A novel procedure for phase separation in dispersive liquid-liquid microextraction based on solidification of the aqueous phase

Author’s Accepted Manuscript A novel procedure for phase separation in dispersive liquid-liquid microextraction based on solidification of the aqueous phase J.G. March, V. Cerdà www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(16)30345-9 http://dx.doi.org/10.1016/j.talanta.2016.05.027 TAL16577

To appear in: Talanta Received date: 5 March 2016 Revised date: 6 May 2016 Accepted date: 10 May 2016 Cite this article as: J.G. March and V. Cerdà, A novel procedure for phase separation in dispersive liquid-liquid microextraction based on solidification of the aqueous phase, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.05.027 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 galley proof before it is published in its final citable 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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A novel procedure for phase separation in dispersive liquid-liquid microextraction based on solidification of the aqueous phase J.G. March∗, V. Cerdà Department of Chemistry, University of Balearic Islands, Carretera de Valldemossa, Km 7,5. 07122 Palma de Mallorca, Illes Balears, Spain

abstract In this paper, an alternative for handling the organic phase after a dispersive liquidliquid microextraction using organic solvents lighter than water is presented. It is based on solidification (at -18 oC) of the aqueous phase obtained after centrifugation, and the decantation, collection and analysis of the liquid organic layer. The extraction of nicotine in toluene, and its determination in eggplant samples was conducted as a proof of concept. The study has been carried out using standards prepared in water and the formation of the dispersion was assisted by sonication. The organic extract was analysed using gas chromatography coupled to mass spectrometry. Satisfactory analytical figures of merit as: limit of detection (0.4 µg L-1, 2 ng g-1 wet sample), limit of quantification (1.2 µg L-1, 6.5 ng g-1 wet sample), within-day precision (RSD = 7 %), and linearity interval (up to 384 µg L-1 nicotine) were achieved. It constituted a contribution to the handling of organic extracts after microextraction processes.

Graphical abstract

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Keywords: Dispersive liquid-liquid microextraction; Nicotine; Eggplant; Gaschromatography. *Corresponding author: tel +34971172504; fax +34971173426. E-mail address: [email protected] 1. Introduction Since dispersive liquid-liquid microextraction (DLLME) was introduced as a useful technique for sample preparation [1], it has experimented a fast development as a consequence of its versatility and applicability to many problems of the analytical chemistry field. Several excellent reviews can be found in the literature [2,3,4]. DLLME is based on the dispersion of tiny droplets of the extraction organic solvent into the aqueous phase and the subsequent phase separation, usually performed by centrifugation. Depending on the density of the extracting organic solvent and aqueous phase, the organic layer is formed either on top or bottom of the centrifuge tube. Initial developments of DLLME were done using organic solvents with a density higher than water [1], and the collection of a microvolume of the sedimented organic layer from the bottom of the centrifuge tube was accomplished by means of a microsyringe. As most of such solvents are toxic chlorinated compounds, low density organic solvents, with a lower toxicity, are often preferred in DLLME methods. In such case, the withdrawal of the organic phase, formed on top of the aqueous phase, requires the use of ingenious home-designed devices. Examples are glass vessels with a capillary [5] or a narrow neck [6,7,8]. Such devices operated the same concept: the two layer system obtained after centrifugation and contained in the vessel is elevated by addition of water (by injection or drop by drop) until the microvolume of organic layer reached the capillary or narrow neck, from where the necessary volume for chemical analysis is taken with a microsyringe. Good analytical features were obtained using these devices, but their in-laboratory manufacture restricts its usage. A similar proposed strategy for

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organic phase collection consisted in the transfer of the organic layer (using a syringe) mixed with considerable aqueous phase to a microtube where a fast separation of the two layers occurred; then the upper organic layer is easily withdrawn [9]. A different strategy developed for the use of low density organic solvents as extractants is the DLLME based on solidification of the floating organic drop (DLLMESFO) using an ice bath and subsequent collection of the solid organic drop with a spatula or similar [10,11,12,13,14,15]. So, this methodology requires organic solvents of melting point higher than 0 OC, which greatly restrict the number of useful organic solvents.

