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Graphical Abstract

Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxxHigh performance liquid chromatographic determination ofultra traces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersiveliquid–liquid microextraction coupled with response surfaceoptimization

Mojtaba Shamsipur∗, Mehrosadat Mirmohammadi

PBA 9683 1

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Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

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Journal of Pharmaceutical and Biomedical Analysis

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Highlights

Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxxHigh performance liquid chromatographic determination ofultra traces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersiveliquid–liquid microextraction coupled with response surface optimization

Mojtaba Shamsipur∗, Mehrosadat Mirmohammadi

• Design of a dispersive liquid–liquid microextraction–HPLC method for assay of antidepressants in urine.• Use of response surface methodology for multivariate optimization of DLLME conditions.• Method development and validation for assay of imipramine and trimipramine in urine samples.• Enrichment factor, linear range and limit of detection of 161.7–186.7, 5–100 ng mL−1 and 0.6 ng mL−1, respectively.• Possibility to determine the residual of drugs four days after using a 30 mg dosage in urine samples.

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

ARTICLE IN PRESSG ModelPBA 9683 1–8

Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

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High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine andtrimipramine in urine samples after their dispersive liquid–liquidmicroextraction coupled with response surface optimization

1

2

3

4

Mojtaba Shamsipur ∗, Mehrosadat MirmohammadiQ15

Department of Chemistry, Razi University, Kermanshah, Iran6

7

a r t i c l e i n f o8

9

Article history:10

Received 22 March 201411

Received in revised form 3 August 201412

Accepted 5 August 201413

Available online xxx14

15

Keywords:16

Dispersive liquid–liquid microextraction17

High-performance liquidchromatography–ultraviolet detection

18

19

Response surface methodology20

Tricyclic antidepressants21

Urine analysis22

a b s t r a c t

Dispersive liquid–liquid microextraction (DLLME) coupled with high performance liquid chromatog-raphy by ultraviolet detection (HPLC–UV) as a fast and inexpensive technique was applied to thedetermination of imipramine and trimipramine in urine samples. Response surface methodology (RSM)was used for multivariate optimization of the effects of seven different parameters influencing the extrac-tion efficiency of the proposed method. Under optimized experimental conditions, the enrichment factorsand extraction recoveries were between 161.7–186.7 and 97–112%, respectively. The linear range andlimit of detection for both analytes found to be 5–100 ng mL−1 and 0.6 ng mL−1, respectively. The relativestandard deviations for 5 ng mL−1 of the drugs in urine samples were in the range of 5.1–6.1 (n = 5). Thedeveloped method was successfully applied to real urine sample analyses.

© 2014 Published by Elsevier B.V.

23

1. Introduction24

Imipramine (IMIP) and trimipramine (TRIM), with the chemical25

structures shown in Fig. 1, are among widely used tricyclic antide-26

pressants (TCAs) throughout the world. Imipramine is mainly used27

in the treatment of major depression and enuresis (inability to28

control urination). It has also been evaluated for use in panic dis-29

order [1]. Trimipramine is chemically similar to other tricyclic30

antidepressants such as imipramine as well as the antipsychotic31

levomepromazine (Nozinan). Trimipramine mechanism of action32

differs from other TCAs. It is only a moderate reuptake inhibitor33

of norepinephrine, and a weak reuptake inhibitor of serotonin and34

dopamine. Its main effects are due to considerable receptor antago-35

nism [2]. However, these drugs possess some side effects and could36

cause toxic accidents to human. It has been reported that even37

selective serotonin reuptake inhibiter (SSRI) with fewer side effects38

could cause toxic accidents, resulted in arousing aggressive charac-39

ter in the patients [3]. The published literature in this respect clearly40

demonstrated that the poisoning trouble could happen by taking41

∗ Corresponding author. Tel.: +98 21 66908032; fax: +98 21 66908030.E-mail address: mshamsipur@yahoo.com (M. Shamsipur).

large amounts of antidepressant drugs, many of such incidents 42

being related to suicides [4]. Antidepressants also have a higher 43

risk of serious cardiovascular side effects [5]. Thus, fast screening 44

test of these drugs in biological fluids is of critical importance. 45

Currently, there are a number of commercially available kits, 46

such as Triage® immunoassay test kit, in the market for the detec- 47

tion of many, but not all, tricyclic antidepressants. Although the 48

tricyclic antidepressants can be well detected by this kit, their 49

detection limits, which are in the range of 1000–4000 nM, are not 50

sufficient for many clinical purposes [4]. Thus, the development of 51

new or improved assay methods for trace levels of antidepressant 52

drugs in biological samples is of increasing interest. The general 53

methods for analyzing psychotropic drugs in different biological 54

samples are based on combining an efficient separation technique 55

with a sensitive detection method. At present, a number of separa- 56

tion/determination techniques, including high-performance liquid 57

chromatography (HPLC) [6,7], capillary electrophoresis (CE) [8] and 58

gas chromatography (GC) [9,10] have been used for the analy- 59

sis of psychotropic drugs. Among those methods, HPLC has been 60

considered as the most efficient and robust specific technique 61

due to some advantages including convenience, simple operation, 62

strong separation ability and wide sample application. Some other 63

detection techniques have also been used to accurately determine 64

the concentration of psychotropic drugs in various samples; these 65

http://dx.doi.org/10.1016/j.jpba.2014.08.0080731-7085/© 2014 Published by Elsevier B.V.

