Accepted Manuscript

‘ Pulsed electromembrane method for simultaneous extraction of drugs with

different properties

Leila Arjomandi-Behzad, Yadollah Yamini, Maryam Rezazadeh

PII: S0003-2697(13)00147-4

DOI: http://dx.doi.org/10.1016/j.ab.2013.03.027

Reference: YABIO 11301

To appear in: Analytical Biochemistry

Received Date: 8 February 2013

Revised Date: 23 March 2013

Accepted Date: 25 March 2013

Please cite this article as: L. Arjomandi-Behzad, Y. Yamini, M. Rezazadeh, ‘ Pulsed electromembrane method for

simultaneous extraction of drugs with different properties, Analytical Biochemistry (2013), doi: http://dx.doi.org/

10.1016/j.ab.2013.03.027

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1

`Pulsed electromembrane method for simultaneous extraction of drugs 1

with different properties 2

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Leila Arjomandi-Behzad, Yadollah Yamini∗, Maryam Rezazadeh 4

Department of Chemistry, Tarbiat Modares University, P. O. Box 14115-175, Tehran, Iran 5

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∗ Corresponding author: Tarbiat Modares University, Department of Chemistry, P.O. Box 14115-175, Tehran, Iran. Tel.: +98 21 82883417; Fax: +98 21 88006544.

E-mail address: yyamini@modares.ac.ir (Y. Yamini).

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Abstract 22

In the present work, a new setup including two cathodes and one anode is designed and 23

employed for the first time for pulsed electromembrane extraction (PEME) of atenolol (ATE) 24

and betaxolol (BET) from water, urine and plasma samples. Since these analytes have 25

different lipophilicities, the composition of supported liquid membrane (SLM) should be 26

optimized for each drug and it is impossible to extract them simultaneously using common 27

electromembrane setups. The SLMs employed for the extraction of BET and ATE were, 28

respectively, pure 2-nitrophenyl octyl ether (NPOE) and a mixture of 90% NPOE and 10% 29

di-(2-ethylhexyl) phosphate (DEHP), which were immobilized in the pores of two different 30

hollow fibers. An electric field of 100 V was applied to transfer the analytes from the sample 31

solution across the SLMs into acidic acceptor solutions with pH 1.0 which were located 32

inside the lumens of hollow fibers. Preconcentration factors in the range of 69-363 and 33

satisfactory repeatabilities (2.2 < RSD% < 7.4) were obtained in different matrices. The 34

method offered a good linearity with correlations of determination (R2) higher than 0.9944 35

and was applied for determination and quantification of the analytes in some real samples. 36

Finally, satisfactory results were obtained. 37

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Keywords: Atenolol; Betaxolol; Pulsed electromembrane extraction; Simultaneous 39

extraction; Urine; Plasma 40

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1. Introduction 47

β-Blockers or β-adrenergic blocking agents are among the most frequently prescribed 48

medicines all over the world that are used in the treatment of disorders such as hypertension, 49

angina pectoris and arrhythmia [1]. Since β-blockers can improve the heart’s ability to relax 50

and exhibit calming neurological effects, decrease anxiety and nervousness and stabilize 51

motor performances, consumption of them by athletes enhances their abilities [1, 2]. Thus, 52

their intake has been forbidden by the Medical Commission of the International Olympic 53

Committee [3]. Therefore, measurement of β-adrenoreceptor antagonists in biofluids may 54

provide useful information in the fields of intoxication, managing the patient compliance with 55

therapy and doping control. 56

Various analytical methods have been developed to determine β-blockers, including 57

spectrofluorimetric techniques [4-6], enzyme-linked immunosorbent assay (ELISA) [7, 8], 58

and chromatographic methods [9-18]. More recently, the use of GC/MS/MS [15], LC/MS/MS 59

[19] and UPLC/MS/MS [20] systems has increased the selectivity and sensitivity of the 60

process of screening β-blocker agents in biological fluids. However, such expensive 61

instruments are available only in a few laboratories. Due to the low concentration of drugs 62

and a high number of interferences present in complicated matrices such as biological fluids, 63

sample preconcentration and cleanup must be carried out before drug determination. A large 64

number of modern sample preparation techniques including solvent-free extraction methods 65

or extraction procedures with a very high sample to solvent ratio, which leads to a high 66

preconcentration factor for analytes, have been introduced. 67

In 2006, Pedersen-Bjergaard et al. reported a novel microextraction technique called 68

electromembrane extraction (EME) [21]. EME can be used to extract ionizable compounds 69

from plasma samples and other complicated biological matrices without protein precipitation 70