Main analytical applications have been developed using hexadecane, 1-

undecanol, 1-dodecanol or 2-dodecanol as extractants. The low volatility of these solvents limits the compatibility with several analytical techniques, such as gas chromatography. In this paper, a novel approach has been developed as an alternative to perform DLLME-SFO, which is based on solidification of the aqueous phase (DLLME-SAP) and subsequent separation of the liquid organic layer from the frozen aqueous phase by simple spilling of the centrifuge tube. According to the proposed methodology, volatile organic solvents (with low melting points) can be used, thus complementing the characteristics of the organic solvents suitable for microextraction procedures based in the separation of a liquid layer from the other in solid state. Then, the choice of the organic solvent is less restricted and, consequently, other requirements as: ability to extract target analytes, toxicity or/and compatibility with the instrumental technique for chemical analysis can be taken in consideration. As a proof of concept nicotine was selected as the analyte. Nicotine is a compound naturally found at very low levels in different plant species, such as eggplants, tomatoes, potatoes, chili, pepper sauce [16], mushrooms [17] and tea [18]. Nicotine has also been determined in some other kind of food samples, such as fish tissue [19]. Due to the well-known toxicity and adverse effects of nicotine on human health, the European Food Safety Authority has proposed temporary maximum residue limits for nicotine in some crops [20]. In this context, the determination of nicotine in edible plants is of evident interest. In this work, nicotine has been determined in eggplants. 2. Experimental

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2.1. Reagents, standard solutions and accessories Analytical grade sodium chloride, hydrochloric acid, ammonia, sodium hydroxide, methanol and ethyl acetate were purchased from Scharlau Chemie (Barcelona, Spain), toluene, nicotine (99.1 %) and quinaldine (>95.0 %) were from Sigma-Aldrich (Madrid, Spain). A stock standard of nicotine containing 660 mg L-1 in water was prepared weekly and stored in dark conditions at room temperature. A working standard of 6.6 mg L-1 was prepared by appropriate dilution each working session. A quinaldine solution containing 92 mg L-1 in 3 mol L-1 HCl was used as internal standard, and it was prepared and stored at 4 oC. A 2.3 mg L-1 working standard was prepared by dilution in 3 mol L-1 HCl. Purified water (resistivity 18.2 M cm) obtained using a Millipore MilliQ system (Bedford, MA, USA), was used to prepare aqueous solutions. Screw-cap glass tubes of 12 mL were obtained from Macherey-Nagel (Düren, Germany). Nylon syringe filters (0.45 µm pore size) were from Scharlau Chemie. A centrifuge Biocen 22R from Ortoalresa (Madrid, Spain) was used for centrifugation. A pH-meter Crison PH25+ from Crison Instruments (Alella, Barcelona, Spain) was used for pH measurements. Glassware was cleaned, rinsed with acetone and heated al 380 oC during 2 hours before use. 2.2. Procedure for dispersive liquid-liquid microextraction based on solidification of the aqueous phase (DLLME-SAP) A schematic diagram for the performance of the procedure is shown in Fig. 1. In a screw-cup tube, 0.1 mL of 2.3 mg L-1 quinaldine solution in 3 mol L-1 HCl, 0.5 mL of 5 mol L-1 ammonia, nicotine aqueous standard solution to reach a final concentration in the range 8 – 384 µg L-1 and water up to 5 mL were added (the pH of the formed ammonium ion – ammonia pH buffer was in the interval 9.9 – 10.1). Then 100 µL of toluene were added, and the tube was stopped. It was placed in the ultrasonic water bath, where a cloudy dispersion was formed. The sonication was maintained 10 min to complete the extraction. After that, the tube was centrifuged at 3500 relative centrifugal force (5865 rpm) during 5 min. Then, the tube containing two clear phases was placed in a freezer at –18 oC for 40 min to ensure solidification of the aqueous phase. Then, the organic phase enriched with nicotine was decanted on a cold (porcelain or glass) plate and quickly transferred, with an automatic pipette, to a microvial for chromatographic