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

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2 M. Shamsipur, M. Mirmohammadi / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

Fig. 1. Structures of IMIP and TRIM.

include flow-injection fluorimetry [11], voltammetry [12], chemi-66

luminescence [13] and HPLC–MS [14].67

However, in many cases, because of matrix interference and68

insufficient instrumental detection limit for traces of psychotropic69

drugs in real biological samples, direct chromatographic separa-70

tion and determination by these methods are very difficult [15].71

Therefore, in order to obtain accurate, reliable and sensitive results,72

an extraction/preconcentration method is required prior to chro-73

matographic separation of psychotropic drugs [2,16]. In fact, the74

use of an appropriate pre-concentration method such as DLLME75

may lead to an increase in the signal to noise ratio of chromato-76

graphic peaks and a consequent decrease in the detection limit of77

the chromatographic methods [2,15,16].78

Recent researches have been directed toward developing effi-79

cient, economical, and miniaturized sample preparation methods.80

One of the most recent modalities of microextraction introduced81

by Assadi and co-workers is dispersive liquid–liquid microextrac-82

tion (DLLME) [17,18]. It is based on a ternary component solvents83

system similar to homogeneous liquid–liquid extraction (HLLE) and84

cloud point extraction (CPE). Rapidity, high enrichment factor, high85

extraction recovery, simplicity of operation and low cost are some86

of the advantages of this method. The performance of DLLME was87

illustrated [17–20].88

Due to the fact that IMIP and TRIM are among the most89

prescribed synchronously antidepressant drugs worldwide and,90

especially in Iran, in the present study, a new DLLME method cou-91

pled to HPLC–UV has been developed for simultaneous extraction,92

preconcentration and determination of their trace amounts in urine93

samples. The important factors influencing the extraction efficiency94

of the system were optimized using the Box–Behnken model as an95

experimental design for response surface methodology (RSM) [21].96

The RSM is a collection of mathematical and statistical techniques97

that are useful for the modeling and analysis of problems in which98

a response of interest is influenced by several variables and the99

objective is to optimize this response. The orthogonal designs are100

used in obtaining regression models of the second-order [21,22].101

The model can predict how a response relates to the values of var-102

ious factors and it usually requires less number of experiments103

compared to a one-factor-at-a-time (OFAT) procedure [23]. The104

developed method was successfully applied to real urine sample105

analysis. Moreover, in order to express the strength of the proposed106

technique, its limits of quantification for IMIP and TRIM were com-107

pared with best of those previously reported in the literature (see108

following sections).109

2. Experimental110

2.1. Reagents and chemicals111

Imipramine (IMIP) and trimipramine (TRIM) with purity of112

>99% and loss on drying <0.3% (based on their certification) were113

kindly supplied by Amin Pharmaceutical Co. (Isfahan, Iran) and 114

Tehran Chemie Pharmaceutical Co. (Tehran, Iran), respectively. 115

All analytical or HPLC grade solvents used and analytical grade 116

reagents dehydrogenated potassium phosphate, sodium chloride, 117

hydrochloric acid and etc. were purchased from Merck (Darmstadt, 118

Germany). Doubly distilled water was used for preparation of aque- 119

ous solutions. 120

All laboratory glassware used were cleaned by soaking in sul- 121

fochromic acid and thoroughly rinsed with doubly distilled water, 122