[22]. In this process, the ionized target analytes are extracted from an aqueous sample into an 71

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organic solvent located in the pores of a porous hollow fiber, and then transported into an 72

aqueous acceptor solution inside the lumen of hollow fiber by applying an electric potential 73

across the SLM. 74

Recently, a new concept of EME named pulsed electromembrane extraction (PEME) 75

was proposed by Rezazadeh et al. [23]. In this method, a simple pulse generator device was 76

applied in combination with a common constant DC power supply to create pulsed voltages. 77

It was shown that the PEME process increases stability by decreasing the thickness of the 78

double layer at the interfaces and improves extractability by eliminating this mass transfer 79

barrier [23]. 80

The conventional EME technique has been employed for selective extraction of ATE in 81

the presence of BET and proporanolol [24]. It has been demonstrated that the use of carriers 82

in the composition of SLM has a strong negative effect on the extraction of nonpolar drugs 83

such as BET. However, the presence of DEHP as an anionic ion-pairing reagent is necessary 84

for the extraction of hydrophilic compounds such as ATE. Therefore, BET could be extracted 85

by the use of only pure NPOE as the SLM while for the extraction of ATE, a mixture of 86

NPOE and DEHP is needed [24]. Consequently, the simultaneous extraction of these analytes 87

is impossible. 88

In the present work, a new PEME setup is used for the first time for simultaneous 89

extraction of different classes of analytes. Herein, two cathodes are employed and SLMs are 90

optimized for each class of analytes. As a result, this system offers suitable conditions for the 91

simultaneous extraction of ATE and BET as model compounds and is applicable for all 92

analytes with such properties. In the present research, effects of different variables on PEME 93

efficiency are studied and optimized. After optimization, the above method followed by a 94

HPLC-UV procedure are applied for the extraction and determination of ATE and BET in 95

pure water, urine and untreated human plasma samples. 96

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2. Experimental 97

2.1. PEME Equipment 98

The equipment used for the PEME procedure is shown in Fig. 1. A 24 ml glass vial was 99

used as the sample compartment. The electrodes applied in this work were platinum wires 100

with the diameter of 0.25 mm, which were obtained from Pars Pelatine (Tehran, Iran). The 101

electrodes were coupled to a power supply model 8760T3 with programmable voltages in the 102

range of 0-600 V and output currents in the range of 0-500 mA purchased from Paya 103

Pajoohesh Pars (Tehran, Iran). A home-made pulse generator was used to set the pulse 104

duration and outage period with a timer in the range of 1 s to 10 min. During the extraction, 105

the PEME unit was stirred at speeds in the range of 0-1250 rpm by a heater-magnetic stirrer 106

model 3001 supplied by Heidolph (Kelheim, Germany) using a 1.5 cm × 0.3 cm magnetic 107

bar. 108

2.2. Chemicals and materials 109

BET and ATE were obtained from Sina Darou (Tehran, Iran) and Sobhan Darou 110

(Tehran, Iran), respectively. The chemical structures of ATE and BET are illustrated in Table 111

1. 2-Nitrophenyl octyl ether (NPOE) and di-(2-ethylhexyl) phosphate (DEHP) were 112

purchased from Fluka (Buchs, Switzerland). All the chemicals used were of analytical-113

reagent grade. The porous hollow fiber applied in the SLM composition was a PPQ3/2 114

polypropylene hollow fiber from Membrana (Wuppertal, Germany) with an inner diameter of 115

0.6 mm, a wall thickness of 200 µm, and a pore size of 0.2 µm. Ultra-pure water was 116

supplied by a Younglin aqua MAx purification system 370 series (Kyounggi-do, Korea). 117

2.3. Biological matrices and standard solutions 118

Drug-free human plasma (blood group A+) was obtained from the Iranian Blood 119

Transfusion Organization (Tehran, Iran). Urine samples were collected from two persons 120

who were under treatment and one person who had not consumed the drugs at all (as a match 121