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analysis using an autosampler. Solvents and glassware were checked periodically for potential nicotine contamination. Experiments were run in triplicate. 2.3. Sample preparation Commercially available eggplants from the local market were analysed. They were washed with hot deionized water to remove eventual external contamination. Two fractions (i.e. peel and pulp) were obtained from each sample using a cutter. Each fraction was crushed using a food blender until a homogeneous shake was obtained. Then, it was stored at -18 oC until use. For analysis, in a tube containing 1.0 g of wet sample, 0.1 mL of quinaldine in 3 mol L-1 hydrochloric acid and 4.3 mL of water were added. Then it was shaken and sonicated for 15 min. After, the tube was centrifuged at 3500 relative centrifugal force (5865 rpm) during 10 min. Then, the solution was filtered and transferred to another centrifuge tube. 0.5 mL of 5 mol L-1 ammonia and 100 µL of toluene were added. Then, the procedure for DLLME-SAP was followed. Nicotine concentrations were calculated assuming a complete extraction of the nicotine contained in the samples. 2.4. Equipment and conditions for gas chromatography-mass spectrometry A Shimadzu GC 2010 gas chromatograph fitted with a TRB-624 fused silica capillary column (30 m, 0.25 mm i.d., 1.40 µm film thickness) purchased from Teknokroma (Barcelona, Spain) and a mass selective detector Shimadzu GCMSQP20105 was used for the analysis of nicotine in the organic extract. Helium X50S supplied by Carburos Metálicos (Barcelona, Spain) was used as carrier gas at a constant flow rate of 1 mL min-1 (linear velocity 35 cm s-1). 1 µL was injected into the inlet in splitless mode at 220

o

C (sampling time 1 min) using a Shimadzu AOC-20i

autosampler. The GC-MS interface and the ion source were at 220 oC. The oven temperature program started at 60 oC for 1 min, was increased at 30 oC min-1 to 230 oC, and maintained for 20 min; fragments were generated by impact ionization at 70 eV; for qualitative analysis of real samples full scan mode in the 50-250 amu range was used; for quantitative analysis m/z = 84 and 143 (base peak of the mass spectra of nicotine and quinaldine, respectively) were selectively monitored (SIM). Working at indicated chromatographic conditions, the tR of quinaldine and nicotine were 12.5 and 13.1 min, respectively.

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A Selecta-3000617 ultrasonic bath (200 W, 40 kHz) purchased from Selecta (Barcelona, Spain) was used for ultrasonication treatments. 3. Results and discussion Experimental variables that influence the area of the chromatographic peak of nicotine were studied. Experiments were carried out with 5 mL of aqueous phase containing 48 µg L-1 nicotine.

3.1. Organic solvent The proposed approach for organic phase collection is applicable when using an organic solvent lighter than water and with a low freezing point. According to these conditions, toluene and ethyl acetate were selected because, according to reported data in the literature, they show appropriate characteristics for liquid-liquid micro-extraction of nicotine [16,21]. Efficient organic solvents for microextraction of nicotine as dichloromethane, trichloromethane or undecanol were not appropriated for the proposed procedure because of their density or freezing point. When using ethyl acetate, a first point to comment is the extensive solubility of the ethyl acetate in the aqueous phase (8.7 % at 20 oC) that considerably diminished the collected volume after extraction, and also can favour the solubility of the organic analyte in the aqueous phase which affects negatively on the extraction efficiency. For example, using a 5 mL aqueous phase and 800 µL ethyl acetate, after extraction, only 230 µL (s = 17) were collected (483 µL could be assumed dissolved in the aqueous phase, some amount was evaporated into the head space of the tube, and also some evaporation can take place during the decantation of the solvent and its transference to a chromatographic microvial). On the other hand, using this solvent, the phase separation obtained by decantation can be considered quantitative because, when the aqueous phase that remains into the tube (after decantation) melted, no visible organic phase was present. When using toluene, the collected volume after extraction is closer to the added volume due to its negligible solubility in water. Depending on the volume added initially, the collected fraction was in the range 40 – 65 % of the initial volume. This