prior to use. All solutions used were filtrated through a 0.2 �m 123

polypropylene filter from Agilent (Waldbronn, Germany) to dis- 124

card granules. Drug-free human urine samples were obtained from 125

healthy male and female volunteers. 126

2.2. Apparatus 127

A HPLC system of Agilent 1200 series equipped with a qua- 128

ternary pump G1311A, an on-line solvent vacuum degasser and 129

a variable wavelength UV detector (G1314B). The Chem32 soft- 130

ware was used to record chromatograms and calculate peak area. 131

Chromatographic separations were performed on Eclipse XDB-C18 132

column (150 mm × 4.6 mm) packed with 5 �m particles (Agilent). 133

A CARY 100 UV–Vis Spectrophotometer (Agilent) was used to 134

obtain the absorption maxima of analytes. The pH of solutions was 135

controlled with a Horiba F12 pH meter (Horiba, Irvine, CA, USA) 136

supplied with a Sentek combined electrode. An ultrasonic system 137

(Hielscher UIP1000hp, Teltow, Germany) was used for ultrasonica- 138

tion of the samples, A Kokosun centrifuge (Model h 11 n, Shizuoka, 139

Japan) was used for centrifugation of the extracts. A Hamilton 140

syringe (2.5 mL) and a SGE microsyringe (SGE Analytical Science, 141

Melbourne, Australia) were used for DLLME procedure. A vac- 142

uum drying oven (Daihan LabTech, Gyeonggi-do, South Korea) was 143

employed for drying the extraction solvents. 144

2.3. Preparation of standard solutions and urine samples 145

Stock solutions of IMIP and TRIM (1000.0 mg mL−1) were pre- 146

pared by dissolving the equivalent of 10 mg of the respective drug 147

related to its free base in 10 mL methanol. They were stored at 148

4 ◦C in refrigerator, protected from light. Working solutions were 149

prepared daily by appropriate dilution of the stock solutions with 150

high-purity deionized water. Aqueous standard solutions were 151

spiked in urine and used to modify the separation/preconcentration 152

conditions of DLLME. 153

Drug-free urine samples were supplied by healthy volunteers 154

not exposed to any drug for at least two months. In addition, some 155

real urine samples were collected from a male patient following 156

courses of treatment with a psychotropic drug including IMIP. The 157

urine samples were separately collected and stored in PTFE flasks 158

at −20 ◦C until analysis. 159

The hydrolysis of frozen urine samples after defrosting at room 160

temperature was carried out as follows. First, 2 mL of 10 M KOH was 161

added to 10 mL of urine sample and then it was hold in 60 ◦C for 162

10 min. Then, the mixture was centrifuged for 10 min at 2000 rpm. 163

The supernatant was transferred to a clean glass beaker and set 164

under a 50 W ultrasound for 5 min. The resulting solution was fil- 165

tered through a 0.2 �m filter and adjusted to pH 8.50 with about 166

1 mL of 6 M HCl solution and, finally, subjected to the DLLME pro- 167

cess. 168

A series of standard solutions, prepared by diluting appropriate 169

aliquots of the stock solution with drug-free urine at the desired 170

pH values were subjected to the optimal DLLME procedure. The 171

calibration curve for each drug was obtained by simple linear 172

regression of the peak area-drug concentration plot and the con- 173

centration of analyte in the sample was calculated based on the 174

resulting calibration curve. A urine sample without any species of 175

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

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Table 1The factors included in the response surface design and the corresponding levels.

Factor Abbreviation Level

Low High

Volume of extraction solvent (�L) VE 10 60Volume of dispersion solvent (mL) VD 0.1 1.5Salt percent %S 0 10Sample pH pH 3 11Centrifuge time (min) tC 1 15Reaction time (s) tR 0 6Centrifuge revolution per minute (rpm) w 1000 3000

