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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.

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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: yyamini@modares.ac.ir (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-a-

chip (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. Bubble-

free 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 EME-

DLLME 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.

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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

Water 220 V

pH 12 Voltage Urine & Plasma 85 V

Saliva 150 V

Spin speed 100 rpm Time 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

LODa LOQ Linearity RSD%

Sample Analyte R2 PFb ER%
(µg L-1) (µg L-1) (µg L-1) (n= 5)

Water Ami 0.1 0.25 0.25-500 0.9959 35.4 70.8 1.9

Imi 0.1 0.5 0.5-500 0.9974 32.1 64.2 2.0

Saliva Ami 0.25 0.5 0.5-500 0.9977 31.2 62.5 2.7

Imi 0.5 1.0 1.0-500 0.9984 28.2 56.4 2.4

Urine Ami 0.5 1.0 1.0-500 0.9987 32.9 65.8 2.9

Imi 0.5 1.0 1.0-500 0.9977 29.0 58.0 3.2

Ami 0.5 1.0 1.0-500 0.9979 29.9 59.9 3.1

Blood plasma
Imi 0.5 1.0 1.0-500 0.9994 21.5 43.0 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)

Analytical method Analyte Matrix Linearity (µg L-1) ER Volume of donor phase (µL) Reference

CEMEa-HPLC-UV Ami Urine 3.0 10.0 10.0-500 36 1000 [24]

b
PEME -HPLC-UV Ami Water 0.5 1.0 1.0-500 - 2500 [49]
c
EM-SPME -GC-FID Ami Water 0.5 2.0 - 11.5 24000 [10]
EM-SPME-GC-FID Ami Urine 1.0 2.5 - 10.4 24000 [10]
EM-SPME-GC-FID Ami Plasma 1.0 5.0 - 5.9 24000 [10]
d
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]
e
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]
e
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
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) Cadded (µg L-1) Cfound (µg L-1) RSD% Error%

Saliva Ami Nda 10.0 10.3 2.1 +2.9

Imi Nd 10.0 9.8 2.5 -2.1

Urine Ami 144.3 150.0 291.0 2.6 -2.2

Imi Nd 150.0 155.8 3.3 +3.9

Blood Plasma Ami Nd 50.0 52.9 3.2 +5.8

Imi Nd 50.0 50.8 3.9 +1.6
a
Not detected

30
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: