- Email: [email protected]
MS
Journal Pre-proof On-disc electromembrane extraction-dispersive liquid-liquid microextraction: A fast and effective method for extraction and determination of ionic target analytes from complex biofluids by GC/MS Monireh Karami, Yadollah Yamini PII:
S0003-2670(20)30059-3
DOI:
https://doi.org/10.1016/j.aca.2020.01.024
Reference:
ACA 237383
To appear in:
Analytica Chimica Acta
Received Date: 16 November 2019 Revised Date:
10 January 2020
Accepted Date: 12 January 2020
Please cite this article as: M. Karami, Y. Yamini, On-disc electromembrane extraction-dispersive liquid-liquid microextraction: A fast and effective method for extraction and determination of ionic target analytes from complex biofluids by GC/MS, Analytica Chimica Acta, https://doi.org/10.1016/ j.aca.2020.01.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier B.V. All rights reserved.
CRediT author statement Monireh Karami: Methodology, Investigation, Validation, Writing-Original draft preparation. Yadollah Yamini: Supervision, Reviewing and Editing
For TOC only
On-disc electromembrane extraction-dispersive liquid-liquid microextraction: A fast and effective method for extraction and determination of ionic target analytes from complex biofluids by GC/MS
Monireh Karami and Yadollah Yamini*1 Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
1
∗
Corresponding author: Tel.: +98 21 82883449; Fax: +98 21 82883460.
E-mail address: [email protected] (Y. Yamini).
1
Abstract In this study, an electromembrane extraction-dispersive liquid-liquid microextraction (EME-DLLME) was performed using a lab-on-a-disc device. It was used for sample microextraction, preconcentration, and quantitative determination of tricyclic antidepressants as model analytes in biofluids. The disc consisted of six extraction units for six parallel extractions. First, 100 µL of a biofluid was used to extract the analytes by the drop-to-drop EME to clean-up the sample. The extraction then was followed by applying the DLLME method to preconcentrate the analytes and make them ready for being analyzed by gas chromatography (GC). Implementing the EME-DLLME method on a chip device brought some significant advantages over the conventional methods, including saving space, cost, and materials as well as low sample and energy consumption. In the designed device, centrifugal force was used to move the fluids in the disc. Both sample preparation methods were performed on the same disc without manual transference of the donor phases for doing the two methods. Scalable centrifugal force made it possible to adjust the injection speed of the organic solvent into the aqueous solution in the DLLME step by changing the spin speed. Spin speed of 100 rpm was used in dispersion step and spin speed of 3500 rpm was used to sediment organic phase in DLLME step. The proposed device provides effective and reproducible extraction using a low volume of the sample solution. After optimization of the effective parameters, an EME-DLLME followed by GC-MS was performed for determination of amitriptyline and imipramine in saliva, urine, and blood plasma samples. The method provides extraction recoveries and preconcentration factors in the range of 43%70.8% and 21.5-35.5 respectively. The detection limits less than 0.5 µg L-1 with the relative standard deviations of the analysis which were found in the range of 1.9%-3.5% (n = 5). The method is suitable for drug monitoring and analyzing biofluids containing low levels of the model analytes. 2
Keywords: Electromembrane extraction; Dispersive liquid-liquid extraction; Lab-on-a-Disc; Centrifugal microfluidic platform; µCD platforms; Tricyclic antidepressant.
1. Introduction Liquid-liquid extraction (LLE) is the most extensively used sample preparation technique in chemistry and biochemistry [1]. Despite its widespread use, traditional LLE suffers from consumption of large quantities of toxic organic solvents and being tedious as well as time consuming. Due to global concerns about environmental pollution; the need to develop environment-friendly practices has been heighted in different areas of research. Attempts in the field of analytical chemistry to develop methods using organic solvents as less as possible led to introduction of different liquid phase microextraction methods (LPME). LPME methods also give rise to minimizing the overall extraction time [2, 3]. Dispersive liquid-liquid microextraction (DLLME) is one of the popular microextraction methods introduced in 2006 by Rezaee et al. [4]. Rapid injection of an appropriate mixture of extraction and disperser solvents into an aqueous donor phase containing the desired analyte/analytes makes a cloudy solution and a multitude of fine droplets of the extraction solvent forms in the solution. Formation of these fine droplets is the most significant advantage of DLLME, which causes the extraction to be obtained very fast [5]. Moreover, the method is compatible with most analytical instruments. The features of being fast and capable of reaching high preconcentration factors make DLLME receive analytical chemists’ interests [5]. However, DLLME suffers from some disadvantages. Perhaps the most serious disadvantage of the method is the lack of sample clean-up because of which this method can be only efficiently applied to simple matrices. Electromembrane extraction (EME) is one of LPME techniques based on electrokinetic migration of ionized analytes from an aqueous donor phase across a supported liquid 3
membrane to an acceptor solution. Since it was reported in 2006 [6], a considerable amount of literature has been published on application of EME [7, 8]. Since EME is a membrane based extraction, it provides a good sample clean-up, which makes this method capable of being directly used in complex sample matrices such as biofluids. The acceptor solution in EME is mostly an aqueous solution. Thus the main drawback of the method is its incompatibility in to gas chromatography (GC) instrument. While GC is faster, cheaper, and simpler than liquid chromatography (LC) and can be easily conjugated with different kinds of detectors such as flame ionization detector (FID) and mass spectrometer (MS). Some attempts have been made so far to transfer the extracted analytes of EME to a GC-compatible phase [9-14]. DLLME is the method which can be properly combined with EME. Combination of EME with DLLME provides a two-step extraction method that benefits from great sample clean-up of EME as well as high preconcentration factors of DLLME. Another advantage is that the extracts can be analyzed by GC. In 2012, Guo developed an electromembrane extraction followed by low-density solvent based ultrasound-assisted emulsification microextraction (EME–LDS-USAEME) using 100 mL of sample solution. [11]. The proposed method showed a good limit of detection and linearity for extraction of trace levels of chlorophenols; however, the two-step extraction method with manual transference of the donor phase can affect repeatability and precision of the extraction and make the method tedious and hard to operate. Moreover, although the method provides very good sample clean-up, it needs a large quantity of a donor phase. Therefore, it seems that the method is not suitable for analysis of biofluids. Seidi et al. reported a method to combine EME and DLLME to determine tricyclic antidepressants in biological samples [12]. They used 24 mL of a donor phase in an EME step with the acceptor solution volume of 10 µL.. However, as it was the case with the previously mentioned work, manual handling and transference of phases can influence repeatability and precision of the method. Also using 24 4
mL of a donor phase still seems too much for some of biological fluids. Some other works have been done to couple EME with DLLME [13, 14]. They had the same drawbacks as were mentioned before. General advantages of miniaturization including the capability to save time, space, cost, and materials lead microfluidic systems to become a necessity in today’s world. Keeping company with many areas of science, the last three decades have seen a growing interest in miniaturization and automation of classical analytical operations. The main motivation for such miniaturization is the development of micro total analysis systems (µTAS) or lab-on-achip (LOC) technology. Furthermore, automation leads to the development of devices for performing laboratory processes which are easy to handle without any need for a skillful operator. To date plenty of attempts have been made to down-scale and implement LLE and also LPME on microfluidic chip devices [15-25]. Research studies could successfully implement LLE-like principals to microfluidic chip devices, employing different microfluidic platforms. There have been some reviews describing different microfluidic and on-chip LLEs [26-30]. Microfluidic compact-disc (µ-CD) platforms, commonly referred to as lab-on-a-disc (LOAD), are one of the microfluidic platforms that provide specific advantages over other on-chip devices [31-33]. LOADs use centrifugal force for flow control. They simply need a rotary motor to create a centrifugal force. Therefore, there is no need for external pumps or power supplies for liquid actuation, which can make a great potential for portability. Bubblefree liquid handling without dead volume and scalable centrifugal forces for sedimentation are some of the other advantages of such a platform. LOADs also offer closed fluidic systems with the capability of parallel sample operation on the same disc [32]. To date several reports have been published in the field of sample preparation using LOADs [34-36]. There are still
5
negligible reports using LOADs for implementation of LLE [37, 38] and using the inherent capability of centrifugal platforms in microextraction methods like DLLME. In the present work, the on-disc EME-DLLME method has been introduced. The method benefits from high clean-up ability of a drop-to-drop EME for extraction of target analytes form biofluids as well as high preconcentration factors and GC compatibility of DLLME. Implementing the EME-DLLME method on a lab-on-a-disc device brings some significant advantages over the conventional methods, including miniaturization and saving time, space, and organic solvents. In addition, in the DLLME step, dispersion of an organic solvent mixture into a donor phase and sedimentation of an extraction solvent can be performed simply by changing the spin speed of the disc. In this work, we investigated the effect of the injection speed of the organic solvent into the aqueous solution on extraction efficiency. Finally, the applicability and performance of the system were demonstrated for human urine and blood plasma by extraction of two tricyclic antidepressants.
2. Experimental 2.1 Chemicals and materials Amitriptyline (Ami) and imipramine (Imi) were kindly provided by Razak Laboratories (Tehran, Iran). The structure of the drugs along with their physicochemical properties are shown in Table1. NaOH, CCl4, and C2HCl3 were purchased from Merck (Darmstadt, Germany). C2Cl4 and 2-nitrophenyl octylether (NPOE) were supplied from Acros (Geel, Belgium) and Fluka (Buchs, Switzerland), respectively. Isopropanol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol was purchased from Caledon (George Town, Ont., Canada). All chemicals were of analytical reagent grade. Deionized water was prepared by a Young Lin aquaMAx purification system 370 series (Seoul, Korea). The
6
Accurel PP 1E polypropylene membrane sheet with a wall thickness of 100 µm and a pore size of 0.1 µm was bought from Membrana (Wuppertal, Germany).
2.2 Standard solutions Stock solutions of the drugs at 1 mg mL−1 as well as a standard solution of the mixture of the drugs containing 50 µg mL−1 of each drug were prepared in methanol and stored in a refrigerator at 4°C. The aqueous working solution was prepared daily by diluting the standard solution at 0.5 µg mL−1 level in ultra-pure water and also in saliva, urine and blood plasma for some investigations.
2.3 Real samples Urine sample. A drug-free urine sample was collected from a healthy volunteer. The sample was stored in a glass vial which was carefully cleaned with hydrochloric acid and rinsed with deionized water and stored at 4 °C to prevent bacterial growth and proteolysis. The urine sample was spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations. A real urine sample was collected from a volunteer treated with Ami. The collection was done 10 hours after taking an Ami tablet comprising 25 mg of the drug. The sample was stored at 4 °C in a clean glass vial and analyzed a day after collection without any further pretreatment or dilution. The sampling procedures were performed according to the guidelines for research ethics. Plasma sample. Frozen drug-free human plasma samples (blood group +B) were obtained from the Iranian Blood Transfusion Organization (Tehran, Iran) and stored at −4 °C. The samples were allowed to thaw at room temperature, and then were shaken and diluted with deionized water (1:4) before extraction. The plasma sample was spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations.
7
Saliva sample. Drug-free saliva samples were collected from a healthy volunteer several times. The sample was stored in a glass vial which was carefully cleaned with hydrochloric acid, rinsed with deionized water, and stored at 4 °C to prevent bacterial growth and proteolysis. The saliva samples were spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations. 100 µL of the saliva sample was exposed to the on-disc EME-DLLME without any pretreatment or dilution. The sampling procedures were performed according to the guidelines for research ethics.
2.4 Chromatographic apparatus Optimization was performed on an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a FID detector. The injector was used in the splitless mode at 300 °C. The column used for separation of the analytes was a Varian wall coated fused silica capillary column (30 m, 0.25 mm, i.d., film thickness 0.25 µm). The FID temperature was fixed at 300 °C. Ultrapure helium and nitrogen (>99.999%) were used as the carrier and makeup gas at 1 mL.min-1 and 25 mL.min-1, respectively. Analysis of the standards related to calibration curves and real samples was performed on an Agilent 7890B GC with a 5977B MS equipped with a split/splitless injector. GC-MS separations were performed using an HP-5MS capillary column (30 m × 0.25 mm) with a film thickness of 0.25 µm. The mass spectrometer was operated in the electron impact ionization mode with an ionizing energy of 70 eV. The interface and ion source temperatures were both set at 300 °C. The oven temperature was initially set at 100 °C for 1 min and then was programed to 200 °C at 100 °C min-1 and was held at this temperature for 4 min. The temperature then increased to 260 ° C at 10 ° C min-1 followed by another increase to 280 ° C at 100 ° C min-1 and was held for 4 min. Helium (99.999%) was used as the carrier gas at a flow rate of 1 mL min-1. The solvent delay time was 10 min. The two model analytes were identified using the NIST database as well. Quantitative determination of the analytes in the standards and samples 8
were performed in the selective ion monitoring (SIM) mode of MS. The monitored ions of the analytes were selected on the basis of good selectivity and high sensitivity and were set as follows: Ami, m/z 58.1, 202.1 and Imi, m/z 58.0, 208.1, 234.0.