interest was used as the blank solution. The amounts of IMIP and176

TRIM were determined after the blank solution was subjected to177

the procedure described for sample. The actual concentrations of178

the analytes were obtained after blank subtraction.179

2.4. Procedures180

To a calibrated 10 mL screw cap glass test tube was added181

1.00 mL of hydrolyzed urine at pH 8.5, 1.00 mL of aqueous sample182

solution of analytes at a concentration level of 5 �g mL−1 and 0.2 mL183

of 25 wt% of NaCl and diluted to 5.00 mL with doubly distilled water.184

After rapidly and vigorously injecting of 0.8 mL of acetonitrile (as185

disperser solvent) containing 50 �L of chloroform (as extraction186

solvent) into sample solution by a syringe, a cloudy solution was187

formed in the test tube (the cloudy state was stable for at least188

2 h) and then the mixture was gently shaken. The separation of189

the phases was achieved by centrifugation at 2000 rpm for 5 min.190

In the case of aqueous samples, a small droplet of chloroform was191

sedimented in the bottom of the conical test tube. While in the192

case of urine samples, a white lipidic compound was sedimented193

in the bottom of the conical test tube, most probably due to the co-194

sedimentation of the matrices (such as carbamide and uric acid)195

in urine at high pH values [15]. However, by ultrasonic irradiation196

of the urine samples before DLLME procedure, these lipid materi-197

als were shattered and the sediment solvent was separated easily198

in the bottom of the conical tube after centrifuging process. The199

volume of sediment phase was determined using a 50 �L microsy-200

ring and found to be 45.7 ± 0.2 �L. Then, 40.0 �L of the sedimented201

phase was removed with a microsyring and placed in a 5 mL screw202

cap glass test tube and dried in a vacuum drying oven at a temper-203

ature of 25 ◦C and pressure of −0.08 MPa. Finally, the object was204

dissolved in 30 �L acetonitrile and injected into the 20 �L injection205

loop of HPLC for ensuing analysis.206

Optimization of different parameters affecting the extraction by207

the DLLME method was performed by a full Box–Behnken design208

[21] using the Minitab® 16.2 statistical software [23]. This response209

surface design was employed with seven plan factors at two levels210

in three blocks. These factors include volume of extraction solvent211

(VE), volume of dispersion solvent (VD), salt percentage (%S), sample212

pH, centrifuge revolutions per minute (w), time of reaction (tR) and213

time of centrifugation (tC). Table 1 depicts the abbreviation and214

levels of each factor employed in the design.215

In order to examine the enrichment factor of each analyte, three216

replicate extractions were performed at optimal conditions from217

urine solutions consisting 5 ng mL−1 IMIP and TRIM. The enrich-218

ment factor (EF) was calculated as the ratio of final concentration219

of analytes in sediment phase (CSed) and its concentration in the220

original solution (C0). In this case, Csed was calculated from the221

calibration curve and C0 was 5 ng mL−1, as:222

EF = Csed

C0223

Percent recovery (%R) was also evaluated from the enrichment 224

factors obtained, as: 225

%R =(

V sed

Vaq

)× EF × 100 226

where Vsed and Vaq are the volumes of sediment phase and aqueous 227

sample, respectively. 228

3. Results and discussion 229

Because urine is a biological fluid and its complex matrix would 230

cause a negative effect on the recovery of analytes under ordinary 231

conditions, the hydrolysis of urine into an appropriate form for 232

DLLME method is a critical requirement [15]. Therefore, each urine 233

sample was basified using 2 M of KOH so that, after hydrolysis, most 234

of interfering compounds such as carbamide, uric acid, calcium salt 235

and some rarely present protein, glucose, calcium phosphate and 236

so on was precipitated out. As mentioned in Section 2.4, by ultra- 237

sonic irradiation of the urine samples before DLLME procedure, it 238

was possible to break down the structure of such lipid materials 239

remaining, which interfere in removal of sediment part. 240

In the design of a DLLME method combined with HPLC–UV for 241

the pre-concentration and determination of TCAs in biological flu- 242

ids, there are several factors that affect the extraction process; these 243

include the nature and volume of extraction and disperser solvents, 244

pH of solution, salt effect and reaction and centrifugation times. In 245

the present study, the selection of best extraction and dispersion 246

solvents was achieved by one-factor-at-a-time (OFAT) experiments 247

and the other parameters were optimized by the response sur- 248

face methodology to reduce the interaction between the effective 249

factors [24,25]. 250

3.1. Selection of extraction solvent 251

Choosing the most suitable extraction solvent is of primary 252

importance for achieving good extractability and selectivity of the 253

target analytes in DLLME. In this context, some factors should be 254

considered: the extraction solvent must be immiscible with water, 255

the solubility of analytes should be higher in the organic phase than 256

the donor phase to promote the extraction of the analytes and the 257

density of the organic solvent must be higher than water and it 258

should be non-volatile in the course of extraction [17]. Therefore, 259

several organic solvents as carbon disulfide (CS2), dichloromethane 260

(CH2Cl2), chlorobenzene (C6H5Cl), carbon tetrachloride (CCl4) and 261

chloroform (CHCl3), which have the above mentioned characteris- 262

tics, were studied. Thus, to a series of 5.0 mL of 100 ng mL−1 of the 263

two drug sample solutions was added 1.00 mL of acetonitrile, as dis- 264

perser solvent, containing 50.0 �L of different extraction solvents, 265

and the recommended procedure was followed. 266

The results revealed that, in the cases of CS2 and CH2Cl2, no 267

cloudy state was observed and also no sediment droplet of extract 268

was formed in the bottom of the tube after centrifugation. This is 269

most possibly due to higher water solubility of these solvents than 270

the other tested solvents [9]. While, in the cases of use of C6H5Cl, 271

CCl4 and CHCl3 as extraction solvents, the cloudy state was occurred 272

and a sediment droplet of extract was formed in the bottom of test 273

tube. However, after HPLC–UV analysis of the resulting extracts, it 274

was found that C6H5Cl has a strong peak at 2.035 min which was 275

partially overlapped with the peak of IMIP at 2.479 min, but not 276

with that of TRIM, observed at 4.754 min, so that the simultaneous 277

detection of the two drugs cannot be performed accurately in this 278

solvent. On the other hand, there was no overlapping problem in the 279

resulting chromatograms in the cases of CCl4 and CHCl3 as extrac- 280

tion solvent and the extraction efficiency of both solvents for IMIP 281

and TRIM was more or less the same. However, the interference of 282

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

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Table 2Response surface design experiments and the responses for IMIP and TRIM.Q4

