBABr and
52
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
Fig. 2. Effect of DES composition (volume of aqueous phase, 1.5 mL; concentration of 17b-estradiol, 10 mg L1; volume of DES phase, 250 mL).
heptanol (1:1, 1:2, and 1:3, respectively). Several absorption bands connected to the methyl and methylene groups of both compounds were identified. The dominant peaks in the region 2960e2858 cm1 arising from (CeH) stretching vibration, existence of the above mentioned groups corroborates the broader band with the minimum at 1467 cm1 dedicated to the eCH2e deformation vibration and also bands of the eCH3 group's deformation vibration that were found at 1458 cm1 and 1380 cm1, respectively. Skeletal vibrations of e(CH2)4e group in heptanol was visible by bands about 740 cm1. As it was evident from Fig. S2 the intensity varied according to the ratio between TBABr and heptanol. The peaks about 3350 cm1 indicated stretching vibrations of the OeH group of the primary alcohol. However, broader character hid the shift due to interaction of the OeH$$$Br type as it was found in mixture of water and TBABr [17]. Absorption bands of n(CeN) asymmetrical and symmetrical stretching vibrations in the region 1059e882 cm1 confirmed the TBABr. Unfortunately, the IR spectroscopy cannot clearly confirm the association between heptanol and TBABr as the differences between the IR spectra of each components and mixtures were insignificant. As it is mentioned above the difference was found mainly in intensity of the absorption bands. Differential scanning calorimetry investigations of the DES (TBABr and heptanol at a 1:2 M ratio) synthesized and initial components were also performed in the range from 30 C to 110 C at a rate of 5 C min1. In the thermograms (Fig. S3), the peaks corresponding to TBABr and heptanol melting were observed, yielding melting temperatures of 105 C and 34 C, respectively. The position of the peak in the graph of the heattemperature dependence confirmed a significant decrease in the melting point value of the mixture as compared with its precursors. The lowering of melting temperatures 10 C was observed for the DESs. From the thermograms obtained, it can be concluded that the thermal behavior of DES was different from the behavior of the individual components.
GC-MS. It was established that 85 ± 2% (m/m) of the original TBABr was dissolved into the aqueous phase. In the aqueous phase dissociation of TBABr was observed and a peak of TBAþ was presented on the chromatogram (Fig. S4). The concentrations of heptanol in organic phase was found to be 78 ± 2% (m/m) (Fig. S5). Moreover, the obtained organic phase was analyzed by the KarlFisher method, and it was established that it contains 7.01 ± 0.07% of water. Thus, the extraction mechanism was associated with the DES decomposition as a consequence of TBABr dissolution into aqueous sample phase and heptanol hydration. From this point of view TBABr can be considered as dispersive agent as in conventional dispersive liquid-liquid microextraction (DLMME), where dispersive solvent is used for organic phase dispersion. However, TBABr as quaternary ammonium salt containing bromide ion can also act as salting out agent and promote mass transfer between the two phases. Both DES before extraction (TAQ1) and top phase after the extraction (TAQ2) were also studied by 1H NMR (Please see electronic Supplementary materials for details). The results obtained are in good agreement with the results obtained by the GC-MS method. To demonstrate above mentioned features, the developed approach was compared with the DLMME procedure using the mixture of heptanol and various polar water-miscible solvents. For this purpose, 250 mL of extraction mixture containing of heptanol and various dispersive solvents (isopropyl alcohol, methanol, acetone or acetonitrile) in ratio 2:1 v/v was injected into 1.5 mL of aqueous analyte solution (10 mg L1) by the syringe. After centrifugation organic phase was analyzed by HPLC-UV. It was shown (Fig. 3), that extraction efficiency obtained for DES was higher than extraction efficiency for the mixtures of heptanol with dispersive solvents studied. The result obtained confirmed fact, that polar organic dispersive solvents increased solubility of E2 in an aqueous phase.
3.3. Extraction mechanism investigation
3.4.1. Effect of DES volume The volume of the DES phase may result in preconcentration or dilution of the extracted analyte. On the one hand the volume of the DES phase should be as small as possible to achieve high enrichment factor. On the other hand, the use small volume of the DES phase can give rise to poor reproducibility. The effect of the DES volume was investigated in the range from 200 to 600 mL. Each measurement was performed three times. By increasing the DES volume, the dilution effect predominated and the analytical signal was decreased (Fig. S6). On the other hand, it is too difficult to withdraw an extraction phase obtained if the applied DES volume
The TBABr is water-soluble component of the DES and it was assumed that DES decomposition was associated with TBABr dissolution into aqueous phase while heptanol was responsible for formation of dispersed organic phase. To determine TBABr and heptanol concentration after extraction 1.5 mL of aqueous phase was rapidly injected by the syringe into the 5 mL polypropylene tube containing 250 mL of DES (TBABr and heptanol at a 1:2 M ratio). After centrifugation the aqueous phase was analyzed by HPLC-UV and the organic phase was analyzed by
3.4. Optimization of extraction procedure
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
53
Fig. 3. Effect of dispersive solvent type (volume of aqueous phase, 1.5 mL; concentration of 17b-estradiol, 10 mg L1).