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could be attributed to evaporation (as for ethyl acetate) as well to a deficient separation of phases. So, after decantation, when the aqueous phase melted, a visible amount of organic solvent remained into the tube due to the retention of part of the toluene on the solid-liquid interface. In order to compare both solvents, experiments with different initial volume of organic solvent, which conducted to the same collected volume, were carried out. It was found that, 230 – 240 µL of organic phase can be collected either, from 800 µL ethyl acetate or 390 µL toluene. However, the area of the chromatographic peak using toluene was almost twofold the area obtained using ethyl acetate with similar relative standard deviation. For this reason, toluene was selected for further experiments. 3.2. pH and buffer concentration Nicotine is a diprotic base that undergoes protonation in acidic media. Consequently, its extraction into an organic solvent is favoured in basic media, independently of the used extraction technique. Adjusting the pH of the aqueous phase from 3 to 12 with HCl or NaOH, it was observed that nicotine was hardly extracted from acidic phases. Extraction improved from pH 7 to 9.5, and for higher pH values, a maximum extraction took place, as similarly has been reported [22]. To adjust the pH for nicotine extraction, ammonia is a recommended base [18,23]; so, a buffer ammonium chloride-ammonia of pH 10 was selected in the present work. Several buffers of total concentration in the range 0.7 – 0.1 mol L-1 were assayed and no significant effect was observed. A 0.5 mol L-1 was selected to develop the analytical application. 3.3. Salt addition This is a typical variable studied in liquid-liquid extraction processes. In most of cases, the analyte extraction efficiency increased with the ionic concentration of the aqueous phase due to the salting-out effect. Depending on the analyte, it has also been reported a salting-in effect due to changes on the viscosity of the solution, which embarrasses the diffusion of analytes towards the organic solvent [24]. For nicotine, the addition of NaCl has been recommended because its presence improved the extraction efficiency using a binary solvent mixture (undecanol and chloroform) [22]. In this work, it was found that NaCl concentration did not affect significantly the analyte peak area in

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the range 1 – 20 g L-1 NaCl. Moreover, for a NaCl concentration higher than 40 g L-1, the aqueous phase did not solidify, and consequently the organic phase collection cannot be performed following the proposed DLLME-SAP procedure. Furthermore, the presence of salt somewhat favour the phase separation, and NaCl addition has been recommended for the extraction of nicotine from fresh vegetables when phases did not show good separation [21].

3.4. Methanol concentration In DLLME, usually a disperser solvent that must be miscible with both organic and aqueous phase is used to increase the rate of the extraction. Nevertheless, the partition constant of the analytes in the system with three solvents can be lower than the corresponding to the binary system. This fact can affect negatively on the sensitivity of the analytical determination. The effect of methanol, which has been widely used as disperser solvent in DLLME procedures, on the area of the peak has been studied to illustrate this effect. As can be seen in Fig. 2, a pronounced diminution of the peak was observed even for low amounts of methanol as 1 %. For this, alternative treatments to assist the extraction were explored, and also the nicotine and quinaldine standards were prepared in water in contrast to reported procedures that recommended standard prepared in polar organic solvents as methanol or acetonitrile. 3.5. Ultrasound application and vortex agitation Application of ultrasound and vortex agitation are efficient treatments to enhance liquid-liquid microextraction [25]. Both treatments were compared. When ultrasound was applied to the extraction system, a cloudy and stable dispersion of fine droplets of the toluene into the aqueous phase was formed in few seconds. Nevertheless, stationary conditions were reached after 15 min of treatment. When vortex agitation was used, comparable results (to ultrasound application) were obtained for vortex times higher than 5 min. Ultrasound application was selected to continue the study because many samples can be processed in parallel. 3.6. Phase separation and aqueous phase freezing