No. Parameter EFIMIP EFTRIM %RIMIP %RTRIM

pH %S VD VE tR tC w

1 3 5 0.1 30 6 8 2000 35.565 36.472 21.339 21.8832 7 5 0.8 50 6 15 2000 195.916 233.819 117.550 140.2923 7 10 1.5 30 3 15 2000 8.308 4.939 4.985 2.9634 11 5 1.5 30 6 8 2000 * * * *5 7 5 0.8 30 3 8 2000 110.707 127.131 66.424 76.2796 7 10 0.8 30 6 8 3000 5.607 * 3.364 *7 7 5 0.1 50 3 8 1000 85.169 79.831 51.101 47.8998 7 0 1.5 30 3 1 2000 15.184 7.567 9.110 4.5409 11 5 0.8 30 3 1 1000 83.204 72.736 49.923 43.642

10 7 0 0.1 30 3 15 2000 19.113 13.348 11.468 8.00911 3 0 0.8 50 3 8 2000 218.017 242.228 130.810 145.33712 3 5 0.8 30 3 15 1000 78.539 78.254 47.123 46.95313 7 5 0.8 10 6 1 2000 14.202 11.508 8.521 6.90514 7 5 0.1 10 3 8 3000 6.098 7.041 3.659 4.22515 7 5 0.8 50 0 1 2000 233.242 247.747 139.945 148.64816 7 5 1.5 10 3 8 1000 * * * *17 7 5 1.5 50 3 8 3000 * * * *18 11 5 0.8 30 3 15 3000 152.698 143.686 91.619 86.21219 3 5 1.5 30 0 8 2000 4.625 * 2.775 *20 3 5 0.8 30 3 1 3000 75.346 84.298 45.208 50.57921 7 5 0.8 10 0 15 2000 * * * *22 7 10 0.1 30 3 1 2000 67.243 69.057 40.346 41.43423 7 0 0.8 30 0 8 3000 51.036 89.291 30.621 53.57524 11 5 0.1 30 0 8 2000 90.080 79.568 54.048 47.74125 3 10 0.8 10 3 8 2000 3.152 * 1.891 *26 7 0 0.8 30 6 8 1000 102.849 108.211 61.709 64.92727 7 5 0.8 30 3 8 2000 57.911 62.488 34.747 37.49328 7 5 0.8 30 3 8 2000 60.858 64.853 36.515 38.91229 7 10 0.8 30 0 8 1000 39.740 261.674 23.844 157.00430 11 0 0.8 10 3 8 2000 * * * *31 11 10 0.8 50 3 8 2000 109.479 77.466 65.688 46.480

white lipoids sediment in DLLME of urine samples was higher in283

CCl4 than that in CHCl3; therefore, chloroform was selected as the284

most suitable extraction solvent for subsequent experiments.285

3.2. Selection of disperser solvent286

The disperser solvent must be miscible in both the extraction287

solvent (organic phase) and the sample solution (aqueous phase).288

Therefore, the ability of acetone, methanol, ethanol, isopropanol289

and acetonitrile as disperser solvent was tested, by extracting a290

series of 5.0 mL of 100 ng mL−1 of the two drug sample solutions291

after addition of 1.00 mL of each candidate disperser solvent con-292

taining 50.0 �L of chloroform, as extraction solvent. In the case of293

methanol, a cloudy state was observed but no sediment droplet294

of extract was found in the bottom of the extraction tube after295

centrifugation. It is apparently because of higher solubility of chlo-296

roform in methanol than the other tried disperser solvents.297

While, the use of ethanol, acetone and acetonitrile as disperser298

solvent not only possessed a good cloudy state but also revealed299

an acceptable sediment phase and proper HPLC results. However,300

a maximum peak area was obtained by using acetonitrile as a dis-301

perser solvent (Fig. 2). This is most likely due to the synergistic302

effect of good compatibility of acetonitrile with aqueous solution303

and low distribution ratio of analyte drugs in the binary mixed304

solution of water and acetonitrile [15]. Therefore, acetonitrile was305

selected as a disperser solvent for further experiments.306

3.3. Optimization of extraction parameters by the response307

surface design308

After performing some preliminary experiments, optimization309

of the extraction conditions for DLLME method was conducted310

using a response surface design of Box–Behnken [21], with seven311

factors as illustrated in Table 2. Two dependent variables of312

extraction recovery (%R) and enrichment factor (EF) were opti- 313

mized with the model. 314

In each experiment, a suitable amount of extraction solvent (VE) 315

was injected into the urine sample containing a given percent of 316

salt (%S) at an adjusted pH. After elapse of a given reaction time 317

(tR) between the extraction and dispersion processes, an intended 318

volume of dispersion solvent (VD) was injected vigorously. Then the 319

cloudy solution was centrifuged in a suitable centrifuge revolution 320

per minute (w, rpm) and given period of time (tC), the sediment 321

phase formed in the bottom of the extraction tube was taken by 322

a microsyring and placed in a clean tube and dried in a vacuum 323

oven. Finally, the sediment was dissolved in 30 �L of acetonitrile 324

and injected into the HPLC column for following analysis. The data 325

Fig. 2. Selection of disperser solvent.

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

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Table 3Estimated regression coefficients and analysis of variance (ANOVA) of the predicted model for R% and EF.