was less than 250 mL. Therefore, a DES volume of 250 mL was selected for further experiments. In this case 100 ± 5 mL of organic phase was separated. 3.4.2. Effect of sample volume The sample volume can influence on the extraction efficiency and enrichment factor (EF). The sample volume was varied from 0.5 to 2.0 mL, where by a volume of 1.5 mL was found to provide the best results in both analytical signal and reproducibility. Therefore, a 1.5 mL sample volume was chosen. The estimated EF was 15. The EF value was estimated as the ratio between the analyte concentration in the separated organic phase (Csop) and the initial concentration of analyte (C0) in the sample phase: EF ¼ Csop/C0. Extraction recovery (ER) value was also calculated as ER ¼ (Csop∙Vsop/C0∙V0) 100%, were Csop and C0 are analyte concentrations in the separated organic phase and in the initial sample phase, respectively; Vsop and V0 are volumes of separated organic phase and sample, respectively. The estimated ER was (95 ± 5) %. 3.5. Quality assurance/quality control To ensure an adequate level of quality assurance and a quality control of measurements, the developed procedure was utilized for the determination of E2 in transdermal gel samples. To obtain calibration curve, eight analyte standard solutions were prepared via the proposed microextraction procedure and the final analysis was performed by HPLC-UV. The results obtained showed that the curve obtained was linear in the range of 0.5e100 mg L1. The regression equation was Y ¼ 53113 C (where Y corresponds to the peak area and C means concentration of E2 expressed in mg L1), the coefficient of determination was 0.9954.
To determine the limit of detection (LOD) of the proposed procedure, initially, a blank sample was analyzed. The LOD was determined based on a signal-to-noise ratio (S/N) of 3. Also, the limit of quantification (LOQ) was determined based on S/N ¼ 10. On this basis, the LOD level was 0.15 mg L1, and the LOQ value was found to be 0.5 mg L1. To evaluate the precision of the developed procedure, the repeatability of the peak areas obtained was investigated for five replicate microextractions and the deionized water sample spiked at a 10 mg L1 level. This parameter was expressed as the relative standard deviation (RSD) and was provided for both the intra- and inter-day precisions. As it can be seen in Table 1S, the relative standard deviations obtained were below 8.07%. To assess the extraction recovery and enrichment factor, the equations presented in section 3.4.2 were applied. Based on the results obtained, the extraction recovery was (95 ± 5) %, and enrichment factor in the value 15 was achieved. To investigate the accuracy of the procedure, the relative recoveries obtained from analysis of real samples spiked with known amounts of E2 under investigation at different concentration levels were calculated. Spiking levels were chosen regarding real content levels and method quantification limits of 15, 30, and 45 mg L1 for transdermal gel 1 (Divigel) and 10, 20, and 30 mg L1 for transdermal gel 2 (Oestrogel). The obtained results are summarized in Table 1. The results obtained showed that the different matrices used for samples had no significant effect on the microextraction efficiency and obtaining high relative recoveries (from 89 to 105%) approved this fact. The mentioned samples were analyzed using reference procedure described in Section 2.8. As it can be seen from Table 1, the analytical results agreed well with the results obtained with reference method [16]. F-values 19.2 indicated insignificant
Table 1 The results of determination of 17b-estradiol (mg L1) in transdermal gel samples (n ¼ 3, P ¼ 0.95, Fk ¼ 19.2, tk ¼ 2.78). Sample
Added
Developed procedure
Reference procedure
F-test
t-test
Recovery, %
1
0 15 30 45 0 10 20 30
36.3 ± 5.8 49.7 ± 6.6 63.4 ± 3.4 77.8 ± 8.5 20.0 ± 2.8 30.6 ± 1.3 39.8 ± 2.5 48.6 ± 4.9
35.0 ± 6.5 48.7 ± 9.1 62.9 ± 10.1 75.1 ± 11.4 21.9 ± 3.3 32.4 ± 8.4 46.6 ± 7.9 52.8 ± 3.4
0.79 0.53 0.11 0.55 0.73 0.03 0.14 2.10
0.29 0.18 0.11 0.38 0.83 0.59 1.94 1.16
89 90 92 105 99 95
2
54
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55
Table 2 Comparison of the proposed procedure with those described in the literature. Sample
Detection
Sample pretreatment
LOD, mg L1
RSD, %
Recovery,%
Total analysis time, min
Reference
Gel Tablets Tablets Gel
HPLC-UV HPLC-FLD DPV HPLC-UV
Dissolution Dissolution Dissolution Dissolution
0.01 0.01 5.5 0.15
1.94 1.89 0.99 6
99.6 101.6 100.5 90e101
26 22 15 (Dissolution) 12
19 20 21 This work
in in in in
20 mL of acetonitrile 75 mL of methanol supporting electrolyte 1.5 mL of water and LLME (250 mL of DES)
HPLC-UV e high-performance liquid chromatography with ultraviolet detection, HPLC-FLD e high-performance liquid chromatography with fluorescence detection, DPV e differential pulse voltammetry, LLME e liquideliquid microextraction.