2.5 Design and fabrication of Lab-on-a-Disc The disc contains 5 layers and also 6 units for 6 parallel extractions. The disc was designed using AutoCAD software (Autodesk, Inc.). A schematic view of the disc and the fabricated disc is shown in Fig. 1A and C. A schematic of one extraction unit is also shown in Fig. 1B. The structure of the disc was cut on a 1 mm-thick poly(methyl methacrylate) (PMMA) sheet using a laser cutting device (Perfect laser Co., Ltd, China). Afterwards, all layers were cleaned with isopropanol, rinsed with DI water, and dried with nitrogen gas. The layers were fixed by chemical-thermal bonding. First, layers 1 and 2 were bonded. After that, a hole (0.7 mm D.) was drilled in the reservoir I of all the 6 units to insert the electrodes. A stainless steel wire (0.5 mm D.) as the electrode was mounted inside the reservoir I (Fig. 1B). The assembly was washed again with isopropanol, rinsed with DI water, and dried with nitrogen gas. A porous polypropylene membrane (about 1.5 × 1.5 cm) was placed above the reservoir I. Then, layers 3, 4, and 5 were respectively bonded to other layers. Before bonding layer 5 to layer 4, a stainless steel wire as an electrode was mounted to layer 5 through a hole drilled in this layer. The bonding procedure was as follows: Several microliters of ethanol was poured on the surface of the PMMA sheet. Then, the second layer was placed above it and aligned to match the right position. The assembly was then placed in an oven with pressure applied on it and was cured at 75 ºC for 40 min. Finally, a piece of a pressure sensitive adhesive (PSA) film was placed on the hole II (Fig. 1) embedded on layer 3.
2.6 On-Disc EME-DLLME procedure
9
Initially, the disc was fixed to the rotor of a homemade centrifugal driver. Before starting the extraction, NPOE was immobilized in the pores of the polypropylene membrane sheet. 100 µL of a 10 mM HCl solution, which is the acceptor solution during the EME process, was injected through the hole I into the reservoir I, so that the solution was placed on the electrode (cathode) mounted in this reservoir. Then, 100 µL of a donor phase (sample solution) was placed in the reservoir II on the polypropylene membrane sheet. The donor phase must be in contact with the electrode (anode) mounted in the last layer. The electrodes were then connected to a DC power supply. The voltages of 85 V for urine and plasma samples and 150 V for saliva samples were applied and extraction was performed for 15 min. The extraction was run without any agitation or convection of the donor solution. During the extraction, the target analytes migrated from the aqueous sample toward the acceptor solution placed in the reservoir I through the SLM. This step provides a proper clean-up for extraction of target analytes from complex matrices like biofluids. After the EME extraction was completed, a DLLME was done in the disc. The dispersive liquid-liquid extraction was implemented on the acceptor phase of the EME step. To do so, there was no need to manually transfer the acceptor phase of the EME step to perform a DLLME step. By rotating the disc, the acceptor phase of the EME step flowed to the extraction chamber for the next step. A mixture of 30 microliter of methanol (the disperser solvent) and 5 microliter of tetrachloroethene (the extraction solvent) was injected to the reservoir III. 5 microliter of a NaOH solution (pH = 12) was also introduced to the reservoir IV to adjust the pH of the donor phase in the DLLME step. Then, the disc rotated counter clockwise with the spin speed of 100 rpm for 2 min. As the disc rotated, centrifugal force caused the fluids to exist in the reservoirs I, III, and IV to flow toward the extraction chamber. It should be noted that the acceptor solution of the EME step, which was placed in the reservoir I, was used as the donor phase in the DLLME step. In this step, the extraction solvent was dispersed in the donor
10
phase (Fig. 2A). After 2 min, the spin speed increased to 3500 rpm and the device kept rotating for 4 min (See the video in ESI). As a result, a phase separation occurred and the extraction solvent, which was denser than the aqueous solution, sedimented at the conical end of the extraction chamber (Fig. 2B). The settled phase (about 2 µL) was then drawn into a Hamilton syringe by perforating the PSA film on the hole II and was injected manually into the GC/MS device. Each extraction unit was used only once.
2.7 Calculation of preconcentration factor, extraction recovery, and relative recovery Preconcentration factor (PF) was defined as the ratio of the final concentration of the analyte in the acceptor phase (Cf,a) to the initial concentration of the analyte (Ci,s). PF =
,
(1)
,
Where Cf,a was calculated from a calibration graph obtained from the direct injection of the standard solutions of the model analyte and Ci,s is the initial concentration of the analyte. The extraction recovery (ER) was defined as the percentage of the mole's number of the analyte extracted to the acceptor phase (nf,a) with respect to the number of moles of the same analyte originally present in the donor phase (ni,s). ,
ER% =
× 100 =
,
,
×
,
× ,
,
× 100
(2)
Relative recovery (RR) was calculated from the following equation: RR% =
× 100
(3)
Error% = RR% - 100
(4)
Where Cfound, Creal, and Cadded are the concentration of the analyte after addition of a known amount of the standard into the real sample, the concentration of the analyte in a real
11
sample, and the concentration of a known amount of the standard which was spiked into the real sample, respectively.
3. Results and discussion In this study, the on-disc EME-DLLME was developed. A schematic of the disc devise is illustrated in Fig. 1. A chip device provides the facility to implement consecutive extractions on a solitary chip with miniaturizing the whole procedure. Moreover, the extraction procedure is easy to handle and could be performed automatically. To show the applicability of the designed disc, two basic drugs were chosen to be extracted from biofluids. Amitriptyline and imipramine were selected as model analytes for this study. To reach the best extraction efficiency, the effective parameters of DLLME and EME were optimized.
3.1 Optimization of DLLME parameters 3.1.1 Extraction solvent: The extraction solvent has a vital role in the extraction efficiency of DLLME. The disc was designed in such a way that the extraction solvent could be collected at the end of the conical shape of the extraction chamber. For this purpose, the extraction solvent must have higher density than the aqueous solution. Therefore, chlorinated solvents are good candidates. On the other hand, considering the chemical resistance of PMMA, most of the chlorinated solvents damage the PMMA disc. Among chlorinated solvents, PMMA offers a substantial chemical resistance to C2Cl4 (perchloroethylene or tetrachloroethene) [39, 40]. As a result, C2Cl4 was chosen as the extraction solvent. 3.1.2 Volume of the extraction solvent: The critical importance of the volume of the extraction solvent in the DLLME efficiency is undeniable. Using a small amount of the extraction solvent could form a good emulsion, but this may not be sufficient to perform a proper extraction. On the other hand, a large quantity of the extraction solvent does not result in the formation of a proper emulsion. Therefore, it is important to find a balance between 12
emulsion formation and achieving good extraction efficiency. To investigate the effect of this parameter, different volumes of C2Cl4 were examined. The results depicted in Fig. 3A show that the response increased by increasing the volume of the extraction solvent up to 5 µL. Further increase in the volume of the extraction solvents led to a decline in the responses, which was probably due to the dilution effect. In volumes lower than 5 µL, the collection of the settled phase was faced with some difficulties. In addition, repeatability of the results was reduced by using a lower volume of the extraction solvent. A volume of 5 µL was used for the subsequent experiments. 3.1.3 Volume of the disperser solvent: DLLME is based on a ternary component solvent system: the aqueous solution, the disperser solvent, and the extraction solvent. These solvents can form an emulsion. A sufficient amount of the disperser solvent is important in complete formation of emulsion, so the volume of the disperser solvent has a critical role in any DLLME procedure. To investigate the effect of the volume of the disperser solvent, a series of volumes were considered. As shown in Fig. 3B, by increasing the volume of the disperser solvent from 10 to 30 µL, an enhancement in the responses can be observed. A further increase in the volume of the disperser solvent has no significant effect on the responses. 3.1.4 pH: In DLLME, the best extraction can be done when the analytes are in their neutral forms. Two worthy tricks to make analytes neutral are changing the pH of the donor phase and adding a proper ion pair agent. In this work, as the EME step is performed first, the donor solution (the acceptor phase of the EME step) was acidified. In this work, 5 microliters of a NaOH solution (pH = 12) was introduced to the reservoir IV to adjust the pH of the donor solution in the DLLME step. 3.1.5 Spin speed: The main advantage of DLLME is formation of fine droplets. Formation of the droplets leads to an increase in the contact area between an extraction 13
solvent and an aqueous solution, which causes faster distribution of the analytes between the extraction solvent and the aqueous solution. As a result a very fast extraction of the analytes into the extraction solvent can occur. To form such droplets, the organic solvent mixture (the mixture of an extraction solvent and a disperser solvent) must be injected quickly into an aqueous solution. The fast injection of the organic solvent mixture can affect the size of the droplets and subsequently the extraction efficiency by increasing the contact area. In the constructed disc, the mixture of the organic solvents are introduced into the reservoir III. The reservoir is connected to the extraction chamber by a channel. As the disc rotates, centrifugal force makes the organic solvent mixture flow to the extraction chamber. The aqueous solution (the acceptor phase of the EME step, discussed in the on-disc EME-DLLME procedure section) flows toward the extraction chamber as well. As a result, the organic solvent mixture is injected into the aqueous solution and dispersion occurs (Fig. 2A). Faster spin speed leads to faster dispersion of the organic solvent mixture into the aqueous solution and the formation of finer droplets. Thus, the spine speed can affect the extraction performance. To investigate this parameter, several spin speeds were examined. Fig. 3C shows the results. Increasing the spin speed up to 100 rpm increases the extraction efficiency as well. By increasing the spin speed more than 100 rpm, no significant increase in the extraction efficiency was observed. To compare the size of the droplets in the dispersion step, the droplets formed at different spin speeds were imaged using a light microscopy (at 40X magnification). As can be seen in Fig. 4, by increasing the spin speed up to 100 rpm, the size of the droplets decreased and no significant change in the size of the droplets was observed at spin speeds higher than 100 rpm. As a result, 100 rpm was chosen as the optimum spin speed of the disc for the dispersion step in DLLME.