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matrix for plotting the calibration curves). Adult patients consumed 100 mg of atenolol orally 122

and their urine samples were collected after 2 h. All samples were stored at 4 ◦C, thawed and 123

shaken before extraction. A stock solution, containing 1 mg/ml of the analyte in methanol, 124

was prepared for each analyte and stored at 4 ◦C protected from light. Working standard 125

solutions were prepared by diluting the above stock solutions with methanol. 126

2.4. HPLC conditions 127

Separation and detection of the target analytes were performed by a Varian HPLC 128

(Walnut Creek, CA, USA) comprising a 9012 HPLC pump, a six-port Cheminert HPLC 129

valve from Valco Instruments (Houston, TX, USA) with a 20 µl sample loop and a Varian 130

9050 UV-Vis detector. Chromatographic data were recorded and analyzed using Chromana 131

software (version 3.6.4). The separations were carried out on an ODS-3 column (250 mm × 132

4.6 mm, with a particle size of 5 µm) from MZ-Analysentechnik (Mainz, Germany). The 133

flow rate of the mobile phase was set at 1.0 ml/min and the detections were performed at a 134

wavelength of 225 nm. The mobile phase consisted of 40% phosphate buffer solution (pH 135

3.5) and 60% methanol. Total analysis time was 10 min. 136

2.5. PEME Procedure 137

Twenty four milliliters of the sample solution, containing the target analytes in ultra-138

pure water, was transferred into the sample vial. To impregnate the pores of hollow fibers 139

with the organic solvents, two pieces of the hollow fibers were cut out (4 cm) and dipped in 140

the organic solvents for 5 s and then the excess amounts of organic solvents were gently 141

wiped away by air blowing using a medical syringe. The upper ends of hollow fibers were 142

connected to two medical needle tips as guiding tubes, which were inserted through the 143

rubber cap of the vial. A 100 mM HCl solution (as an acceptor phase) was introduced into the 144

lumens of hollow fibers by a microsyringe and then the lower ends of hollow fibers were 145

mechanically sealed. Two platinum cathodes were introduced into the lumens of fibers. Both 146

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fibers, containing the cathodes, the SLMs and the acceptor solution, were then moved into the 147

sample solution. The anode was transferred directly into the sample solution. The electrodes 148

were subsequently coupled to a power supply, which was connected to a pulse generator. 149

After the extraction was completed, the acceptor solutions were collected by a 25 µl 150

microsyringe and mixed together in a microtube. Twenty µl of the mixture was injected into a 151

HPLC apparatus for further analysis. 152

2.6. Calculation of preconcentration factor, extraction recovery and relative recovery 153

Preconcentration factor (PF) was defined as the ratio of final analyte concentration in 154

the acceptor phase (Cf,a) to initial concentration of analyte in the sample solution (Ci,s): 155

156

Wherein, Cf,a was calculated from a calibration graph obtained via the direct injection of ATE 157

and BET standard solutions (5–100 mg/l) into a 100 mM HCl solution. 158

Relative recovery (RR%) was calculated by the following equation: 159

160

Wherein, Cfound, Creal, and Cadded are, respectively, the concentration of analyte after the 161

addition of a known amount of the standard into the real sample, the concentration of analyte 162

in the real sample, and the concentration of a known amount of the standard spiked into the 163

real sample. 164

3. Results and discussion 165

To achieve maximum extraction recoveries in the determination of ATE and BET, the 166

effective parameters on PEME including applied voltage, extraction time, stirring rate, pHs of 167

donor and acceptor phases, pulse duration and outage period were optimized. All 168

optimizations were done in ultra-pure water containing both of ATE and BET. 169

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According to a previous study on the extraction of target drugs using EME method, 170

NPOE and a mixture of 90% NPOE and 10% DEHP were used as SLMs for the extraction of 171

BET and ATE, respectively [24]. 172

3.1. Effects of extraction time and voltage 173

It has been proven that the flux of analytes in EME process is affected by the 174

magnitude of applied voltage which provides the main driving force for the extraction 175

procedure [26]. Thus, applying a voltage across a SLM is an important factor to be 176

considered for efficient extraction of basic drugs. In three-phase microextraction, mass 177

transfer is a time-dependent process; so, time is another parameter which can affect the flux 178

of analytes. Due to their antagonistic effects, the simultaneous investigation of extraction 179

time and applied voltage leads to a more accurate optimal point. Extraction of drugs was 180

studied at different EME durations, ranging from 5 min to 25 min, and applied electric 181

potentials, which were varied in the range of 50-200 V. In order to optimize these parameters, 182