difference in precision between both methods at the 95% confidence level. t-values 2.78 indicated insignificant difference between the results obtained using these methods (n ¼ 3). The chromatogram of transdermal gel sample (Divigel) is shown in Fig. S7.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.03.038. References
3.6. Comparison of the developed procedure with other procedures Table 2 shows figures of merit for the proposed procedure and those of other procedures reported for the determination of E2 in pharmaceutical samples. As it can be seen, LOD of the proposed procedure is comparable with those reported for the other procedures. The proposed procedure is characterized by lower consumption of organic solvents and therefore offers low waste production. Moreover, the total analysis time is less in comparison with the developed procedures based on HPLC-UV [19], HPLC-FLD [20] and DPV [21]. 4. Conclusion A really effective liquid-liquid microextraction approach based on deep eutectic solvent decomposition was suggested for the first time. Above all, it was revealed and confirmed that extraction mechanism was associated with the DES decomposition as a consequence of TBABr dissolution into aqueous sample phase and heptanol hydration. This methodology can be considered as a mode of dispersive liquid-liquid microextraction. It is known, that the addition of dispersive solvents in conventional DLLME can decrease the partition coefficients of some analytes into the extraction solvents [18]. Unlike conventional DLLME, in the developed procedure the hydrogen bond acceptor (TBABr) during DES dissolution and decomposition on the one hand acted as the dispersive agent, on the other hand it promoted mass transfer between aqueous and organic phases as a salting out agent. As a result, two simultaneously occurring effects (organic phase dispersion and salting out effect) provided efficient extraction of E2 with good extraction recovery (95 ± 5%), excellent reproducibility (6%) and short time (5 min). Comparative studies have indicated that the developed approach is a reliable analytical tool with wide potential applications in analysis of various sample matrixes and it has the opportunity to be coupled with other instrumental methods. Acknowledgements This work was supported by the Russian Foundation for Basic Research (project no. 18-33-20004) and by Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic (VEGA 1/0010/15). Scientific research was performed using the equipment of the Research Park of St. Petersburg State University (Chemical Analysis and Materials Research Centre, Centre for Magnetic Resonance, Centre of Thermal Analysis and Calorimetry and the Chemistry Educational Centre of Research Park of St. Petersburg State University).
[1] A.P. Abbott, D. Boothby, G. Capper, D.L. Davies, R.K. Rasheed, Deep Eutectic Solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids, J. Am. Chem. Soc. 126 (2004) 9142e9147, https:// doi.org/10.1021/ja048266j. [2] B. Tang, K.H. Row, Recent developments in deep eutectic solvents in chemical sciences, Monatshefte Fur Chemie 144 (2013) 1427e1454, https://doi.org/ 10.1007/s00706-013-1050-3. [3] A. Shishov, A. Bulatov, M. Locatelli, S. Carradori, V. Andruch, Application of deep eutectic solvents in analytical chemistry, in: A review, Microchemical Journal, vol. 135, 2017, pp. 33e38, https://doi.org/10.1016/j.microc.2017.07. 