Term IMIP TRIM

Coeff. for EF Coeff. for %R Regression and ANOVA factors for EF and %R Coeff. for EF Coeff. for %R Regression and ANOVA factors for EF and %R

P F P F

pH 1.5223 0.9134 0.861 0.76 −8.039 −4.8234 0.395 0.76%S 10.5889 6.3533 0.189 3.02 14.5334 8.72 0.099 3.02VD 12.4556 −7.4733 0.321 0.85 12.1425 −7.2855 0.369 0.85VE 75.0813 45.0488 0 62.07 79.1801 47.508 0.000 62.07tR 33.0446 19.8268 0.002 22.43 47.5954 28.5572 0.000 22.43tC 9.6723 5.8034 0.26 1.75 11.8699 7.1219 0.202 1.75w 13.4703 8.0822 0.133 1.31 10.546 6.3276 0.268 1.31pH × pH −7.1219 −4.2731 0.516 7.83 18.0455 10.8273 0.137 7.83%S × %S 17.1532 10.2919 0.125 2.42 12.2507 −7.3504 0.301 2.42VD × VD 65.9702 39.5821 0 1.14 78.9741 47.3845 0.000 1.14VE × VE 5.7506 3.4504 0.609 28.7 5.7172 3.4303 0.637 28.7tR × tR 23.6893 14.2136 0.046 0.23 37.5014 22.5008 0.006 0.23tC × tC 22.5954 13.5573 0.05 9.9 18.595 11.157 0.127 9.9w × w 16.5478 −9.9287 0.141 2.57 13.0187 −7.8112 0.277 2.57pH × %S 12.8356 7.7014 0.322 1.26 8.3925 5.0355 0.545 1.26pH × VD 11.3179 −6.7907 0.547 1.38 18.7739 11.2644 0.358 1.38pH × VE −4.9675 −2.9805 0.698 0.38 12.9035 −7.7421 0.356 0.38pH × tR −5.5143 −3.3086 0.751 0.89 −8.3261 −4.9957 0.657 0.89pH × tC 0.0921 0.0553 0.994 0.9 −0.761 −0.4566 0.956 0.9pH × w −5.8423 −3.5054 0.649 0.2 10.2101 −6.126 0.463 0.2%S × VD −7.2263 −4.3358 0.633 0 −6.4155 −3.8493 0.695 0%S × VE 10.1856 6.1114 0.43 0.56 8.2721 4.9632 0.551 0.56%S × tR 21.6451 12.9871 0.103 0.16 31.4732 18.8839 0.033 0.16%S × tC 3.2305 1.9383 0.831 0.37 1.4679 0.8807 0.928 0.37%S × w 7.1059 4.2635 0.58 5.34 −2.9398 −1.7639 0.831 5.34VD × VE 44.0224 26.4134 0.03 0.01 48.5733 29.144 0.027 0.01VD × tR 25.1566 15.0939 0.196 0.05 37.7802 22.6681 0.078 0.05VD × tC −5.8089 −3.4853 0.701 5.78 −4.2708 −2.5625 0.794 5.78VD × w 18.1928 10.9157 0.337 3.5 16.0136 9.6082 0.431 3.5VE × tR 18.4404 11.0643 0.229 0.07 15.9107 −9.5464 0.332 0.07VE × tC −4.0386 −2.4231 0.788 0.65 10.3891 −6.2335 0.524 0.65VE × w 9.8793 5.9276 0.571 0.99 13.3072 7.9843 0.480 0.99tR × tC 27.8784 −16.727 0.076 0.42 31.8713 19.1228 0.061 0.42tR × w 32.0712 19.2427 0.02 0.52 −25.068 15.0408 0.082 0.52tC × w −3.9187 −2.3512 0.76 3.98 −6.772 −4.0632 0.625 3.98