pulse duration and outage period were set at 15 s and 5 s, respectively, and the analytes were 183

extracted from a neutral sample solution, which was agitated by stirring at a rate of 700 rpm, 184

into a 100 mM HCl solution. The normalized peak area of each run was selected as a 185

response objective for the study [27, 28]. To normalize the peak areas for ATE and BET, first 186

all the experiments were performed and then the peak area of each analyte was divided by the 187

analyte’s smallest peak area which was obtained in all of the experiments. The normalized 188

peak areas were subsequently added for each run and used in the calculation of total 189

normalized peak area. The results are summarized in Fig. 2A. According to this figure, it can 190

be observed that the normalized peak area increases by increasing the extraction time up to 191

20 min and then reaches a plateau. The results also demonstrate that, as the voltage increases, 192

the extractabilities of both ATE and BET decrease. This phenomenon is most probably 193

caused by a slight increase in the pH of acceptor solution due to electrolysis, which leads to 194

9

the deprotonation of analytes and their back-extraction into the organic phases. Therefore, the 195

extraction efficiency was improved by decreasing the applied electric potential and increasing 196

the extraction time. Finally, a 100 V electric potential was applied for 20 min for the rest of 197

the work. 198

3.2. Effect of donor and acceptor phases pH 199

During the rest of optimization process, effect of different pH values of the donor and 200

acceptor solutions was investigated. It was shown that the ratio of total ionic concentration of 201

the donor phase to that of the acceptor phase, which is defined as ion balance (χ), affects the 202

flux over the membrane [29, 30]. The flux may decline with an increase in the above ratio as 203

described by theoretical models [30]. To study the effect of this parameter, HCl concentration 204

in the donor phase was changed from 0 mM (i.e. ultra-pure water) to 100 mM while it was 205

altered in the range of 1–100 mM in the acceptor phases (Fig. 2B). Maximum amounts of 206

drugs were extracted when the concentration of HCl in the acceptor phases was 100 mM and 207

ultra-pure water (pH 6.5) was used as the sample solution. As it was expected, a maximum 208

extraction was obtained for a minimum value of χ. On the other hand, to pass the analytes 209

through the electric field, it was necessary to convert the drugs into their ionized forms. Since 210

ATE and BET has relatively strong basic properties [25], it seems that they are completely 211

protonated in ultra-pure water. However, a decrease in the concentration of HCl in the 212

acceptor phases resulted in partial deprotonation of the analytes and accelerated their back-213

diffusion into the donor solution. Therefore, a 100 mM HCl solution was chosen as a suitable 214

acceptor solution for the rest of the study. 215

3.3. Effect of stirring rate 216

Sample stirring is also an important parameter in PEME process. Stirring rate plays a 217

substantial role in enhancing the kinetics and efficiency of extraction through increasing the 218

mass transfer and reducing the thickness of double layer around the SLM [29]. Because of the 219

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outage period in PEME [23], the latter affects the extraction efficiency very significantly. To 220

acquire the best extraction recoveries, the effect of this parameter was investigated in the 221

range of 0–1250 rpm. The highest extraction efficiency was achieved at the maximum stirring 222

speed (Fig. 2C). However, it was anticipated that extractability would diminish at very high 223

stirring rates due to the formation of vortex in the sample solution; it appeared that the 224

decreasing effect of stirring rate on the thickness of double layer was more prominent in 225

PEME technique. On the other hand, the use of three electrodes and two pieces of hollow 226

fibers in the sample solution decreased the vortex intensity to some extent and reduced its 227

negative effect on the extraction efficiency. Thus, a stirring rate of 1250 rpm was selected for 228

the subsequent experiments. 229

3.4 Effects of pulse duration and outage period 230

The main difficulties of EME technique are bubble formation at the electrodes, 231

instability problems, punctuation of the SLM and appearance of sparks at relatively high 232

applied voltages [21, 31-35]. Once the voltage is applied, all ionic species in the solution 233

move toward the electrodes and form a charged double layer on both sides of the SLM, which 234

leads to a mass transfer resistance, and some sparks may be observed at relatively long 235

extraction times or high applied voltages. Pulsed voltage could be an alternative to overcome 236

all these drawbacks. The duration of pulse was long enough for the analytes to migrate from 237

the sample solution through the SLM into the acceptor phase; on the other hand, it was too 238

short, so that the thickness of boundary layer was minimized. At the outage step, there was no 239

electric field while the sample solution was being stirred, so the double layer disappeared. 240