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In order to accomplish a clear separation of the dispersed phase, 5 min of centrifugation at 3500 relative centrifugal force were required. After phase separation at room temperature, the centrifuge tube was exposed at -18 oC. After 40 min the aqueous phase was completely solidified, enabling the separation of the liquid organic phase by simple decantation on a as glass or porcelain surface. The deposited drops can be handled using an automatic pipette, or similar. It is worth to mention that, depending on the ionic concentration of the aqueous phase and/or the presence of a disperser solvent, this temperature and time values should be readjusted. In order to minimize evaporation, the surface of deposition of the drops was previously cooled at -18 oC. According to the proposed DLLME-SAP procedure, the separation of a microvolume of organic phase without risk of carryover of aqueous phase is very simple, and does not require any special device for phase separation and collection. 3.7. Volume of the extraction solvent This is a factor with a relevant effect on the analytical signal. It should be as small as possible in order to achieve a higher analytical signal. However, it should be a sufficient amount to carry out the chemical analysis. For operational advantages, the chromatographic analysis was carried out using an autosampler. The autosampler vials used in this work required a collected volume of at least 25 µL. It is worth to mention that in DLLME the recovered extractant volume after extraction can be significantly lower than the initially added volume due to the partial dissolution of the organic solvent into the aqueous phase, as well as uncontrolled evaporation. In this study it was found that the fraction of the added volume (in the range 50 -150 µL) that was collected ranged from 50 - 60%. When processing real samples, this percentage decreased up to 35 % in some cases. On the basis of such results, in order to assure a volume enough for injection using the autosampler, 100 µL of toluene were selected to stablish the calibration graph and to develop the analytical application. 3.8. Validation data The extraction and proposed technique for the collection of the organic phase was evaluated for quantitative analysis. Working as indicated in the procedure and

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conditions for gas chromatography, a calibration graph in the range of 6 – 384 g L-1 nicotine was established, being the fitted linear equation as it follows:

Peak ratio (nicotine/quinaldine) = 0.00 (±0.03) + 0.0089 (±0.0002) [nicotine]

(Standard deviation of the slope and the ordinate were calculated from the standard deviation of y-residuals sy/x=0.06; n=8; R2=0.998. Concentration of nicotine is expressed as g L-1 in the aqueous initial phase).

Limit of detection (LOD) and limit of quantification (LOQ) were estimated as the nicotine concentration giving peaks with signal to noise ratio of 3 and 10. Computed values were 0.4 and 1.2 g L-1 (2 and 6.5 ng g-1 wet sample), respectively. Within-day and between-day precision, calculated from 6 standards containing 24 g L-1 nicotine, were 7 % and 13 %, respectively. For a robustness study, seven variables were selected, namely: volume of donor and acceptor phases, ammonia concentration, sonication and centrifugation times and freezing time and temperature. Eight experiments were run with deliberate deviations of  5 % about stipulated values in the procedure for such variables, according to the Placked-Burman experimental design. Found differences were compared with the critical difference associated to the standard deviation corresponding to the within-day precision study for a 0.05 significance level [26]. From this study, no significant difference was found, and consequently, the proposed method can be considered robust. Using nicotine standards in toluene and assuming constant the volume of the organic phase, the enrichment factor and extraction efficiency, corresponding to 7 levels of concentration in the interval 12 – 384 g L-1 nicotine were estimated. The average value for the enrichment factor was 12.3 (0.9), which corresponded to an extraction efficiency of 25 % (2). Such relatively low values are due, by one hand, to the affinity of nicotine for the aqueous phase, and by the other hand, to the relatively high volume of organic phase selected (100 L) to enable automatic injection. A recovery study at two nicotine spiked concentration levels was carried out. As can be seen from Supplementary Table S1, the relative recovery values obtained were in