3.2 Optimization of EME parameters
14
In this study, the EME step is a drop-to-drop electromembrane extraction. Based on the literature [12, 41], NPOE was used as the supported liquid membrane (SLM). NPOE was directly injected by a syringe on the surface of polyprpylen sheet. 100 µL of the sample solution (the donor phase) comprising the target analytes was placed on the polypropylene membrane sheet and remained stagnant during the extraction. 100 µL of an aqueous solution was also used as the acceptor phase. Other effective parameters were optimized as follows: 3.2.1 Donor and acceptor phase solutions: EME is a microextraction method which uses an electrical field as a driving force in extraction of ionized compounds. The extraction mechanism is mainly based on electrokinetic migration. Neutral compounds will not be affected by the electrical field, so to extract target compounds, they must be in their ionic form [6, 42]. By changing the pH value, acidic and basic compounds can be converted to their ionic or neutral forms. Since the pH values for ionization of basic compounds are different from the ones for acidic compounds, by adjusting a proper pH value, EME would be able to selectively extract acidic or basic compounds. This is one of the advantages of the electromembrane microextraction method. Moreover, the pH level of donor and acceptor phases plays a pivotal role in the EME extraction performance. Therefore, this parameter should be optimized to reach the highest extraction recoveries. To reach the optimum value of this parameter, the pH values of the donor and acceptor phases were studied by changing the concentration of HCl in the range of 0-10 mM and 0-30 mM in the donor and acceptor phases, respectively. The results for Ami and Imi are presented in Fig. 5A and Fig. S1. In this work, two basic drugs were used as the model analytes. The sample solution (the donor phase) should be acidic enough so that the target analytes would carry a net positive charge. Additionally, the acceptor solution should be acidic enough to prevent deprotonation of drugs during the extraction and thus their back-diffusion toward the donor phase [7, 24]. As can be seen in Fig. 5A, the best results were obtained when the 15
concentration of HCl in the acceptor phase was 10 mM and no HCl was added to the donor phase (0 mM HCl). A decrease in the extraction efficiency was observed by increasing the concentration of H+ in both the donor and acceptor phases. Using 10 mM HCl in the donor phase and 20 and 30 mM HCl in the acceptor phase resulted in instability in the SLM and bubble formation on the electrodes. All of these indicated that high concentration of H+ led to an increase in the current level and subsequently to an increase in probability of electrolysis reactions in both the donor and acceptor phases. 3.2.2 Voltage and time: As already mentioned, an electrical field is the main driving force in EME. It has been proven that the migration of analytes across the SLM is affected by the magnitude of the applied voltage [43, 44]. Thus, this parameter has an important role that must be investigated. In this work, first, a voltage in the range of 70-240V was applied to the system and ultra-pure water was used to prepare the working solutions. It was observed that 220 V was the optimum voltage in that media (Fig. S2). The goal of this work is to extract some model analytes from biological fluids. It is known that biological fluids like urine and blood plasma contain a high concentration of salts. In EME, as voltage is applied across an SLM, all ionic species in the donor and acceptor solutions move electrokinetically toward their relative electrodes. As a result, a boundary layer of ions is formed at the interface on both sides of the SLM. The existence of a high concentration of ionic species in biological fluids leads to a significant increase in the number of ions migrating through SLM at a given moment. So the thickness of the boundary layer and the current level increase. This also results in Joule heating and therefore instability of SLM [7]. As the electrical field is the main driving force in EME and increasing the magnitude of the applied voltage increases the current level, the applied voltage used in the aqueous media is not usable in biological fluids. Therefore, the optimization of the voltage was repeated in the urine, blood plasma, and saliva media again. Since the mass transfer is time-dependent, a simultaneous investigation of time 16
and applied voltage was performed. The results of the effects of the applied voltage on the extraction efficiency of Ami and Imi in the urine media are shown in Fig. 5B and Fig. S3, respectively. The best extractability of the model analytes in the urine samples were obtained at 85 V for 15 min. As can be seen, by increasing the applied voltage and time, the extraction efficiency increased as well. At 85 V after 20 min, Joule heating and instability of the SLM led to a decrease in the extraction efficiency. By performing the extraction at 105 V for 5 min, the same conditions were observed. Therefore, it was not possible to continue the extraction at 105 V more than 5 min. The same results were observed in the blood plasma media too. Thus, 85 V for 15 min was chosen as the optimum values in both the urine and blood plasma media (Fig. S4). A set of experiments were conducted on the saliva samples (Fig. S5) and 150 V for 15 min was selected as the optimum applied voltage and time. 3.2.3 Investigation of applying a pulsed voltage. As it was reported in previous conventional EME works, applying constant voltages, during relatively long extraction times and/or under high applied voltages pose some problems [45, 46]. To overcome these problems, using a pulsed voltage was proposed by Yamani’s research group in 2012 [47]. In this work, the suitability of using pulsed voltage for a drop-to-drop EME was investigated. In this work, outage duration cannot have any significant influence on the thickness of the charged double layer, but it decreases the whole applied voltage time instead, and so the extraction efficiency decreases (Fig. S6). The detailed results can be found in the supporting information.
3.3 Method evaluation To validate the on-disc EME-DLLME system under optimized conditions (Table 2), Ami and Imi were spiked in the drug-free urine, blood plasma, saliva, and also pure water at different concentrations. Table 3 provides the summery of the method evaluation results.
17
Calculations were made based on equations 1-4. The results showed that the on-disc EMEDLLME system can be effectively employed for analysis of model analytes even in complicated matrices such as biofluids. A good linearity was obtained with a coefficient of determination (R2) more than 0.9959. The limits of detection (LOD) of the on-disc device were in the range of 0.1-0.5 µg L-1 for Ami and Imi in different biofluids with relative standard deviations (RSDs) lower than 3.5%. A comparison of the analytical performance of the proposed method with other analytical reports is presented in Table 4. Although the proposed method used much less donor solution than other methods, it reached LODs which were comparable with other methods. In addition, integration of two sample preparation methods (EME and DLLME) in one chip provides a semiautomatic chip device with easy handling, fast operating, good repeatability and reproducibility at a lower cost and with a smaller amount of the donor solution and organic solvent as well. The method also benefits from the advantages of both analytical methods. Consequently, the proposed method can be used for extraction and determination of low levels of Ami and Imi in different biofluids.
3.4 Validation of the on-disc EME-DLLME method for analysis of Ami and Imi in some biofluids To evaluate the practical suitability of the proposed method for determination of drugs in real biological samples, Ami and Imi as model drugs were determined in three biofluids (urine, blood plasma, and saliva). The results are presented in Table 5. Urine sample: The on-disc system was used for the analysis of the model drugs in the urine sample of a patient undergoing therapy with Ami to assess the applicability of the method. Sample preparation is already explained in the “real sample” section. In summary, a urine sample was collected after 10 hours of taking an Ami tablet comprising 25 mg of the drug. Accuracy was evaluated as the parameter of error %, so the urine sample was spiked with 150 µgL-1 Ami and Imi. RSD% values indicated acceptable precision related to the 18
proposed system. Furthermore, as can be seen in Table 5, error% was less than +3.9%. Thus accuracy was acceptable, and no significant matrix effect was observed for determination of the model drugs using the proposed on-disc system. A typical chromatogram, obtained from the extraction of the urine sample before and after the addition of a certain concentration of the model drugs, is illustrated in Fig. 6. Blood plasma: The validity of the on-disc system was assessed by extracting the model drugs from a blood plasma sample. As described before, the blood plasma sample was diluted 1:4 before extraction. The blood plasma samples were spiked with 50 µgL-1 of Ami and Imi. The results shown in Fig. S7 and Table 5 indicate efficient extraction of the analytes. During the EME step, it was seen that the SLM showed good stability. Saliva: Applicability of the method was assessed in the saliva samples as well. Sample preparation is already explained in the “real sample” section. A saliva sample was spiked with 10 µg L-1 of Ami and Imi. As Table 3 shows, the method has acceptable accuracy with error% less than +2.7. Acceptable precision based on RSD% values was also achieved. A topical chromatogram of the blank and spiked saliva sample is shown in Fig. S8. Furthermore, as can be seen in Table 5, error% was less than +2.9, accuracy was acceptable, and no significant matrix effect was observed for determination of the model drugs using the proposed on-disc system.
4. Conclusions The present work demonstrated the design of a lab-on-a-disc device for performing a combination of two LPME methods, EME and DLLME. The system was simple and the component had a low cost, as each extraction unit was used only once. The system benefits from advantages of both microextraction methods as well as advantages of miniaturization on a lab-on-a-disc device. Using a drop-to-drop EME, the great clean-up potential of EME can be used for extraction of target analytes from complex matrices. Utilizing a DLLME also 19
brings good preconcentration factors and GC compatibility of the final extracted phase. Implementing the EME-DLLME method on a lab-on-a-disc device has some significant advantages over the conventional method. Performing the extraction method on a chip device makes it possible to use a small amount of the donor solution, which is not possible in conventional extraction methods. EME and DLLME processes are performed on the same disc, so there is no need to manually transfer the acceptor solution of EME to conduct a DLLME step. Using a centrifugal platform, the organic solvent mixture can be automatically injected into the donor solution in the DLLME step by simply rotating the disc. Thanks to the scalable centrifugal force, injection can be performed with different speeds. In this work, we showed that the injection speed could influence extraction efficiency by affecting the size of the droplets. Finally, sedimentation of the extraction phase occurs by increasing the spin speed. The system has the potential to run fully-automatically and be miniaturized more by changing the detection method, which can be considered as the main limitation of such systems for miniaturization. More studies focusing on fully automated on-disc extraction and development of a sample-to-answer system are in progress.
References [1] F.F. Cantwell, M. Losier, Chapter 11 Liquid—liquid extraction, Analytical Chemistry, Elsevier, 2002, pp. 297-340.
Comprehensive
[2] S. Tang, H. Zhang, H.K. Lee, Advances in sample extraction, Anal. Chem. 88 (2016) 228-249. [3] A. Sarafraz-Yazdi, A. Amiri, Liquid-phase microextraction, TRAC, Trends Anal. Chem. 29 (2010) 1-14. [4] M. Rezaee, Y. Assadi, M.-R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, Determination of organic compounds in water using dispersive liquid–liquid microextraction, J. Chromatogr. A 1116 (2006) 1-9. [5] M. Rezaee, Y. Yamini, M. Faraji, Evolution of dispersive liquid–liquid microextraction method, J. Chromatogr. A 1217 (2010) 2342-2357. [6] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrokinetic migration across artificial liquid membranes: New concept for rapid sample preparation of biological fluids, J. Chromatogr. A 1109 (2006) 183-190. 20
[7] Y. Yamini, S. Seidi, M. Rezazadeh, Electrical field-induced extraction and separation techniques: Promising trends in analytical chemistry – A review, Anal. Chim. Acta 814 (2014) 1-22. [8] A. Gjelstad, S. Pedersen-Bjergaard, Electromembrane extraction—Three-phase electrophoresis for future preparative applications, Electrophoresis 35 (2014) 2421-2428. [9] C. Basheer, J. Lee, S. Pedersen-Bjergaard, K.E. Rasmussen, H.K. Lee, Simultaneous extraction of acidic and basic drugs at neutral sample pH: A novel electro-mediated microextraction approach, J. Chromatogr. A 1217 (2010) 6661-6667. [10] M. Rezazadeh, Y. Yamini, S. Seidi, B. Ebrahimpour, Electromembrane surrounded solid phase microextraction: A novel approach for efficient extraction from complicated matrices, J. Chromatogr. A 1280 (2013) 16-22. [11] L. Guo, H.K. Lee, Electro membrane extraction followed by low-density solvent based ultrasound-assisted emulsification microextraction combined with derivatization for determining chlorophenols and analysis by gas chromatography–mass spectrometry, J. Chromatogr. A 1243 (2012) 14-22. [12] S. Seidi, Y. Yamini, M. Rezazadeh, Combination of electromembrane extraction with dispersive liquid–liquid microextraction followed by gas chromatographic analysis as a fast and sensitive technique for determination of tricyclic antidepressants, J. Chromatogr. B 913914 (2013) 138-146. [13] L. Arjomandi-Behzad, Y. Yamini, M. Rezazadeh, Extraction of pyridine derivatives from human urine using electromembrane extraction coupled to dispersive liquid–liquid microextraction followed by gas chromatography determination, Talanta 126 (2014) 73-81. [14] M. Khajeh, S. Pedersen-Bjergaard, M. Bohlooli, A. Barkhordar, M. GhaffariMoghaddam, Maghemite nanoparticle-decorated hollow fiber electromembrane extraction combined with dispersive liquid–liquid microextraction for determination of thymol from Carum copticum, J. Sci. Food Agr. 97 (2017) 1517-1523. [15] P.A.L. Wijethunga, Y.S. Nanayakkara, P. Kunchala, D.W. Armstrong, H. Moon, Onchip drop-to-drop liquid microextraction coupled with real-time concentration monitoring technique, Anal. Chem. 83 (2011) 1658-1664. [16] N. Wang, S. Mao, W. Liu, J. Wu, H. Li, J.-M. Lin, Online monodisperse droplets based liquid–liquid extraction on a continuously flowing system by using microfluidic devices, RSC Adv. 4 (2014) 11919-11926. [17] I. Vural Gürsel, S.K. Kurt, J. Aalders, Q. Wang, T. Noël, K.D.P. Nigam, N. Kockmann, V. Hessel, Utilization of milli-scale coiled flow inverter in combination with phase separator for continuous flow liquid–liquid extraction processes, Chem. Eng. J. 283 (2016) 855-868. [18] S.K. Kurt, I. Vural Gürsel, V. Hessel, K.D.P. Nigam, N. Kockmann, Liquid–liquid extraction system with microstructured coiled flow inverter and other capillary setups for single-stage extraction applications, Chem. Eng. J. 284 (2016) 764-777. [19] N.J. Petersen, J.S. Pedersen, N.N. Poulsen, H. Jensen, C. Skonberg, S.H. Hansen, S. Pedersen-Bjergaard, On-chip electromembrane extraction for monitoring drug metabolism in real time by electrospray ionization mass spectrometry, Analyst 137 (2012) 3321-3327. [20] P. Mary, V. Studer, P. Tabeling, Microfluidic droplet-based liquid−liquid extraction, Anal. Chem. 80 (2008) 2680-2687.
21
[21] M.D. Ramos Payán, H. Jensen, N.J. Petersen, S.H. Hansen, S. Pedersen-Bjergaard, Liquid-phase microextraction in a microfluidic-chip – High enrichment and sample clean-up from small sample volumes based on three-phase extraction, Anal. Chim. Acta 735 (2012) 46-53. [22] P. Znidarsic-Plazl, I. Plazl, Steroid extraction in a microchannel system-mathematical modelling and experiments, Lab Chip 7 (2007) 883-889. [23] N.J. Petersen, S.T. Foss, H. Jensen, S.H. Hansen, C. Skonberg, D. Snakenborg, J.r.P. Kutter, S. Pedersen-Bjergaard, On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection, Anal. Chem. 83 (2010) 44-51. [24] Y.A. Asl, Y. Yamini, S. Seidi, B. Ebrahimpour, A new effective on chip electromembrane extraction coupled with high performance liquid chromatography for enhancement of extraction efficiency, Anal. Chim. Acta 898 (2015) 42-49. [25] M. Karami, Y. Yamini, Y. Abdossalami Asl, M. Rezazadeh, On-chip pulsed electromembrane extraction as a new concept for analysis of biological fluids in a small device, J. Chromatogr. A 1527 (2017) 1-9. [26] K. Wang, G. Luo, Microflow extraction: A review of recent development, Chem. Eng. Sci. 169 (2017) 18-33. [27] C. Xu, T. Xie, Review of microfluidic liquid–liquid extractors, Ind. Eng. Chem. Res. 56 (2017) 7593-7622. [28] D. Ciceri, J.M. Perera, G.W. Stevens, The use of microfluidic devices in solvent extraction, J. Chem. Technol. Biotechnol. 89 (2014) 771-786. [29] N. Assmann, A. Ładosz, P. Rudolf von Rohr, Continuous micro liquid-liquid extraction, Chem. Eng. Technol. 36 (2013) 921-936. [30] B.C. Giordano, D.S. Burgi, S.J. Hart, A. Terray, On-line sample pre-concentration in microfluidic devices: A review, Anal. Chim. Acta 718 (2012) 11-24. [31] T. Vilkner, D. Janasek, A. Manz, Micro total analysis systems. Recent developments, Anal. Chem. 76 (2004) 3373-3386. [32] M. Madou, J. Zoval, G. Jia, H. Kido, J. Kim, N. Kim, LAB ON A CD, Annu. Rev. Biomed. Eng. 8 (2006) 601-628. [33] C. Li, X. Dong, J. Qin, B. Lin, Rapid nanoliter DNA hybridization based on reciprocating flow on a compact disk microfluidic device, Anal. Chim. Acta 640 (2009) 9399. [34] M. Vázquez, D. Brabazon, F. Shang, J.O. Omamogho, J.D. Glennon, B. Paull, Centrifugally-driven sample extraction, preconcentration and purification in microfluidic compact discs, TRAC,Trends Anal. Chem. 30 (2011) 1575-1586. [35] E. Duffy, R. Padovani, X. He, R. Gorkin, E. Vereshchagina, J. Ducrée, E. Nesterenko, P.N. Nesterenko, D. Brabazon, B. Paull, M. Vázquez, New strategies for stationary phase integration within centrifugal microfluidic platforms for applications in sample preparation and pre-concentration, Anal. Methods 9 (2017) 1998-2006. [36] G. Duffy, I. Maguire, B. Heery, P. Gers, J. Ducrée, F. Regan, ChromiSense: A colourimetric lab-on-a-disc sensor for chromium speciation in water, Talanta 178 (2018) 392399.
22
[37] A. Kazarine, M.C.R. Kong, E.J. Templeton, E.D. Salin, Automated liquid–liquid extraction by pneumatic recirculation on a centrifugal microfluidic platform, Anal. Chem. 84 (2012) 6939-6943. [38] L. Clime, D. Brassard, M. Geissler, T. Veres, Active pneumatic control of centrifugal microfluidic flows for lab-on-a-chip applications, Lab Chip 15 (2015) 2400-2411. [39] a.R.P.C. eplastics, USA. [40] D.O.M.M. CORNELL UNIVERSITY USA, Sevier Lab. [41] S.S.H. Davarani, A.M. Najarian, S. Nojavan, M.-A. Tabatabaei, Electromembrane extraction combined with gas chromatography for quantification of tricyclic antidepressants in human body fluids, Anal. Chim. Acta 725 (2012) 51-56. [42] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Electrokinetic migration across artificial liquid membranes: tuning the membrane chemistry to different types of drug substances, J. Chromatogr. A 1124 (2006) 29-34. [43] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Simulation of flux during electromembrane extraction based on the Nernst–Planck equation, J. Chromatogr. A 1174 (2007) 104-111. [44] A. Gjelstad, T.M. Andersen, K.E. Rasmussen, S. Pedersen-Bjergaard, Microextraction across supported liquid membranes forced by pH gradients and electrical fields, J. Chromatogr. A 1157 (2007) 38-45. [45] M. Balchen, L. Reubsaet, S. Pedersen-Bjergaard, Electromembrane extraction of peptides, J. Chromatogr. A 1194 (2008) 143-149. [46] M. Eskandari, Y. Yamini, L. Fotouhi, S. Seidi, Microextraction of mebendazole across supported liquid membrane forced by pH gradient and electrical field, S. J. Pharm. Biomed. Anal 54 (2011) 1173-1179. [47] M. Rezazadeh, Y. Yamini, S. Seidi, A. Esrafili, Pulsed electromembrane extraction: A new concept of electrically enhanced extraction, J. Chromatogr. A 1262 (2012) 214- 218. [48] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Electromembrane extraction of basic drugs from untreated human plasma and whole blood under physiological pH conditions, Anal. Bioanal. Chem. 393 (2009) 921-928. [49] M. Rezazadeh, Y. Yamini, S. Seidi, L. Arjomandi-Behzad, Voltage-step pulsed electromembrane as a novel view of electrical field-induced liquid-phase microextraction, J. Chromatogr. A 1324 (2014) 21-28. [50] M. Bazregar, M. Rajabi, Y. Yamini, Z. Saffarzadeh, A. Asghari, Tandem dispersive liquid–liquid microextraction as an efficient method for determination of basic drugs in complicated matrices, J. Chromatogr. A 1429 (2016) 13-21. [51] S.S.H. Davarani, A. Morteza-Najarian, S. Nojavan, A. Pourahadi, M.B. Abbassi, Twophase electromembrane extraction followed by gas chromatography-mass spectrometry analysis, J. Sep. Sci. 36 (2013) 736-743.
23
Table 1. Chemical structures, pKa and log P of the model analytes Chemical structure
Compound
Abbreviation
pKa
log P
Amitriptyline
Ami
9.4
4.9
Imipramine
Imi
9.2
4.2
24
Table 2. Optimum conditions for extraction of model analytes by the on-disc EME-DLLME system
DLLME
EME
Extractive solvent
C2Cl4
Supported liquid membrane (SLM)
NPOE
Volume of extractive solvent
5 µL
Donor phase solution
Without adding HCl
Volume of disperser solvent
30 µL
Acceptor phase solution
10 mM HCl
pH
Spin speed
12
100 rpm
Voltage
Time
Water
220 V
Urine & Plasma
85 V
Saliva
150 V 15 min
25
Table 3. Analytical performance of on-disc EME-DLLME for determination of Ami and Imi in water, saliva, urine, and blood plasma samples
Sample
Analyte
LODa
LOQ
Linearity
(µg L-1)
(µg L-1)
(µg L-1)
R2
PFb
ER%
RSD% (n= 5)
Water
Ami Imi
0.1 0.1
0.25 0.5
0.25-500 0.5-500
0.9959 0.9974
35.4 32.1
70.8 64.2
1.9 2.0
Saliva
Ami Imi
0.25 0.5
0.5 1.0
0.5-500 1.0-500
0.9977 0.9984
31.2 28.2
62.5 56.4
2.7 2.4
Urine
Ami Imi
0.5 0.5
1.0 1.0
1.0-500 1.0-500
0.9987 0.9977
32.9 29.0
65.8 58.0
2.9 3.2
Blood plasma
Ami Imi
0.5 0.5
1.0 1.0
1.0-500 1.0-500
0.9979 0.9994
29.9 21.5
59.9 43.0
3.1 3.5
a) LODs of blood plasma is related to 1:4 diluted sample. b) Preconcentration factor at 50 µg L-1 of analytes
26
27
Table 4. Comparison of figures of merit of on-disc EME-DLLME with other methods for determination of Ami and Imi LOD (µg L-1)
LOQ (µg L-1)
Urine
3.0
Ami
Water
EM-SPME -GC-FID
Ami
EM-SPME-GC-FID EM-SPME-GC-FID
Linearity (µg L-1)
ER
Volume of donor phase (µL)
Reference
10.0
10.0-500
36
1000
[24]
0.5
1.0
1.0-500
-
2500
[49]
Water
0.5
2.0
-
11.5
24000
[10]
Ami
Urine
1.0
2.5
-
10.4
24000
[10]
Ami
Plasma
1.0
5.0
-
5.9
24000
[10]
EME-DLLME _GC-FID
Ami
Water
0.25
2.0
2.0-500
-
24000
[12]
EME-DLLME_GC-FID
Ami
Urine
3.0
10.0
10.0-500
-
24000
[12]
EME-DLLME_GC-FID
Ami
Plasma
15.0
40.0
40.0-500
-
24000
[12]
TDLLME -HPLC-UV
Ami
Plasma
1.0
-
3.0-5000
8000
[50]
On-Disc EME-DLLME-GC-MS
Ami
Water
0.1
0.25
0.25-500
70.8
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Saliva
0.25
0.5
0.5-500
62.5
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Urine
0.5
1.0
1.0-500
65.8
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Plasma
0.5
1.0
1.0-500
59.9
100
This work
EME-GC-FID
Imi
Water
0.35
-
2.0-1500
-
2100
[41]
EME-GC-FID
Imi
Urine
0.40
-
2.0-1500
-
2100
[41]
EME-GC-FID
Imi
Plasma
0.50
-
2.0-1500
-
2100
[41]
Two-phase EME-GS-MS
Imi
Water
0.10
-
1.0-500
-
1200
[51]
TDLLME -HPLC-UV
Imi
Plasma
0.9
-
3.0-5000
-
8000
[50]
On-Disc EME-DLLME-GC-MS
Imi
Water
0.1
0.5
0.5-500
64.2
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Saliva
0.5
1.0
1.0-500
56.4
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Urine
0.5
1.0
1.0-500
58.1
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Plasma
0.5
1.0
1.0-500
43.1
100
This work
Analytical method
Analyte
Matrix
CEMEa-HPLC-UV
Ami
b
PEME -HPLC-UV c
d
e
e
a
On chip electromembrane extraction
b
Pulsed electromembrane extraction
28
c
Electromembrane solid phase microextraction
d
Electromembrane extraction- Dispersive liquid-liquid microextraction
e
Tandem dispersive liquid–liquid microextraction
29
Table 5. Determination of Ami and Imi in the real samples using on-disc EME-DLLME.
Sample
Analyte
Creal (µg L-1)
Saliva
Ami
Nda
10.0
10.3
2.1
+2.9
Imi
Nd
10.0
9.8
2.5
-2.1
Ami
144.3
150.0
291.0
2.6
-2.2
Imi
Nd
150.0
155.8
3.3
+3.9
Ami
Nd
50.0
52.9
3.2
+5.8
Imi
Nd
50.0
50.8
3.9
+1.6
Urine
Blood Plasma
a
Cadded (µg L-1)
Not detected
30
Cfound (µg L-1)
RSD%
Error%
Figure captions Fig. 1. A) A schematic view of the designed LOD device consists of five layers. B) A schematic of one of the extraction units in Layer 1. C) Fabricated LOD device. Fig. 2. Desperation (A) and sedimentation of the extraction solvent (B) in the DLLME step. For clarity, a colored aqueous solution was used. Fig. 3. Optimization of the effective parameters in the DLLME step. In this step, C2Cl4 and methanol were used as the extraction and disperser solvents, respectively. A) Volume of the extraction solvent. B) Volume of the disperser solvent. C) Spin speed. In the EME step, 180 V was applied for 15 min without adding any acid or base to the donor and acceptor phases. Fig. 4. Effect of spin speed on the size of the formed droplets in the DLLME step. Fig. 5. Optimization of the effective parameters in the EME step. A) Composition of the donor and acceptor phases. B) Effect of applied voltage at different extraction times. For the DLLME step, the optimized parameters were used. Fig. 6. A typical GC/MS chromatogram obtained from the extraction of the model analytes from the plasma samples before and after spiking 50 µg L-1 of the analytes. The GC separation conditions were reported in section 2.4.
31
Fig. 1.
32
Fig. 2.
33
Fig. 3.
34
Fig. 4.
35
Fig. 5. 36
37
Fig. 6.