summarized in Table 2 show the %R and EF responses obtained for326

31 experiments designed by using the MINITAB software.327

Table 3 contains the estimated regression coefficients and the328

analysis of variance (ANOVA) of the effects for each term in the329

model. This table summarizes the linear terms, the squared terms,330

and the interactions. A P-value less than 0.05 in this table indi-331

cates the statistical significance of an effect at 95% confidence level.332

Small P-value of VE and tR, for EFIMIP, %RIMIP, %RTRIM and EFTRIM indi-333

cate that volume of extracting solvent (VE) and time of reaction334

(tR) effects are statistically significant. V2D and t2

R show significant335

effects on EFIMIP, %RIMIP, EFTRIM and %RTRIM while t2C influences only336

%RIMIP and %RTRIM. Effects of VD × VE on EFIMIP, %RIMIP, EFTRIM and337

%RTRIM, tR × w on EFIMIP and %RIMIP and %S × tR on EFTRIM, %RTRIM are338

statistically significant because of their small P-values.339

As it is seen from Fig. 3, the curvatures of the resulting plots340

of EF and %R values against VD and VE and tR and w, for both IMIP341

and TRIM cases, indicate the existence of interaction between the342

factors affecting the DLLME process.343

3.4. Optimization by the response surface design344

To assess whether the residuals are normally distributed,345

goodness-of-fit test (Anderson–Darling) statistics [23,25] was346

applied. The results showed the large P-values.347

For both EFIMIP and %RIMIP (P = 0.849) and EFTRIM and %RTRIM348

(P = 0.543), which exhibited that the residuals followed a normal349

distribution.350

The optimization plots shown in Fig. 4 also indicate the pre-351

dicted conditions for the optimum point and the desirability of352

the prediction. Each individual plot in this figure shows the way 353

by which each factor influences the %R and EF values and their 354

composition. It is expected that increasing VD should increase the 355

recovery. However, at higher VD values, neither a stable cloudy 356

solution nor a sediment phase were formed and the solution 357

approached a single phase at too high acetonitrile volumes, most 358

possibly because chloroform dissolves in high amounts of acetoni- 359

trile [15]. 360

3.5. Analytical performances 361

Ten replicate experiments were performed at the predicted 362

optimum conditions by the response surface model: i.e., a mix- 363

ture containing 1.0 mL drug solution at level 0.5 mg L−1, 1.0 mL 364

hydrolyzed urine at pH 8.5, 0.2 mL 25% sodium chloride and 2.8 mL 365

distilled water was prepared. Then 0.8 mL acetonitrile containing 366

50 �L chloroform was injected to this mixture and centrifuged at 367

2000 rpm for 5 min. The extraction of 100 ng mL−1 of IMIP and 368

TRIM thus carried out under the optimal experimental conditions 369

resulted in extraction recoveries of 97% and 112% for IMIP and TRIM, 370

respectively. From the results obtained, it can be concluded that the 371

prediction of the model is desirable and the recovery is quantitative. 372

The calibration curves were drawn utilizing 7 spiking levels 373

of drugs in concentrations range of 5–100 ng mL−1, with respect 374

to each analyte in urine sample. For each level three replicate 375

extractions at optimal conditions were performed and two calibra- 376

tion curves were drawn. The corresponding regression equations, 377

coefficients of determination (R2), dynamic linear ranges (DLR) and 378

the limits of detection (LOD) were calculated and shown in Table 4. 379

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

ARTICLE IN PRESSG ModelPBA 9683 1–8

6 M. Shamsipur, M. Mirmohammadi / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

Fig. 3. Interaction between factors for IMIP (A) and TRIM (B).

Limits of detection were calculated as the minimum concentra-380

tion providing chromatographic signals 3 times higher than the381

background noise. LODs were determined in hydrolyzed urine.382

In order to examine the enrichment factor of each analyte, three383

replicate extractions were performed at optimal conditions from384

urine samples consisting 5 ng mL−1 IMIP and TRIM. The enrich-385

ment factor was calculated as the ratio of the final concentration of386

analytes in sedimented phase and its concentration in the original 387

solution. The standard solutions of TCAs were prepared in acetoni- 388

trile as solvent and the calibration curves were drawn over the 389

range of 6–100 ng mL−1. Finally, the actual concentration of each 390

analyte in acetonitrile was calculated from this calibration curves 391

and the enrichment factors are determined and summarized in 392

Table 4. 393

Table 4Quantitative analytical data for DLLME–HPLC–UV detection of IMIP and TRIM in urine samples.

Drug DLR (ng mL−1) Regression equation R2 LOD (ng mL−1) RSD (%, n = 5) EF Recovery (%)

IMIP 2–100 Y = 1587.84X − 0.14 0.9951 0.6 5.1 161.7 97TRIM 2–100 Y = 1391.45X − 1.12 0.9946 0.6 6.1 186.7 112

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

ARTICLE IN PRESSG ModelPBA 9683 1–8

M. Shamsipur, M. Mirmohammadi / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx 7

Table 5Residual of IMIP in urine samples of a patient at given times after taking IMIP.

Tablet use IMIP content after 5 h (ng mL−1) IMIP content after 24 h (ng mL−1) IMIP content after 48 h (ng mL−1) IMIP content after 54 h (ng mL−1)

First night 10 mg 21 8 – –Second night 10 mg 74 8 – –Third night 30 mg 105 71 12 8

Table 6Comparison of LOD of different methods for determination of IMIP and TRIM.

Method Sample matrix LOD (ng mL−1) Ref.

SPE–GC–NPDa Blood 37 [2]CE/TOF-MSb Plasma 1–5 [4]HE-LPME–HPLC–UVc Aqueous solution 1.5–2.1 [6]Spectrometry Pharmaceutical preparations 70–110 [8]HPLC–MSd Spiked serum samples 0.23 and 3.3 [14]CE Plasma and urine 44–153 [8]SPE-HPLC–UVe Human serum 1691 [7]DLLME–HPLC–UV Urine 0.6 This work

a Solid phase extraction–gas chromatography–nitrogen–phosphorus detection.b Capillary electrophoresis-time of flight mass spectrometry.c Hollow fiber-based liquid phase microextraction–high performance liquid chromatography–UV detection.d High performance liquid chromatography–mass spectrometry.e Solid phase extraction–high performance liquid chromatography–UV detection.

3.6. Application of proposed method to real urine samples394

The optimized DLLME method was applied to the extraction395

and determination of IMIP in real samples. For this purpose, we396

used the urine samples from a mail patient following the courses397

of treatment with the psychotropic drug and analyzed them by the398

proposed DLLME method. At first, we analyzed his urine when no399

drug was taken, as blank, and the results showed no measurable400

signal for the drug. Then he took a 10 mg tablet of IMIP for the401

first night and his urine analyzed after 5 and 24 h. After that, in the402

second night, he took another tablet and again his urine analyzed403

after 5 and 24 h passed from the second night. Then he used three404

10 mg tablets at the third night, and the drug content of his urine405

after time periods of 5, 24, 48 and 54 h was determined by the pro-406

posed method. The amounts of IMIP content of the patient’s urine407

analyzed by the proposed method are shown in Table 5.408

The results confirm that the peak concentration occurs within409

about 6 h after oral administration [26]. Since the half-life of IMIP is410

approximately 24 h [27], the drug can be accumulated in the body411

and release gradually [28].412

Fig. 5 displays the peaks for the real sample 5 h after using the413

first 10 mg tablet of IMIP, the blank and a blank which was spiked414

IMIP at level of 50 ng mL−1. As seen, it shows that the urinary excre-415

tion has not changed IMIP in part, although it has other active416

metabolites [29].417

Fig. 4. The optimization plot for the central composite design, including seven inde-pendent and four dependent variables.

Fig. 5. Chromatogram of blank (a, blue) overlaid with real sample 5 h after using thefirst 10 mg IMIP tablet (b, red) and the blank spiked at 50 ng mL−1 IMIP concentration Q3level (c, green). (For interpretation of the references to color in this figure legend,the reader is referred to the web version of the article.)

3.7. Comparison of most sensitive detection methods for IMIP and 418

TRIM 419

Table 6 is compared the LODs of the proposed method with those 420

of the most sensitive methods previously reported in the literature 421

[2,4,6–8,14]. As it is quite clear from Table 6, the limit of detec- 422

tion of the proposed method shows a significant improvement over 423

that of the previously reported methods. In addition, the proposed 424

method provided high recoveries and enrichment factors within a 425

very short period of time. 426

4. Conclusions 427

The proposed method provided high recovery and enrichment 428

factor within a very short time. Solvent consumption was greatly 429

reduced compared to conventional LLE and low detection limits 430

were achieved. Up to now, the DLLME method has not been com- 431

prehensively used for biological samples such as urine, plasma and 432

hair, as it had thought that this method is not well compatible for 433

extraction from biological samples. On the other hand, because of 434

the interaction of matrix components in biological samples with 435

organic analysts, the samples needed several dilutions. For this rea- 436

son, this method has been mostly applied to environmental water 437

Please cite this article in press as: M. Shamsipur, M. Mirmohammadi, High performance liquid chromatographic determination of ultratraces of two tricyclic antidepressant drugs imipramine and trimipramine in urine samples after their dispersive liquid–liquid microex-traction coupled with response surface optimization, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.08.008

ARTICLE IN PRESSG ModelPBA 9683 1–8

8 M. Shamsipur, M. Mirmohammadi / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

samples or biological samples spiked with high concentration lev-438

els of organic analytes. While, in the proposed method, we were439

able to use DLLME directly in analysis of real urine samples after440

hydrolyzing and applying an ultrasonic to break down the possi-441

ble matrix interferences. Based on the reports in some articles [19],442

imipramine must have a concentration about 600–850 (ng mL−1) in443

urine to be determined by common analytical methods; however,444

this concentration range is considerably higher than that of the445

therapeutic and toxicity dosage which are established as 150–250446

and >450 ng mL−1, respectively. By the proposed DLLME method447

we could determine these drugs in patients’ urine samples, at least448

5 h after utilizing the first 10 mg dosage that was about 21 ng mL−1,449

which is much less than the toxic dose. Besides, according to these450

results, it will be possible to determine the residual of drugs four451

days after using a 30 mg dosage in urine. The proposed method452

could be of assistance of the coroners to detect the suicides and453

significant self-mutilation, or other self-injurious or destructive454

behavior with TCA drugs.455

Acknowledgements456

This work was financially supported by Razi University andQ2457

Islamic Azad University, Shahreza Branch. The authors are grate-458

ful to Dr. M. Hoghughi in Amin Pharmaceutical Co. (Isfahan, Iran)459

for his kind assistance.460

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