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the range 88 – 103 %) with RSD 3.2 – 5 %. The average recovery (95 %) was not significantly different than 100 (p=0.06). Therefore, it can be assumed that the eggplant matrix did not interfere significantly the nicotine determination, as it has also been found using different extraction techniques [16]. 3.9. Analysis of eggplant samples The extraction of nicotine from the vegetal sample and the later liquid-liquid extraction were based on reported recommendations for nicotine determination by GC. A reported treatment for eggplants [21] is: lixiviation with ammonia, liquid-liquid extraction with toluene and centrifugation. In this work the sequence was slightly modified in order to achieve cleaner chromatograms. The lixiviation was carried out in hydrochloric acid media where nicotine is very soluble as the protonated form, and before basification with ammonia, the sample was filtered. Five commercial samples were analysed as indicated in sample preparation and procedure for DLLME-SAP. For all reported samples, the presence of nicotine was confirmed by a qualitative analysis in scan mode. The quantitative obtained results are summarized in Table 1. As can be seen, the found concentrations were in the range 6.5 – 11.2 ng g-1, in agreement with reported values [16]. As far as we know, this is the first study where peel and pulp from eggplants have been analysed separately. From this reduced statistical sample it can be inferred that nicotine concentration in eggplant peel is significantly higher than nicotine content in pulp eggplant (the paired t-test lead to p=0.015), being the average values in pulp and peel, 7.0 and 9.2, respectively. 4. Conclusions The present work demonstrated that the separation and collection of an organic phase lighter than water after DLLME can be performed satisfactorily by simple decantation after freezing of the aqueous phase. This simple handling (DLLME-SAP) represents a new contribution to the separation of phases after liquid-liquid extraction based on solidification processes. The developed technique is easy and cheap, and the use of special home-made devices is not required. In some aspects, DLLME-SAP can be considered complementary to DLLME-SFO. DLLME-SAP is recommendable when

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organic solvents lighter than water with low melting points have been selected for extraction; most of them are volatile and useful for GC analysis. DLLME-SFO requires organic solvents with high melting points (that solidify in ice bath) with problematic use in GC, but with easier handling due to its lower volatility and toxicity.

Acknowledgements Financial support from ‘Ministerio de Economía y Competitividad’ Spanish Government (Grant CTQ2013-47461-R) is acknowledged.

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C.C. Chang, S.Y. Wei, S.D. Huang, Improved solvent collection system for a dispersive liquid-liquid microextraction of organochlorine pesticides from water using low-density organic solvent, J. Sep. Sci. 34 (2011) 837-843.

[10] X. You, S. Wang, F. Liu, K. Shi, Ultrasound-assisted surfactant-enhanced emulsification microextraction based on the solidification of a floating organic droplet used for the simultaneous determination of six fungicide residues in juices and red wine, J. Chromatogr. A 1300 (2013) 64-69. [11] X. You, Z. Xing, F. Liu, X. Zhang, Air-assisted liquid-liquid microextraction by solidifying the floating organic droplets for the rapid determination of seven fungicide residues in juice samples, Anal. Chim. Acta 875 (2015) 54-60. [12] J. Martin, J. L. Santos, I. Aparicio, E. Alonso, Determination of hormones, a plasticizer, preservatives, perfluoroalkylated compounds, and a flame retardant in water samples by ultrasound-assisted dispersive liquid-liquid microextraction based on the solidification of a floating organic drop, Talanta 143 (2015) 335-343. [13] T. Asadollahi, S. Dadfarnia, A. M. H. Shabani, Separation/preconcentration and determination of vanadium with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) and electrothermal atomic absorption spectrometry, Talanta 82 (2010) 208-212. [14] M.I. Leong, S.D. Huang, Dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with gas chromatography with electron-capture or mass spectrometry detection, J. Chromatogr. A 1211 (2008) 812. [15] M. Hu, L. Wu, Y. Song, Z. Li, Q. Ma, H. Zhang, Z. Wang, Microwave-assisted liquid-liquid microextraction based on solidification of floating organic droplet for the determination of triazines in honey samples, Anal. Methods 7 (2015) 91149120.