015. [4] L. Nugbienyo, A. Shishov, S. Garmonov, L. Moskvin, V. Andruch, A. Bulatov, Flow method based on liquid-liquid extraction using deep eutectic solvent for the spectrofluorimetric determination of procainamide in human saliva, Talanta 168 (2017) 307e312. https://doi.org/10.1039/c3ay40284c. [5] S.C. Cunha, J.O. Fernandes, Extraction techniques with deep eutectic solvents, Trac. Trends Anal. Chem. 105 (2018) 225e239, https://doi.org/10.1016/ j.trac.2018.05.001. [6] A. Shishov, N. Volodina, D. Nechaeva, S. Gagarinova, A. Bulatov, Deep eutectic solvents as a new kind of dispersive solvent for dispersive liquid-liquid microextraction, RSC Adv. 8 (2018) 38146e38149, https://doi.org/10.1039/ c8ra07300g. [7] A.Y. Shishov, M.V. Chislov, D.V. Nechaeva, L.N. Moskvin, A.V. Bulatov, A new approach for microextraction of non-steroidal anti-inflammatory drugs from human urine samples based on in-situ deep eutectic mixture formation, J. Mol. Liq. 272 (2018) 738e745, https://doi.org/10.1016/j.molliq.2018.10.006. [8] A. Shishov, N. Volodina, D. Nechaeva, S. Gagarinova, A. Bulatov, An automated homogeneous liquid-liquid microextraction based on deep eutectic solvent for the HPLC-UV determination of caffeine in beverages, Microchem. J. 144 (2019) 469e473. [9] D.J.G.P. Van Osch, L.F. Zubeir, A. Van Den Bruinhorst, M.A.A. Rocha, M.C. Kroon, Hydrophobic deep eutectic solvents as water-immiscible extractants, Green Chem. 17 (2015) 4518e4521. http://doi.org/10.1039/c5gc01451d. [10] S.M. Yousefi, F. Shemirani, S.A. Ghorbanian, Hydrophobic deep eutectic solvents in developing microextraction methods based on solidification of floating drop: application to the trace HPLC/FLD determination of PAHs, Chromatographia 81 (2018) 1201e1211. http://doi.org/10.1007/s10337-0183548-7. [11] S. Zhu, J. Zhou, H. Jia, H. Zhang, Liquideliquid microextraction of synthetic pigments in beverages using a hydrophobic deep eutectic solvent, Food Chem. 243 (2018) 351e356. http://doi.org/10.1016/j.foodchem.2017.09.141. [12] A. Florindo, L.C. Branco, I.M. Marrucho, Development of hydrophobic deep eutectic solvents for extraction of pesticides from aqueous environments, Fluid Phase Equilib. 448 (2017) 135e142. https://doi.org/10.1016/j.fluid.2017. 04.002. [13] S. Pozdniakova, M. Guitart-Mampel, G. Garrabou, G. Di Benedetto, Y. Ladilov, V. Regitz-Zagrosek, 17b-Estradiol reduces mitochondrial cAMP content and cytochrome oxidase activity in a phosphodiesterase 2-dependent manner, Br. J. Pharmacol. 175 (2018) 3876e3890, https://doi.org/10.1111/bph.14455. [14] H. Kuhl, Pharmacology of estrogens and progestogens: influence of different routes of administration, Climacteric 8 (2005) 3e63, https://doi.org/10.1080/ 13697130500148875. [15] J. Poon, A. Katsifis, M. Fulham, Alternate HPLC method for the analysis of tetrabutylammonium hydroxide in [18F]fluorodeoxythymidine (FLT), J. Liq. Chromatogr. Relat. Technol. 40 (2017) 667e670. [16] X. Du, X. Wang, Y. Li, F. Ye, Q. Dong, Ch Huang, Determination of estrone and 17b-estradiol in water samples using dispersive liquid-liquid microextraction followed by LC, Chromatographia 71 (2009) 405e410. https://doi.org/10. 1365/s10337-009-1455-7. [17] H. Kleeberg, IR-Spectroscopic investigation of the hydration of tetrabutylammonium bromide in methylene chloride, J. Solut. Chem. (1986) 169e176. https://doi.org/10.1007/BF00646288.
A. Shishov et al. / Analytica Chimica Acta 1065 (2019) 49e55 [18] J. Regueiro, M. Llompart, C. Garcia-Jares, J.C. Garcia-Monteagudo, R. Cela, Ultrasound-assisted emulsification-microextraction of emergent contaminants and pesticides in environmental waters, J. Chromatogr. A 1190 (2008) 27e38. https://doi.org/10.1016/j.chroma.2008.02.091. , L. Matysova , J. Sícha, [19] L. Havlíkov a, L. Nov akova P. Solich, Determination of estradiol and its degradation products by liquid chromatography, J. Chromatogr. A 1119 (2006) 216e223, https://doi.org/10.1016/j.chroma.
55
2006.01.085. [20] B. Yilmaz, Y. Kadioglu, Determination of 17 b-estradiol in pharmaceutical preparation by UV spectrophotometry and high performance liquid chromatography methods, Arab. J. Chem. 10 (2017) S1422eS1428, https://doi.org/ 10.1016/j.arabjc.2013.04.018. [21] B. Salcı, I. Biryol, Voltammetric investigation of b - estradiol, J. Pharm. Biomed. Anal. 28 (2002) 753e759, https://doi.org/10.1016/S0731-7085(01)00676-8.