38
Highlights A lab-on-a-disc device was designed for sequential performing of EME and DLLME. All of DLLME steps can be fulfilled on the chip by simply controlling the spin speed. EME-DLLME was used for extraction of tricyclic antidepressants in biofluids. The effective parameters on the EME and DLLME were investigated and optimized. The chip is easy to handle without need to any skillful operator. The device provides effective and reproducible extraction using a low volume of sample.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0003-2670(20)30059-3
DOI:
https://doi.org/10.1016/j.aca.2020.01.024
Reference:
ACA 237383
To appear in:
Analytica Chimica Acta
Received Date: 16 November 2019 Revised Date:
10 January 2020
Accepted Date: 12 January 2020
Please cite this article as: M. Karami, Y. Yamini, On-disc electromembrane extraction-dispersive liquid-liquid microextraction: A fast and effective method for extraction and determination of ionic target analytes from complex biofluids by GC/MS, Analytica Chimica Acta, https://doi.org/10.1016/ j.aca.2020.01.024. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier B.V. All rights reserved.
CRediT author statement Monireh Karami: Methodology, Investigation, Validation, Writing-Original draft preparation. Yadollah Yamini: Supervision, Reviewing and Editing
For TOC only
On-disc electromembrane extraction-dispersive liquid-liquid microextraction: A fast and effective method for extraction and determination of ionic target analytes from complex biofluids by GC/MS
Monireh Karami and Yadollah Yamini*1 Department of Chemistry, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
1
∗
Corresponding author: Tel.: +98 21 82883449; Fax: +98 21 82883460.
E-mail address: [email protected] (Y. Yamini).
1
Abstract In this study, an electromembrane extraction-dispersive liquid-liquid microextraction (EME-DLLME) was performed using a lab-on-a-disc device. It was used for sample microextraction, preconcentration, and quantitative determination of tricyclic antidepressants as model analytes in biofluids. The disc consisted of six extraction units for six parallel extractions. First, 100 µL of a biofluid was used to extract the analytes by the drop-to-drop EME to clean-up the sample. The extraction then was followed by applying the DLLME method to preconcentrate the analytes and make them ready for being analyzed by gas chromatography (GC). Implementing the EME-DLLME method on a chip device brought some significant advantages over the conventional methods, including saving space, cost, and materials as well as low sample and energy consumption. In the designed device, centrifugal force was used to move the fluids in the disc. Both sample preparation methods were performed on the same disc without manual transference of the donor phases for doing the two methods. Scalable centrifugal force made it possible to adjust the injection speed of the organic solvent into the aqueous solution in the DLLME step by changing the spin speed. Spin speed of 100 rpm was used in dispersion step and spin speed of 3500 rpm was used to sediment organic phase in DLLME step. The proposed device provides effective and reproducible extraction using a low volume of the sample solution. After optimization of the effective parameters, an EME-DLLME followed by GC-MS was performed for determination of amitriptyline and imipramine in saliva, urine, and blood plasma samples. The method provides extraction recoveries and preconcentration factors in the range of 43%70.8% and 21.5-35.5 respectively. The detection limits less than 0.5 µg L-1 with the relative standard deviations of the analysis which were found in the range of 1.9%-3.5% (n = 5). The method is suitable for drug monitoring and analyzing biofluids containing low levels of the model analytes. 2
Keywords: Electromembrane extraction; Dispersive liquid-liquid extraction; Lab-on-a-Disc; Centrifugal microfluidic platform; µCD platforms; Tricyclic antidepressant.
1. Introduction Liquid-liquid extraction (LLE) is the most extensively used sample preparation technique in chemistry and biochemistry [1]. Despite its widespread use, traditional LLE suffers from consumption of large quantities of toxic organic solvents and being tedious as well as time consuming. Due to global concerns about environmental pollution; the need to develop environment-friendly practices has been heighted in different areas of research. Attempts in the field of analytical chemistry to develop methods using organic solvents as less as possible led to introduction of different liquid phase microextraction methods (LPME). LPME methods also give rise to minimizing the overall extraction time [2, 3]. Dispersive liquid-liquid microextraction (DLLME) is one of the popular microextraction methods introduced in 2006 by Rezaee et al. [4]. Rapid injection of an appropriate mixture of extraction and disperser solvents into an aqueous donor phase containing the desired analyte/analytes makes a cloudy solution and a multitude of fine droplets of the extraction solvent forms in the solution. Formation of these fine droplets is the most significant advantage of DLLME, which causes the extraction to be obtained very fast [5]. Moreover, the method is compatible with most analytical instruments. The features of being fast and capable of reaching high preconcentration factors make DLLME receive analytical chemists’ interests [5]. However, DLLME suffers from some disadvantages. Perhaps the most serious disadvantage of the method is the lack of sample clean-up because of which this method can be only efficiently applied to simple matrices. Electromembrane extraction (EME) is one of LPME techniques based on electrokinetic migration of ionized analytes from an aqueous donor phase across a supported liquid 3
membrane to an acceptor solution. Since it was reported in 2006 [6], a considerable amount of literature has been published on application of EME [7, 8]. Since EME is a membrane based extraction, it provides a good sample clean-up, which makes this method capable of being directly used in complex sample matrices such as biofluids. The acceptor solution in EME is mostly an aqueous solution. Thus the main drawback of the method is its incompatibility in to gas chromatography (GC) instrument. While GC is faster, cheaper, and simpler than liquid chromatography (LC) and can be easily conjugated with different kinds of detectors such as flame ionization detector (FID) and mass spectrometer (MS). Some attempts have been made so far to transfer the extracted analytes of EME to a GC-compatible phase [9-14]. DLLME is the method which can be properly combined with EME. Combination of EME with DLLME provides a two-step extraction method that benefits from great sample clean-up of EME as well as high preconcentration factors of DLLME. Another advantage is that the extracts can be analyzed by GC. In 2012, Guo developed an electromembrane extraction followed by low-density solvent based ultrasound-assisted emulsification microextraction (EME–LDS-USAEME) using 100 mL of sample solution. [11]. The proposed method showed a good limit of detection and linearity for extraction of trace levels of chlorophenols; however, the two-step extraction method with manual transference of the donor phase can affect repeatability and precision of the extraction and make the method tedious and hard to operate. Moreover, although the method provides very good sample clean-up, it needs a large quantity of a donor phase. Therefore, it seems that the method is not suitable for analysis of biofluids. Seidi et al. reported a method to combine EME and DLLME to determine tricyclic antidepressants in biological samples [12]. They used 24 mL of a donor phase in an EME step with the acceptor solution volume of 10 µL.. However, as it was the case with the previously mentioned work, manual handling and transference of phases can influence repeatability and precision of the method. Also using 24 4
mL of a donor phase still seems too much for some of biological fluids. Some other works have been done to couple EME with DLLME [13, 14]. They had the same drawbacks as were mentioned before. General advantages of miniaturization including the capability to save time, space, cost, and materials lead microfluidic systems to become a necessity in today’s world. Keeping company with many areas of science, the last three decades have seen a growing interest in miniaturization and automation of classical analytical operations. The main motivation for such miniaturization is the development of micro total analysis systems (µTAS) or lab-on-achip (LOC) technology. Furthermore, automation leads to the development of devices for performing laboratory processes which are easy to handle without any need for a skillful operator. To date plenty of attempts have been made to down-scale and implement LLE and also LPME on microfluidic chip devices [15-25]. Research studies could successfully implement LLE-like principals to microfluidic chip devices, employing different microfluidic platforms. There have been some reviews describing different microfluidic and on-chip LLEs [26-30]. Microfluidic compact-disc (µ-CD) platforms, commonly referred to as lab-on-a-disc (LOAD), are one of the microfluidic platforms that provide specific advantages over other on-chip devices [31-33]. LOADs use centrifugal force for flow control. They simply need a rotary motor to create a centrifugal force. Therefore, there is no need for external pumps or power supplies for liquid actuation, which can make a great potential for portability. Bubblefree liquid handling without dead volume and scalable centrifugal forces for sedimentation are some of the other advantages of such a platform. LOADs also offer closed fluidic systems with the capability of parallel sample operation on the same disc [32]. To date several reports have been published in the field of sample preparation using LOADs [34-36]. There are still
5
negligible reports using LOADs for implementation of LLE [37, 38] and using the inherent capability of centrifugal platforms in microextraction methods like DLLME. In the present work, the on-disc EME-DLLME method has been introduced. The method benefits from high clean-up ability of a drop-to-drop EME for extraction of target analytes form biofluids as well as high preconcentration factors and GC compatibility of DLLME. Implementing the EME-DLLME method on a lab-on-a-disc device brings some significant advantages over the conventional methods, including miniaturization and saving time, space, and organic solvents. In addition, in the DLLME step, dispersion of an organic solvent mixture into a donor phase and sedimentation of an extraction solvent can be performed simply by changing the spin speed of the disc. In this work, we investigated the effect of the injection speed of the organic solvent into the aqueous solution on extraction efficiency. Finally, the applicability and performance of the system were demonstrated for human urine and blood plasma by extraction of two tricyclic antidepressants.
2. Experimental 2.1 Chemicals and materials Amitriptyline (Ami) and imipramine (Imi) were kindly provided by Razak Laboratories (Tehran, Iran). The structure of the drugs along with their physicochemical properties are shown in Table1. NaOH, CCl4, and C2HCl3 were purchased from Merck (Darmstadt, Germany). C2Cl4 and 2-nitrophenyl octylether (NPOE) were supplied from Acros (Geel, Belgium) and Fluka (Buchs, Switzerland), respectively. Isopropanol was obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol was purchased from Caledon (George Town, Ont., Canada). All chemicals were of analytical reagent grade. Deionized water was prepared by a Young Lin aquaMAx purification system 370 series (Seoul, Korea). The
6
Accurel PP 1E polypropylene membrane sheet with a wall thickness of 100 µm and a pore size of 0.1 µm was bought from Membrana (Wuppertal, Germany).
2.2 Standard solutions Stock solutions of the drugs at 1 mg mL−1 as well as a standard solution of the mixture of the drugs containing 50 µg mL−1 of each drug were prepared in methanol and stored in a refrigerator at 4°C. The aqueous working solution was prepared daily by diluting the standard solution at 0.5 µg mL−1 level in ultra-pure water and also in saliva, urine and blood plasma for some investigations.
2.3 Real samples Urine sample. A drug-free urine sample was collected from a healthy volunteer. The sample was stored in a glass vial which was carefully cleaned with hydrochloric acid and rinsed with deionized water and stored at 4 °C to prevent bacterial growth and proteolysis. The urine sample was spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations. A real urine sample was collected from a volunteer treated with Ami. The collection was done 10 hours after taking an Ami tablet comprising 25 mg of the drug. The sample was stored at 4 °C in a clean glass vial and analyzed a day after collection without any further pretreatment or dilution. The sampling procedures were performed according to the guidelines for research ethics. Plasma sample. Frozen drug-free human plasma samples (blood group +B) were obtained from the Iranian Blood Transfusion Organization (Tehran, Iran) and stored at −4 °C. The samples were allowed to thaw at room temperature, and then were shaken and diluted with deionized water (1:4) before extraction. The plasma sample was spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations.
7
Saliva sample. Drug-free saliva samples were collected from a healthy volunteer several times. The sample was stored in a glass vial which was carefully cleaned with hydrochloric acid, rinsed with deionized water, and stored at 4 °C to prevent bacterial growth and proteolysis. The saliva samples were spiked with the standard solution of the mixture of the drugs to obtain the desired concentrations. 100 µL of the saliva sample was exposed to the on-disc EME-DLLME without any pretreatment or dilution. The sampling procedures were performed according to the guidelines for research ethics.
2.4 Chromatographic apparatus Optimization was performed on an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a FID detector. The injector was used in the splitless mode at 300 °C. The column used for separation of the analytes was a Varian wall coated fused silica capillary column (30 m, 0.25 mm, i.d., film thickness 0.25 µm). The FID temperature was fixed at 300 °C. Ultrapure helium and nitrogen (>99.999%) were used as the carrier and makeup gas at 1 mL.min-1 and 25 mL.min-1, respectively. Analysis of the standards related to calibration curves and real samples was performed on an Agilent 7890B GC with a 5977B MS equipped with a split/splitless injector. GC-MS separations were performed using an HP-5MS capillary column (30 m × 0.25 mm) with a film thickness of 0.25 µm. The mass spectrometer was operated in the electron impact ionization mode with an ionizing energy of 70 eV. The interface and ion source temperatures were both set at 300 °C. The oven temperature was initially set at 100 °C for 1 min and then was programed to 200 °C at 100 °C min-1 and was held at this temperature for 4 min. The temperature then increased to 260 ° C at 10 ° C min-1 followed by another increase to 280 ° C at 100 ° C min-1 and was held for 4 min. Helium (99.999%) was used as the carrier gas at a flow rate of 1 mL min-1. The solvent delay time was 10 min. The two model analytes were identified using the NIST database as well. Quantitative determination of the analytes in the standards and samples 8
were performed in the selective ion monitoring (SIM) mode of MS. The monitored ions of the analytes were selected on the basis of good selectivity and high sensitivity and were set as follows: Ami, m/z 58.1, 202.1 and Imi, m/z 58.0, 208.1, 234.0.