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[16] K. Shrivas, D.K. Patel, Liquid-phase microextraction combined with gas chromatography mass spectrometry for rapid determination of nicotine in onedrop of nightshades vegetables and commercial food products, Food Chem. 122 (2010) 314-318. [17] C. Müller, F. Bracher, F. Plössl, Determination of nicotine in dried mushrooms by a modified QuEChERS approach and GC-MS-MS, Chromatographia 73 (2011) 807-811. [18] C. Thräne, C. Isemer, U.H. Engelhardt, Determination of nicotine in tea (Camellia sinensis) by LC-ESI-MS/MS using a modified QuEChERS method, Eur. Food Res. Technol. 241 (2015) 227-232. [19] Y.W. Chang, H.P. Nguyen, M. Chang, S.R. Burket, B.W. Brooks, K.A. Schug, Determination of nicotine and its metabolites accumulated in fish tissue using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, J. Sep. Sci. 38 (2015) 2414-2422. [20] Commission Regulation (EU) No 401/2015 of 25 February 2015 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for 8-hydroxyquinoline, cyproconazole, cyprodinil, fluopyram, nicotine, pendimethalin, penthiopyrad and trifloxystrobin in or on certain products. https://www.fsai.ie/uploadedFiles/Legislation/Food_Legisation_Links/Pesticides_ Residues_in_food/Reg2015_401.pdf. Accessed 4 March 2016. [21] B. Siegmund, E. Leitner, W. Pfannhauser, Development of a simple sample preparation technique for gas chromatographic-mass spectrometric determination of nicotine in edible nightshades (Solanaceae), J. Chromatogr. A 840 (1999) 249260. [22] X. Wang, Y. Wang, X. Zou, Y. Cao, Improved dispersive liquid-liquid microextraction based on the solidification of floating organic droplet method with a binary mixed solvent applied for determination of nicotine and cotinine in urine, Anal. Methods 6 (2014) 2384-2389. [23] L.Q. Sheng, L. Ding, H.W. Tong, G.P. Yong, X.Z. Zhou, S.M. Liu, Determination of nicotine-related alkaloids in tobacco and cigarette smoke by GC-FID, Chromatographia 62 (2005) 63-68.

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[24] D.A. Lambropoulou, T.A. Albanis, Application of solvent microextraction in a single drop for the determination of new antifouling agents in waters, J. Chromatogr. A 1049 (2004) 17-23. [25] V. Andruch, M. Burdel, L. Kocúrová, J. Šandrejová, I.S. Balogh, Application of ultrasonic irradiation and vortex agitation in solvent microextraction, Trends Anal. Chem. 49 (2013) 1-19. [26] S.R.L. Ellison, V.J. Barwick, T.J. Duguid Farrant, Practical statistics for the analytical scientist, 2nd ed., The Royal Society of Chemistry Publishing, Cambridge, 2009.

Fig. 1. Schematic diagram of the proposed DLLME-SAP. (a) Dispersion of toluene in the aqueous phase. (b) Separated layers by centrifugation. (c) Solidification of the aqueous phase at autosampler

-18 oC. (d) Decantation of the organic layer, transfer to an vial

and

chromatographic

analysis.

16

100000

Peak area

80000 60000 40000 20000 0 0

2

4

6

8

10

12

% Methanol (w/w)

Fig. 2. Influence of methanol concentration on the peak area of nicotine. Extraction conditions:

initial

aqueous

phase,

5

mL;

nicotine

48

µg

L-1;

pH=10.0;

ammonium/ammonia total concentration, 0.5 M; initial organic phase, 100 µL toluene. Other conditions, as in the procedure.

Table 1 Found nicotine in eggplant samples. Pulp

Peel

Sample No

Nicotine (ng g-1)

SD a

Nicotine (ng g-1)

SD a

1

6.5

0.5

7.9

0.6

2

6.7

0.4

8.4

0.5

3

8.0

0.6

10.0

0.4

4

6.8

0.5

11.2

0.8

5

6.8

0.6

8.5

0.6

a

n=3

17

Highlights



A novel technique for collection the organic phase in DLLME is described.



The technique is based on freezing of the aqueous phase.



The organic phase is collected by simple decantation without specific devices.



Useful organic solvent must have lower density than water and low melting points.



On the basis of the organic solvent, the technique is complementary to DLLMESFO.