Pulse duration and outage period were applied repeatedly after each other until the extraction 241

time was spent. Therefore, the total extraction time was not reduced and the extraction system 242

had enough time to reach a steady state; but each pulse was too short to minimize the 243

instability problems. To study the effects of pulse duration and outage period, the duration of 244

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pulse duration was changed from 5 s to 25 s while the length of outage step was altered in the 245

range of 2–10 s. As shown in Fig. 2D, maximum extraction is obtained when the durations of 246

pulse and outage period are 15 s and 2 s, respectively. 247

As a consequence, the optimal conditions were attained via using ultra-pure water and a 248

100 mM HCl solution as donor and acceptor phases, respectively, and applying a 100 V 249

electric potential for 20 min. The sample solution was agitated by stirring at a rate of 1250 250

rpm and the durations of pulse and outage period were 15 s and 2 s, respectively. Also, the 251

SLM composition was pure NPOE for BET and a mixture of 90% NPOE and 10% DEHP for 252

ATE. 253

3.5 Method validation 254

To verify the practical applicability of the proposed PEME technique, calibration 255

curves were plotted for ultra-pure water, drug-free urine and plasma samples under the 256

optimized extraction conditions and figures of merit for the method were evaluated. The 257

results are summarized in Table 2. To this end, the samples were spiked with the drugs and 258

the extraction was accomplished after the dilution of urine (1:3) and plasma samples (1:14) 259

with ultra-pure water. The pH values of all samples were adjusted by dropwise addition of 260

NaOH and/or HCl solutions, so that the final pH of samples was 6.5. As can be seen from 261

Table 2, this system has the potential for simultaneous extraction and determination of target 262

analytes with admissible preconcentration factors in the ranges of 69-160 and 236-363 for 263

ATE and BET, respectively, in different matrices. Satisfactory limits of detection were 264

obtained (LODs < 10 ng/ml) in this technique, which explained the ability of this new set-up 265

to analyze trace amounts of drugs with different properties. The linearity of PEME method 266

was studied up to 1000 ng/ml and correlations of determination higher than 0.9944 with 267

acceptable repeatabilities (RSDs < 7.4%) were achieved. Some experiments were designed to 268

compare the performance of PEME with that of conventional EME. Conventional EME was 269

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carried out for the extraction of analytes from pure water, human plasma and urine samples. 270

All samples were prepared as described above and optimal conditions for PEME were 271

applied unless for pulse duration and outage period. With this aim, pulse duration and outage 272

period were eliminated and a 100 V electric potential was applied continuously for 20 min to 273

force the analytes to transfer from the neutral sample solution into the acidic acceptor phase. 274

Results, given in Table 2, show that PEME offers higher preconcentration factors and better 275

limits of detection which confirm the benefits of this technique compared to conventional 276

EME. Table 3 demonstrates that unlike expensive and uncommon instruments such as LC-277

MS/MS, PEME technique exhibits a wide linear range, high sensitivity, and acceptable 278

repeatabilities. Specially, the preconcentration factors of this method make it more efficient 279

for the cleanup and determination of these analytes in biofluids. Relatively short extraction 280

time and improved sample cleanup facilitate the analysis of these drugs by common 281

instruments like HPLC-UV. Also, in comparison with SPE, EME eliminate possible carry-282

over problems because the hollow fiber is not expensive and can be discarded after each 283

extraction. Selecting an appropriate organic solvent, EME can create a high sensitivity as 284

well as high cleanup whereas SPE has not selectivity and causes crowded chromatograms 285

after extraction of the analytes from complex matrices. The main disadvantage of the 286

proposed method is its small throughput (3 samples per hour). Although the extraction time 287

was relatively long, the sheer smallness of the developed EME system lends itself to 288

extraction in parallel. Along with simplicity, PEME has a high sensitivity and provides 289

efficient cleanup in complex matrices. Meanwhile, the consumption of organic solvents in 290

this technique is minimized. 291

3.6 Analysis of real samples 292

In order to investigate matrix effects and the applicability of the proposed technique for 293

extraction from real samples, some final experiments were carried out on different urine 294

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samples taken from volunteers who had consumed ATE tablets (for details, see section 2.3). 295

Urine samples were diluted 1:3 with ultra-pure water and their pHs were adjusted to 6.5 by 296

dropwise addition of NaOH solution. Then, 24 ml of each solution was transferred into the 297

sample vial and the extraction process was performed three times for each sample under 298

optimal conditions. The obtained results are illustrated in Table 4. The relative standard 299

deviations (RSDs%) were within the range of 2.2–7.4%. Also, to examine the accuracy of 300

the proposed method, 500 ng/ml of each analyte was spiked into the samples, PEME was 301

carried out and the relative recoveries were calculated (Table 3). All non-spiked and spiked 302

chromatograms of the real samples are depicted in Fig. 3. As seen in this figure, relative 303

recoveries for the spiked samples are in an acceptable range (97–107%) and there is no 304

significant difference between the media, used to plot the calibration curves, and the real 305

sample matrices. Therefore, calibration curves can be employed directly to calculate the 306

amounts of drugs in samples. 307

4. Conclusions 308

The present work reveals the feasibility of simultaneous extraction of two β-blocker 309

drugs with different polarities across appropriate SLMs by applying a new concept of EME 310

technique named PEME. To achieve this goal, a new setup with two cathodic hollow fibers 311

was designed. The proposed method was successfully developed for the extraction and 312

determination of ATE and BET in water and in human urine and plasma samples. Compared 313

to the existing methods for extraction of such drugs, this technique showed several 314

advantages. The stability and the extraction efficiency were improved relative to conventional 315

EME [24]. Also, the analysis of two drugs with different properties became possible in 316

addition to providing EME advantages such as minimum consumption of organic solvents, 317

short extraction time, efficient sample clean-up and very simple and inexpensive equipment. 318

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The suggested method may become a very powerful and innovative sample preparation 319

technique for drug analysis in different complex biological matrices in the future. 320

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Table Captions 486

Table 1. Chemical structures and amounts of pKa, logP and therapeutic concentration (TC) 487

for ATE and BET. 488

489

Table 2. Figures of merit for the process of EME-HPLC-UV, for the extraction and 490

determination of analytes in drug-free water, plasma and urine samples. 491

492

Table 3. Comparison of the proposed method with other analytical techniques for extraction 493

and determination of ATE and BET in different samples. 494

495

Table 4. Determination of ATE and BET in different urine samples. 496

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Figure legends 507

Fig. 1. A schematic illustration of the equipment used for simultaneous extraction of ATE 508

and BET by PEME process. Different parts of the set-up are represented in the figure. 509

Fig. 2. Effects of applied voltage and extraction time (A), HCl concentration in the donor and 510

acceptor phases (B), stirring rate (C) and pulse duration and outage period (D), on the 511

extraction efficiencies of ATE and BET. Spiked concentrations for both of ATE and BET: 512

500 ng/ml: 500 ng/ml; SLMs: NPOE and a mixture of 90% NPOE+10% DEHP; sample 513

volume: 24 ml; extraction time: 20 min. 514

Fig. 3. Chromatograms obtained after performing (A) PEME process with (a) a non-spiked 515

plasma sample, (b) a plasma sample spiked at a concentration of 25 ng/ml of the drugs and 516

(B) PEME process with (a) a non-spiked urine sample (b) a urine sample spiked at a 517

concentration of 500 ng/ml of the drugs. 1: ATE, 2: BET. 518

519

520

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Table 1. Chemical structures and amounts of pKa, logP and therapeutic concentration (TC) for ATE and BET.

Chemical structure Name of compound

IUPAC name pKaa Log pa TC

(ng/ml)

Atenolol 4-[2-Hydroxy–3-[(1–methylethyl) amino]propoxy]benzeneacetamide

9.6 0.23 410-870

Betaxolol 1-[4-[2-(Cyclopropylmethoxy)ethyl]phenoxy]-3-[(1–methylethyl)amino]-2–propanol

9.4 2.81 5-50

a Reference [25]

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Table 2. Figures of merit for the process of EME-HPLC-UV, for the extraction and determination of analytes in drug-free water, plasma and urine samples.

PEME Conventional EME

Sample Analyte LOD (ng/ml)

Linear range

(ng/ml) R2 PFa RSD%b

LOD

(ng/ml)

Linear range

(ng/ml) R2 PFa RSD%b

ATE 5 10-1000 0.9995 160 3.6 10 25-1000 0.9972 83 4.5 Water BET 1 5-1000 0.9988 363 4.5 5 10-1000 0.9989 280 6.6 ATE 10 15-1000 0.9988 86 7.4 15 25-1000 0.9978 42 4.9

Plasma BET 2 5-1000 0.9983 245 2.2 5 10-1000 0.9960 179 8.3 ATE 10 25-1000 0.9974 69 3.9 25 40-1000 0.9973 31 5.5

Urine BET 2 5-1000 0.9944 236 4.9 7 10-1000 0.9983 170 3.4

a Preconcentration factors at 25 ng/ml and 50 ng/ml for PEME and conventional EME, respectively.

b For five replicate measurements.

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Table 3. Comparison of the proposed method with other analytical techniques for extraction and determination of ATE and BET in different samples.

Ref RSD%

LOD

(ng/ml) R2 Linear range

(ng/ml) Extraction time

(min) Sample Analyte Analytical techniquea

[36] 5.05 27 0.995 50-400 45 Plasma ATE SPE-CZE [37] �18.5 0.017 �0.997 0.04-1000 30 Water ATE SPE-LC/MS/MS [37] �18.5 0.75 �0.997 0.04-1000 30 Water BET SPE-LC /MS/MS [38] 8.5 10 0.992 50-1250 - Plasma ATE SPE-LC /MS [38] 8.6 2.5 0.996 2.5-62.5 - Plasma BET SPE-LC/MS [39] 4.7 6 0.997 25-1000 35 Plasma ATE LLE-HPLC/UV [39] 4.12 10 0.996 25-1000 35 Plasma BET LLE-HPLC/UV [40] 4 7 0.995 25-5000 50 Water ATE PC-HFLE-CE [41] 17.2 - 0.992 25-1500 60 Plasma ATE HF-LPME-LC/MS [42] 20� 0.0015 0.999 0.1-200 - Water ATE MIP-LC-MS [42] 10� 0.003 0.998 0.1-200 - Water BET MIP-LC/MS [43] 5.2 - 0.998 0.015-140 - Water ATE SPE-UPLC/MS [43] 6.8 - 0.998 0.015-140 - Water BET SPE-UPLC/MS [44] 15.2 0.013 0.989 0.013-0.8 �30 Water ATE SPE-UPLC/Ms [20] �9.9 - 0.999 10-2000 - Eye tissue ATE LLE-LC-MS/MS [20] 9.42 - 0.999 10-2000 - Eye tissue BET LLE-LC/MS/MS [45] 4.6 - 0.993 50-750 30 Plasma ATE LLE-GC/MS [46] �5 50 0.996 50-500 - Urine ATE SPE-CE-UV [46] �5 50 0.999 50-500 - Urine BET SPE-CE-UV [47] 7.1 - 0.980 10-2050 - Plasma ATE SPE-LC/MS/MS [48] �10 - 0.999 10.6–6000 - Water, plant ATE SPE-LC/MS/MS [24] 6.4 2 0.997 10-5000 15 Water ATE EME-HPLC-UV

This work 3.6 5 0.999 10-1000 20 Water ATE PEME-HPLC-UV This work 7.4 10 0.999 15-1000 20 Plasma ATE PEME-HPLC-UV This work 3.9 10 0.997 25-1000 20 Urine ATE PEME-HPLC-UV This work 4.5 1 0.999 5-1000 20 Water BET PEME-HPLC-UV This work 2.2 2 0.998 5-1000 20 Plasma BET PEME-HPLC-UV This work 4.9 2 0.994 5-1000 20 Urine BET PEME-HPLC-UV

a Hollow fiber (HF), liquid-phase microextraction (LPME), liquid–liquid extraction (LLE), liquid chromatography (LC), mass spectrometry (MS), solid-phase extraction (SPE), molecularly imprinted polymer (MIP), polymer-coated hollow fiber microextraction (PC-HFME), capillary electrophoresis (CE), capillary zone electrophoresis (CZE), ultra-performance liquid chromatography (UPLC), electromembrane extraction (EME), pulsed electromembrane extraction (PEME).

26

1

Table 4. Determination of ATE and BET in different urine samples. Sample Analyte Creal

(ng/ml) Cadded

(ng/ml)

Cfound

(ng/ml)

RSD% (n = 3)

RR%

ATE 651 500 994 3.6 97 Urine 1 BET

nda 500 491 4.3 98

ATE 414 500 933 5.1 104 Urine 2 BET nd 500 519 4.9 107

a Not detected. 2

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