2.5 Design and fabrication of Lab-on-a-Disc The disc contains 5 layers and also 6 units for 6 parallel extractions. The disc was designed using AutoCAD software (Autodesk, Inc.). A schematic view of the disc and the fabricated disc is shown in Fig. 1A and C. A schematic of one extraction unit is also shown in Fig. 1B. The structure of the disc was cut on a 1 mm-thick poly(methyl methacrylate) (PMMA) sheet using a laser cutting device (Perfect laser Co., Ltd, China). Afterwards, all layers were cleaned with isopropanol, rinsed with DI water, and dried with nitrogen gas. The layers were fixed by chemical-thermal bonding. First, layers 1 and 2 were bonded. After that, a hole (0.7 mm D.) was drilled in the reservoir I of all the 6 units to insert the electrodes. A stainless steel wire (0.5 mm D.) as the electrode was mounted inside the reservoir I (Fig. 1B). The assembly was washed again with isopropanol, rinsed with DI water, and dried with nitrogen gas. A porous polypropylene membrane (about 1.5 × 1.5 cm) was placed above the reservoir I. Then, layers 3, 4, and 5 were respectively bonded to other layers. Before bonding layer 5 to layer 4, a stainless steel wire as an electrode was mounted to layer 5 through a hole drilled in this layer. The bonding procedure was as follows: Several microliters of ethanol was poured on the surface of the PMMA sheet. Then, the second layer was placed above it and aligned to match the right position. The assembly was then placed in an oven with pressure applied on it and was cured at 75 ºC for 40 min. Finally, a piece of a pressure sensitive adhesive (PSA) film was placed on the hole II (Fig. 1) embedded on layer 3.
2.6 On-Disc EME-DLLME procedure
9
Initially, the disc was fixed to the rotor of a homemade centrifugal driver. Before starting the extraction, NPOE was immobilized in the pores of the polypropylene membrane sheet. 100 µL of a 10 mM HCl solution, which is the acceptor solution during the EME process, was injected through the hole I into the reservoir I, so that the solution was placed on the electrode (cathode) mounted in this reservoir. Then, 100 µL of a donor phase (sample solution) was placed in the reservoir II on the polypropylene membrane sheet. The donor phase must be in contact with the electrode (anode) mounted in the last layer. The electrodes were then connected to a DC power supply. The voltages of 85 V for urine and plasma samples and 150 V for saliva samples were applied and extraction was performed for 15 min. The extraction was run without any agitation or convection of the donor solution. During the extraction, the target analytes migrated from the aqueous sample toward the acceptor solution placed in the reservoir I through the SLM. This step provides a proper clean-up for extraction of target analytes from complex matrices like biofluids. After the EME extraction was completed, a DLLME was done in the disc. The dispersive liquid-liquid extraction was implemented on the acceptor phase of the EME step. To do so, there was no need to manually transfer the acceptor phase of the EME step to perform a DLLME step. By rotating the disc, the acceptor phase of the EME step flowed to the extraction chamber for the next step. A mixture of 30 microliter of methanol (the disperser solvent) and 5 microliter of tetrachloroethene (the extraction solvent) was injected to the reservoir III. 5 microliter of a NaOH solution (pH = 12) was also introduced to the reservoir IV to adjust the pH of the donor phase in the DLLME step. Then, the disc rotated counter clockwise with the spin speed of 100 rpm for 2 min. As the disc rotated, centrifugal force caused the fluids to exist in the reservoirs I, III, and IV to flow toward the extraction chamber. It should be noted that the acceptor solution of the EME step, which was placed in the reservoir I, was used as the donor phase in the DLLME step. In this step, the extraction solvent was dispersed in the donor
10
phase (Fig. 2A). After 2 min, the spin speed increased to 3500 rpm and the device kept rotating for 4 min (See the video in ESI). As a result, a phase separation occurred and the extraction solvent, which was denser than the aqueous solution, sedimented at the conical end of the extraction chamber (Fig. 2B). The settled phase (about 2 µL) was then drawn into a Hamilton syringe by perforating the PSA film on the hole II and was injected manually into the GC/MS device. Each extraction unit was used only once.
2.7 Calculation of preconcentration factor, extraction recovery, and relative recovery Preconcentration factor (PF) was defined as the ratio of the final concentration of the analyte in the acceptor phase (Cf,a) to the initial concentration of the analyte (Ci,s). PF =
,
(1)
,
Where Cf,a was calculated from a calibration graph obtained from the direct injection of the standard solutions of the model analyte and Ci,s is the initial concentration of the analyte. The extraction recovery (ER) was defined as the percentage of the mole's number of the analyte extracted to the acceptor phase (nf,a) with respect to the number of moles of the same analyte originally present in the donor phase (ni,s). ,
ER% =
× 100 =
,
,
×
,
× ,
,
× 100
(2)
Relative recovery (RR) was calculated from the following equation: RR% =
× 100
(3)
Error% = RR% - 100
(4)
Where Cfound, Creal, and Cadded are the concentration of the analyte after addition of a known amount of the standard into the real sample, the concentration of the analyte in a real
11
sample, and the concentration of a known amount of the standard which was spiked into the real sample, respectively.
3. Results and discussion In this study, the on-disc EME-DLLME was developed. A schematic of the disc devise is illustrated in Fig. 1. A chip device provides the facility to implement consecutive extractions on a solitary chip with miniaturizing the whole procedure. Moreover, the extraction procedure is easy to handle and could be performed automatically. To show the applicability of the designed disc, two basic drugs were chosen to be extracted from biofluids. Amitriptyline and imipramine were selected as model analytes for this study. To reach the best extraction efficiency, the effective parameters of DLLME and EME were optimized.
3.1 Optimization of DLLME parameters 3.1.1 Extraction solvent: The extraction solvent has a vital role in the extraction efficiency of DLLME. The disc was designed in such a way that the extraction solvent could be collected at the end of the conical shape of the extraction chamber. For this purpose, the extraction solvent must have higher density than the aqueous solution. Therefore, chlorinated solvents are good candidates. On the other hand, considering the chemical resistance of PMMA, most of the chlorinated solvents damage the PMMA disc. Among chlorinated solvents, PMMA offers a substantial chemical resistance to C2Cl4 (perchloroethylene or tetrachloroethene) [39, 40]. As a result, C2Cl4 was chosen as the extraction solvent. 3.1.2 Volume of the extraction solvent: The critical importance of the volume of the extraction solvent in the DLLME efficiency is undeniable. Using a small amount of the extraction solvent could form a good emulsion, but this may not be sufficient to perform a proper extraction. On the other hand, a large quantity of the extraction solvent does not result in the formation of a proper emulsion. Therefore, it is important to find a balance between 12
emulsion formation and achieving good extraction efficiency. To investigate the effect of this parameter, different volumes of C2Cl4 were examined. The results depicted in Fig. 3A show that the response increased by increasing the volume of the extraction solvent up to 5 µL. Further increase in the volume of the extraction solvents led to a decline in the responses, which was probably due to the dilution effect. In volumes lower than 5 µL, the collection of the settled phase was faced with some difficulties. In addition, repeatability of the results was reduced by using a lower volume of the extraction solvent. A volume of 5 µL was used for the subsequent experiments. 3.1.3 Volume of the disperser solvent: DLLME is based on a ternary component solvent system: the aqueous solution, the disperser solvent, and the extraction solvent. These solvents can form an emulsion. A sufficient amount of the disperser solvent is important in complete formation of emulsion, so the volume of the disperser solvent has a critical role in any DLLME procedure. To investigate the effect of the volume of the disperser solvent, a series of volumes were considered. As shown in Fig. 3B, by increasing the volume of the disperser solvent from 10 to 30 µL, an enhancement in the responses can be observed. A further increase in the volume of the disperser solvent has no significant effect on the responses. 3.1.4 pH: In DLLME, the best extraction can be done when the analytes are in their neutral forms. Two worthy tricks to make analytes neutral are changing the pH of the donor phase and adding a proper ion pair agent. In this work, as the EME step is performed first, the donor solution (the acceptor phase of the EME step) was acidified. In this work, 5 microliters of a NaOH solution (pH = 12) was introduced to the reservoir IV to adjust the pH of the donor solution in the DLLME step. 3.1.5 Spin speed: The main advantage of DLLME is formation of fine droplets. Formation of the droplets leads to an increase in the contact area between an extraction 13
solvent and an aqueous solution, which causes faster distribution of the analytes between the extraction solvent and the aqueous solution. As a result a very fast extraction of the analytes into the extraction solvent can occur. To form such droplets, the organic solvent mixture (the mixture of an extraction solvent and a disperser solvent) must be injected quickly into an aqueous solution. The fast injection of the organic solvent mixture can affect the size of the droplets and subsequently the extraction efficiency by increasing the contact area. In the constructed disc, the mixture of the organic solvents are introduced into the reservoir III. The reservoir is connected to the extraction chamber by a channel. As the disc rotates, centrifugal force makes the organic solvent mixture flow to the extraction chamber. The aqueous solution (the acceptor phase of the EME step, discussed in the on-disc EME-DLLME procedure section) flows toward the extraction chamber as well. As a result, the organic solvent mixture is injected into the aqueous solution and dispersion occurs (Fig. 2A). Faster spin speed leads to faster dispersion of the organic solvent mixture into the aqueous solution and the formation of finer droplets. Thus, the spine speed can affect the extraction performance. To investigate this parameter, several spin speeds were examined. Fig. 3C shows the results. Increasing the spin speed up to 100 rpm increases the extraction efficiency as well. By increasing the spin speed more than 100 rpm, no significant increase in the extraction efficiency was observed. To compare the size of the droplets in the dispersion step, the droplets formed at different spin speeds were imaged using a light microscopy (at 40X magnification). As can be seen in Fig. 4, by increasing the spin speed up to 100 rpm, the size of the droplets decreased and no significant change in the size of the droplets was observed at spin speeds higher than 100 rpm. As a result, 100 rpm was chosen as the optimum spin speed of the disc for the dispersion step in DLLME.
3.2 Optimization of EME parameters
14
In this study, the EME step is a drop-to-drop electromembrane extraction. Based on the literature [12, 41], NPOE was used as the supported liquid membrane (SLM). NPOE was directly injected by a syringe on the surface of polyprpylen sheet. 100 µL of the sample solution (the donor phase) comprising the target analytes was placed on the polypropylene membrane sheet and remained stagnant during the extraction. 100 µL of an aqueous solution was also used as the acceptor phase. Other effective parameters were optimized as follows: 3.2.1 Donor and acceptor phase solutions: EME is a microextraction method which uses an electrical field as a driving force in extraction of ionized compounds. The extraction mechanism is mainly based on electrokinetic migration. Neutral compounds will not be affected by the electrical field, so to extract target compounds, they must be in their ionic form [6, 42]. By changing the pH value, acidic and basic compounds can be converted to their ionic or neutral forms. Since the pH values for ionization of basic compounds are different from the ones for acidic compounds, by adjusting a proper pH value, EME would be able to selectively extract acidic or basic compounds. This is one of the advantages of the electromembrane microextraction method. Moreover, the pH level of donor and acceptor phases plays a pivotal role in the EME extraction performance. Therefore, this parameter should be optimized to reach the highest extraction recoveries. To reach the optimum value of this parameter, the pH values of the donor and acceptor phases were studied by changing the concentration of HCl in the range of 0-10 mM and 0-30 mM in the donor and acceptor phases, respectively. The results for Ami and Imi are presented in Fig. 5A and Fig. S1. In this work, two basic drugs were used as the model analytes. The sample solution (the donor phase) should be acidic enough so that the target analytes would carry a net positive charge. Additionally, the acceptor solution should be acidic enough to prevent deprotonation of drugs during the extraction and thus their back-diffusion toward the donor phase [7, 24]. As can be seen in Fig. 5A, the best results were obtained when the 15
concentration of HCl in the acceptor phase was 10 mM and no HCl was added to the donor phase (0 mM HCl). A decrease in the extraction efficiency was observed by increasing the concentration of H+ in both the donor and acceptor phases. Using 10 mM HCl in the donor phase and 20 and 30 mM HCl in the acceptor phase resulted in instability in the SLM and bubble formation on the electrodes. All of these indicated that high concentration of H+ led to an increase in the current level and subsequently to an increase in probability of electrolysis reactions in both the donor and acceptor phases. 3.2.2 Voltage and time: As already mentioned, an electrical field is the main driving force in EME. It has been proven that the migration of analytes across the SLM is affected by the magnitude of the applied voltage [43, 44]. Thus, this parameter has an important role that must be investigated. In this work, first, a voltage in the range of 70-240V was applied to the system and ultra-pure water was used to prepare the working solutions. It was observed that 220 V was the optimum voltage in that media (Fig. S2). The goal of this work is to extract some model analytes from biological fluids. It is known that biological fluids like urine and blood plasma contain a high concentration of salts. In EME, as voltage is applied across an SLM, all ionic species in the donor and acceptor solutions move electrokinetically toward their relative electrodes. As a result, a boundary layer of ions is formed at the interface on both sides of the SLM. The existence of a high concentration of ionic species in biological fluids leads to a significant increase in the number of ions migrating through SLM at a given moment. So the thickness of the boundary layer and the current level increase. This also results in Joule heating and therefore instability of SLM [7]. As the electrical field is the main driving force in EME and increasing the magnitude of the applied voltage increases the current level, the applied voltage used in the aqueous media is not usable in biological fluids. Therefore, the optimization of the voltage was repeated in the urine, blood plasma, and saliva media again. Since the mass transfer is time-dependent, a simultaneous investigation of time 16
and applied voltage was performed. The results of the effects of the applied voltage on the extraction efficiency of Ami and Imi in the urine media are shown in Fig. 5B and Fig. S3, respectively. The best extractability of the model analytes in the urine samples were obtained at 85 V for 15 min. As can be seen, by increasing the applied voltage and time, the extraction efficiency increased as well. At 85 V after 20 min, Joule heating and instability of the SLM led to a decrease in the extraction efficiency. By performing the extraction at 105 V for 5 min, the same conditions were observed. Therefore, it was not possible to continue the extraction at 105 V more than 5 min. The same results were observed in the blood plasma media too. Thus, 85 V for 15 min was chosen as the optimum values in both the urine and blood plasma media (Fig. S4). A set of experiments were conducted on the saliva samples (Fig. S5) and 150 V for 15 min was selected as the optimum applied voltage and time. 3.2.3 Investigation of applying a pulsed voltage. As it was reported in previous conventional EME works, applying constant voltages, during relatively long extraction times and/or under high applied voltages pose some problems [45, 46]. To overcome these problems, using a pulsed voltage was proposed by Yamani’s research group in 2012 [47]. In this work, the suitability of using pulsed voltage for a drop-to-drop EME was investigated. In this work, outage duration cannot have any significant influence on the thickness of the charged double layer, but it decreases the whole applied voltage time instead, and so the extraction efficiency decreases (Fig. S6). The detailed results can be found in the supporting information.
3.3 Method evaluation To validate the on-disc EME-DLLME system under optimized conditions (Table 2), Ami and Imi were spiked in the drug-free urine, blood plasma, saliva, and also pure water at different concentrations. Table 3 provides the summery of the method evaluation results.
17
Calculations were made based on equations 1-4. The results showed that the on-disc EMEDLLME system can be effectively employed for analysis of model analytes even in complicated matrices such as biofluids. A good linearity was obtained with a coefficient of determination (R2) more than 0.9959. The limits of detection (LOD) of the on-disc device were in the range of 0.1-0.5 µg L-1 for Ami and Imi in different biofluids with relative standard deviations (RSDs) lower than 3.5%. A comparison of the analytical performance of the proposed method with other analytical reports is presented in Table 4. Although the proposed method used much less donor solution than other methods, it reached LODs which were comparable with other methods. In addition, integration of two sample preparation methods (EME and DLLME) in one chip provides a semiautomatic chip device with easy handling, fast operating, good repeatability and reproducibility at a lower cost and with a smaller amount of the donor solution and organic solvent as well. The method also benefits from the advantages of both analytical methods. Consequently, the proposed method can be used for extraction and determination of low levels of Ami and Imi in different biofluids.
3.4 Validation of the on-disc EME-DLLME method for analysis of Ami and Imi in some biofluids To evaluate the practical suitability of the proposed method for determination of drugs in real biological samples, Ami and Imi as model drugs were determined in three biofluids (urine, blood plasma, and saliva). The results are presented in Table 5. Urine sample: The on-disc system was used for the analysis of the model drugs in the urine sample of a patient undergoing therapy with Ami to assess the applicability of the method. Sample preparation is already explained in the “real sample” section. In summary, a urine sample was collected after 10 hours of taking an Ami tablet comprising 25 mg of the drug. Accuracy was evaluated as the parameter of error %, so the urine sample was spiked with 150 µgL-1 Ami and Imi. RSD% values indicated acceptable precision related to the 18
proposed system. Furthermore, as can be seen in Table 5, error% was less than +3.9%. Thus accuracy was acceptable, and no significant matrix effect was observed for determination of the model drugs using the proposed on-disc system. A typical chromatogram, obtained from the extraction of the urine sample before and after the addition of a certain concentration of the model drugs, is illustrated in Fig. 6. Blood plasma: The validity of the on-disc system was assessed by extracting the model drugs from a blood plasma sample. As described before, the blood plasma sample was diluted 1:4 before extraction. The blood plasma samples were spiked with 50 µgL-1 of Ami and Imi. The results shown in Fig. S7 and Table 5 indicate efficient extraction of the analytes. During the EME step, it was seen that the SLM showed good stability. Saliva: Applicability of the method was assessed in the saliva samples as well. Sample preparation is already explained in the “real sample” section. A saliva sample was spiked with 10 µg L-1 of Ami and Imi. As Table 3 shows, the method has acceptable accuracy with error% less than +2.7. Acceptable precision based on RSD% values was also achieved. A topical chromatogram of the blank and spiked saliva sample is shown in Fig. S8. Furthermore, as can be seen in Table 5, error% was less than +2.9, accuracy was acceptable, and no significant matrix effect was observed for determination of the model drugs using the proposed on-disc system.
4. Conclusions The present work demonstrated the design of a lab-on-a-disc device for performing a combination of two LPME methods, EME and DLLME. The system was simple and the component had a low cost, as each extraction unit was used only once. The system benefits from advantages of both microextraction methods as well as advantages of miniaturization on a lab-on-a-disc device. Using a drop-to-drop EME, the great clean-up potential of EME can be used for extraction of target analytes from complex matrices. Utilizing a DLLME also 19
brings good preconcentration factors and GC compatibility of the final extracted phase. Implementing the EME-DLLME method on a lab-on-a-disc device has some significant advantages over the conventional method. Performing the extraction method on a chip device makes it possible to use a small amount of the donor solution, which is not possible in conventional extraction methods. EME and DLLME processes are performed on the same disc, so there is no need to manually transfer the acceptor solution of EME to conduct a DLLME step. Using a centrifugal platform, the organic solvent mixture can be automatically injected into the donor solution in the DLLME step by simply rotating the disc. Thanks to the scalable centrifugal force, injection can be performed with different speeds. In this work, we showed that the injection speed could influence extraction efficiency by affecting the size of the droplets. Finally, sedimentation of the extraction phase occurs by increasing the spin speed. The system has the potential to run fully-automatically and be miniaturized more by changing the detection method, which can be considered as the main limitation of such systems for miniaturization. More studies focusing on fully automated on-disc extraction and development of a sample-to-answer system are in progress.
References [1] F.F. Cantwell, M. Losier, Chapter 11 Liquid—liquid extraction, Analytical Chemistry, Elsevier, 2002, pp. 297-340.
Comprehensive
[2] S. Tang, H. Zhang, H.K. Lee, Advances in sample extraction, Anal. Chem. 88 (2016) 228-249. [3] A. Sarafraz-Yazdi, A. Amiri, Liquid-phase microextraction, TRAC, Trends Anal. Chem. 29 (2010) 1-14. [4] M. Rezaee, Y. Assadi, M.-R. Milani Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, Determination of organic compounds in water using dispersive liquid–liquid microextraction, J. Chromatogr. A 1116 (2006) 1-9. [5] M. Rezaee, Y. Yamini, M. Faraji, Evolution of dispersive liquid–liquid microextraction method, J. Chromatogr. A 1217 (2010) 2342-2357. [6] S. Pedersen-Bjergaard, K.E. Rasmussen, Electrokinetic migration across artificial liquid membranes: New concept for rapid sample preparation of biological fluids, J. Chromatogr. A 1109 (2006) 183-190. 20
[7] Y. Yamini, S. Seidi, M. Rezazadeh, Electrical field-induced extraction and separation techniques: Promising trends in analytical chemistry – A review, Anal. Chim. Acta 814 (2014) 1-22. [8] A. Gjelstad, S. Pedersen-Bjergaard, Electromembrane extraction—Three-phase electrophoresis for future preparative applications, Electrophoresis 35 (2014) 2421-2428. [9] C. Basheer, J. Lee, S. Pedersen-Bjergaard, K.E. Rasmussen, H.K. Lee, Simultaneous extraction of acidic and basic drugs at neutral sample pH: A novel electro-mediated microextraction approach, J. Chromatogr. A 1217 (2010) 6661-6667. [10] M. Rezazadeh, Y. Yamini, S. Seidi, B. Ebrahimpour, Electromembrane surrounded solid phase microextraction: A novel approach for efficient extraction from complicated matrices, J. Chromatogr. A 1280 (2013) 16-22. [11] L. Guo, H.K. Lee, Electro membrane extraction followed by low-density solvent based ultrasound-assisted emulsification microextraction combined with derivatization for determining chlorophenols and analysis by gas chromatography–mass spectrometry, J. Chromatogr. A 1243 (2012) 14-22. [12] S. Seidi, Y. Yamini, M. Rezazadeh, Combination of electromembrane extraction with dispersive liquid–liquid microextraction followed by gas chromatographic analysis as a fast and sensitive technique for determination of tricyclic antidepressants, J. Chromatogr. B 913914 (2013) 138-146. [13] L. Arjomandi-Behzad, Y. Yamini, M. Rezazadeh, Extraction of pyridine derivatives from human urine using electromembrane extraction coupled to dispersive liquid–liquid microextraction followed by gas chromatography determination, Talanta 126 (2014) 73-81. [14] M. Khajeh, S. Pedersen-Bjergaard, M. Bohlooli, A. Barkhordar, M. GhaffariMoghaddam, Maghemite nanoparticle-decorated hollow fiber electromembrane extraction combined with dispersive liquid–liquid microextraction for determination of thymol from Carum copticum, J. Sci. Food Agr. 97 (2017) 1517-1523. [15] P.A.L. Wijethunga, Y.S. Nanayakkara, P. Kunchala, D.W. Armstrong, H. Moon, Onchip drop-to-drop liquid microextraction coupled with real-time concentration monitoring technique, Anal. Chem. 83 (2011) 1658-1664. [16] N. Wang, S. Mao, W. Liu, J. Wu, H. Li, J.-M. Lin, Online monodisperse droplets based liquid–liquid extraction on a continuously flowing system by using microfluidic devices, RSC Adv. 4 (2014) 11919-11926. [17] I. Vural Gürsel, S.K. Kurt, J. Aalders, Q. Wang, T. Noël, K.D.P. Nigam, N. Kockmann, V. Hessel, Utilization of milli-scale coiled flow inverter in combination with phase separator for continuous flow liquid–liquid extraction processes, Chem. Eng. J. 283 (2016) 855-868. [18] S.K. Kurt, I. Vural Gürsel, V. Hessel, K.D.P. Nigam, N. Kockmann, Liquid–liquid extraction system with microstructured coiled flow inverter and other capillary setups for single-stage extraction applications, Chem. Eng. J. 284 (2016) 764-777. [19] N.J. Petersen, J.S. Pedersen, N.N. Poulsen, H. Jensen, C. Skonberg, S.H. Hansen, S. Pedersen-Bjergaard, On-chip electromembrane extraction for monitoring drug metabolism in real time by electrospray ionization mass spectrometry, Analyst 137 (2012) 3321-3327. [20] P. Mary, V. Studer, P. Tabeling, Microfluidic droplet-based liquid−liquid extraction, Anal. Chem. 80 (2008) 2680-2687.
21
[21] M.D. Ramos Payán, H. Jensen, N.J. Petersen, S.H. Hansen, S. Pedersen-Bjergaard, Liquid-phase microextraction in a microfluidic-chip – High enrichment and sample clean-up from small sample volumes based on three-phase extraction, Anal. Chim. Acta 735 (2012) 46-53. [22] P. Znidarsic-Plazl, I. Plazl, Steroid extraction in a microchannel system-mathematical modelling and experiments, Lab Chip 7 (2007) 883-889. [23] N.J. Petersen, S.T. Foss, H. Jensen, S.H. Hansen, C. Skonberg, D. Snakenborg, J.r.P. Kutter, S. Pedersen-Bjergaard, On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection, Anal. Chem. 83 (2010) 44-51. [24] Y.A. Asl, Y. Yamini, S. Seidi, B. Ebrahimpour, A new effective on chip electromembrane extraction coupled with high performance liquid chromatography for enhancement of extraction efficiency, Anal. Chim. Acta 898 (2015) 42-49. [25] M. Karami, Y. Yamini, Y. Abdossalami Asl, M. Rezazadeh, On-chip pulsed electromembrane extraction as a new concept for analysis of biological fluids in a small device, J. Chromatogr. A 1527 (2017) 1-9. [26] K. Wang, G. Luo, Microflow extraction: A review of recent development, Chem. Eng. Sci. 169 (2017) 18-33. [27] C. Xu, T. Xie, Review of microfluidic liquid–liquid extractors, Ind. Eng. Chem. Res. 56 (2017) 7593-7622. [28] D. Ciceri, J.M. Perera, G.W. Stevens, The use of microfluidic devices in solvent extraction, J. Chem. Technol. Biotechnol. 89 (2014) 771-786. [29] N. Assmann, A. Ładosz, P. Rudolf von Rohr, Continuous micro liquid-liquid extraction, Chem. Eng. Technol. 36 (2013) 921-936. [30] B.C. Giordano, D.S. Burgi, S.J. Hart, A. Terray, On-line sample pre-concentration in microfluidic devices: A review, Anal. Chim. Acta 718 (2012) 11-24. [31] T. Vilkner, D. Janasek, A. Manz, Micro total analysis systems. Recent developments, Anal. Chem. 76 (2004) 3373-3386. [32] M. Madou, J. Zoval, G. Jia, H. Kido, J. Kim, N. Kim, LAB ON A CD, Annu. Rev. Biomed. Eng. 8 (2006) 601-628. [33] C. Li, X. Dong, J. Qin, B. Lin, Rapid nanoliter DNA hybridization based on reciprocating flow on a compact disk microfluidic device, Anal. Chim. Acta 640 (2009) 9399. [34] M. Vázquez, D. Brabazon, F. Shang, J.O. Omamogho, J.D. Glennon, B. Paull, Centrifugally-driven sample extraction, preconcentration and purification in microfluidic compact discs, TRAC,Trends Anal. Chem. 30 (2011) 1575-1586. [35] E. Duffy, R. Padovani, X. He, R. Gorkin, E. Vereshchagina, J. Ducrée, E. Nesterenko, P.N. Nesterenko, D. Brabazon, B. Paull, M. Vázquez, New strategies for stationary phase integration within centrifugal microfluidic platforms for applications in sample preparation and pre-concentration, Anal. Methods 9 (2017) 1998-2006. [36] G. Duffy, I. Maguire, B. Heery, P. Gers, J. Ducrée, F. Regan, ChromiSense: A colourimetric lab-on-a-disc sensor for chromium speciation in water, Talanta 178 (2018) 392399.
22
[37] A. Kazarine, M.C.R. Kong, E.J. Templeton, E.D. Salin, Automated liquid–liquid extraction by pneumatic recirculation on a centrifugal microfluidic platform, Anal. Chem. 84 (2012) 6939-6943. [38] L. Clime, D. Brassard, M. Geissler, T. Veres, Active pneumatic control of centrifugal microfluidic flows for lab-on-a-chip applications, Lab Chip 15 (2015) 2400-2411. [39] a.R.P.C. eplastics, USA. [40] D.O.M.M. CORNELL UNIVERSITY USA, Sevier Lab. [41] S.S.H. Davarani, A.M. Najarian, S. Nojavan, M.-A. Tabatabaei, Electromembrane extraction combined with gas chromatography for quantification of tricyclic antidepressants in human body fluids, Anal. Chim. Acta 725 (2012) 51-56. [42] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Electrokinetic migration across artificial liquid membranes: tuning the membrane chemistry to different types of drug substances, J. Chromatogr. A 1124 (2006) 29-34. [43] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Simulation of flux during electromembrane extraction based on the Nernst–Planck equation, J. Chromatogr. A 1174 (2007) 104-111. [44] A. Gjelstad, T.M. Andersen, K.E. Rasmussen, S. Pedersen-Bjergaard, Microextraction across supported liquid membranes forced by pH gradients and electrical fields, J. Chromatogr. A 1157 (2007) 38-45. [45] M. Balchen, L. Reubsaet, S. Pedersen-Bjergaard, Electromembrane extraction of peptides, J. Chromatogr. A 1194 (2008) 143-149. [46] M. Eskandari, Y. Yamini, L. Fotouhi, S. Seidi, Microextraction of mebendazole across supported liquid membrane forced by pH gradient and electrical field, S. J. Pharm. Biomed. Anal 54 (2011) 1173-1179. [47] M. Rezazadeh, Y. Yamini, S. Seidi, A. Esrafili, Pulsed electromembrane extraction: A new concept of electrically enhanced extraction, J. Chromatogr. A 1262 (2012) 214- 218. [48] A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, Electromembrane extraction of basic drugs from untreated human plasma and whole blood under physiological pH conditions, Anal. Bioanal. Chem. 393 (2009) 921-928. [49] M. Rezazadeh, Y. Yamini, S. Seidi, L. Arjomandi-Behzad, Voltage-step pulsed electromembrane as a novel view of electrical field-induced liquid-phase microextraction, J. Chromatogr. A 1324 (2014) 21-28. [50] M. Bazregar, M. Rajabi, Y. Yamini, Z. Saffarzadeh, A. Asghari, Tandem dispersive liquid–liquid microextraction as an efficient method for determination of basic drugs in complicated matrices, J. Chromatogr. A 1429 (2016) 13-21. [51] S.S.H. Davarani, A. Morteza-Najarian, S. Nojavan, A. Pourahadi, M.B. Abbassi, Twophase electromembrane extraction followed by gas chromatography-mass spectrometry analysis, J. Sep. Sci. 36 (2013) 736-743.
23
Table 1. Chemical structures, pKa and log P of the model analytes Chemical structure
Compound
Abbreviation
pKa
log P
Amitriptyline
Ami
9.4
4.9
Imipramine
Imi
9.2
4.2
24
Table 2. Optimum conditions for extraction of model analytes by the on-disc EME-DLLME system
DLLME
EME
Extractive solvent
C2Cl4
Supported liquid membrane (SLM)
NPOE
Volume of extractive solvent
5 µL
Donor phase solution
Without adding HCl
Volume of disperser solvent
30 µL
Acceptor phase solution
10 mM HCl
pH
Spin speed
12
100 rpm
Voltage
Time
Water
220 V
Urine & Plasma
85 V
Saliva
150 V 15 min
25
Table 3. Analytical performance of on-disc EME-DLLME for determination of Ami and Imi in water, saliva, urine, and blood plasma samples
Sample
Analyte
LODa
LOQ
Linearity
(µg L-1)
(µg L-1)
(µg L-1)
R2
PFb
ER%
RSD% (n= 5)
Water
Ami Imi
0.1 0.1
0.25 0.5
0.25-500 0.5-500
0.9959 0.9974
35.4 32.1
70.8 64.2
1.9 2.0
Saliva
Ami Imi
0.25 0.5
0.5 1.0
0.5-500 1.0-500
0.9977 0.9984
31.2 28.2
62.5 56.4
2.7 2.4
Urine
Ami Imi
0.5 0.5
1.0 1.0
1.0-500 1.0-500
0.9987 0.9977
32.9 29.0
65.8 58.0
2.9 3.2
Blood plasma
Ami Imi
0.5 0.5
1.0 1.0
1.0-500 1.0-500
0.9979 0.9994
29.9 21.5
59.9 43.0
3.1 3.5
a) LODs of blood plasma is related to 1:4 diluted sample. b) Preconcentration factor at 50 µg L-1 of analytes
26
27
Table 4. Comparison of figures of merit of on-disc EME-DLLME with other methods for determination of Ami and Imi LOD (µg L-1)
LOQ (µg L-1)
Urine
3.0
Ami
Water
EM-SPME -GC-FID
Ami
EM-SPME-GC-FID EM-SPME-GC-FID
Linearity (µg L-1)
ER
Volume of donor phase (µL)
Reference
10.0
10.0-500
36
1000
[24]
0.5
1.0
1.0-500
-
2500
[49]
Water
0.5
2.0
-
11.5
24000
[10]
Ami
Urine
1.0
2.5
-
10.4
24000
[10]
Ami
Plasma
1.0
5.0
-
5.9
24000
[10]
EME-DLLME _GC-FID
Ami
Water
0.25
2.0
2.0-500
-
24000
[12]
EME-DLLME_GC-FID
Ami
Urine
3.0
10.0
10.0-500
-
24000
[12]
EME-DLLME_GC-FID
Ami
Plasma
15.0
40.0
40.0-500
-
24000
[12]
TDLLME -HPLC-UV
Ami
Plasma
1.0
-
3.0-5000
8000
[50]
On-Disc EME-DLLME-GC-MS
Ami
Water
0.1
0.25
0.25-500
70.8
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Saliva
0.25
0.5
0.5-500
62.5
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Urine
0.5
1.0
1.0-500
65.8
100
This work
On-Disc EME-DLLME-GC-MS
Ami
Plasma
0.5
1.0
1.0-500
59.9
100
This work
EME-GC-FID
Imi
Water
0.35
-
2.0-1500
-
2100
[41]
EME-GC-FID
Imi
Urine
0.40
-
2.0-1500
-
2100
[41]
EME-GC-FID
Imi
Plasma
0.50
-
2.0-1500
-
2100
[41]
Two-phase EME-GS-MS
Imi
Water
0.10
-
1.0-500
-
1200
[51]
TDLLME -HPLC-UV
Imi
Plasma
0.9
-
3.0-5000
-
8000
[50]
On-Disc EME-DLLME-GC-MS
Imi
Water
0.1
0.5
0.5-500
64.2
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Saliva
0.5
1.0
1.0-500
56.4
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Urine
0.5
1.0
1.0-500
58.1
100
This work
On-Disc EME-DLLME-GC-MS
Imi
Plasma
0.5
1.0
1.0-500
43.1
100
This work
Analytical method
Analyte
Matrix
CEMEa-HPLC-UV
Ami
b
PEME -HPLC-UV c
d
e
e
a
On chip electromembrane extraction
b
Pulsed electromembrane extraction
28
c
Electromembrane solid phase microextraction
d
Electromembrane extraction- Dispersive liquid-liquid microextraction
e
Tandem dispersive liquid–liquid microextraction
29
Table 5. Determination of Ami and Imi in the real samples using on-disc EME-DLLME.
Sample
Analyte
Creal (µg L-1)
Saliva
Ami
Nda
10.0
10.3
2.1
+2.9
Imi
Nd
10.0
9.8
2.5
-2.1
Ami
144.3
150.0
291.0
2.6
-2.2
Imi
Nd
150.0
155.8
3.3
+3.9
Ami
Nd
50.0
52.9
3.2
+5.8
Imi
Nd
50.0
50.8
3.9
+1.6
Urine
Blood Plasma
a
Cadded (µg L-1)
Not detected
30
Cfound (µg L-1)
RSD%
Error%
Figure captions Fig. 1. A) A schematic view of the designed LOD device consists of five layers. B) A schematic of one of the extraction units in Layer 1. C) Fabricated LOD device. Fig. 2. Desperation (A) and sedimentation of the extraction solvent (B) in the DLLME step. For clarity, a colored aqueous solution was used. Fig. 3. Optimization of the effective parameters in the DLLME step. In this step, C2Cl4 and methanol were used as the extraction and disperser solvents, respectively. A) Volume of the extraction solvent. B) Volume of the disperser solvent. C) Spin speed. In the EME step, 180 V was applied for 15 min without adding any acid or base to the donor and acceptor phases. Fig. 4. Effect of spin speed on the size of the formed droplets in the DLLME step. Fig. 5. Optimization of the effective parameters in the EME step. A) Composition of the donor and acceptor phases. B) Effect of applied voltage at different extraction times. For the DLLME step, the optimized parameters were used. Fig. 6. A typical GC/MS chromatogram obtained from the extraction of the model analytes from the plasma samples before and after spiking 50 µg L-1 of the analytes. The GC separation conditions were reported in section 2.4.
31
Fig. 1.
32
Fig. 2.
33
Fig. 3.
34
Fig. 4.
35
Fig. 5. 36
37
Fig. 6.
38
Highlights A lab-on-a-disc device was designed for sequential performing of EME and DLLME. All of DLLME steps can be fulfilled on the chip by simply controlling the spin speed. EME-DLLME was used for extraction of tricyclic antidepressants in biofluids. The effective parameters on the EME and DLLME were investigated and optimized. The chip is easy to handle without need to any skillful operator. The device provides effective and reproducible extraction using a low volume of sample.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: