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Accepted Manuscript

Simultaneous extraction and determination of albendazole and triclabendazole by


a novel syringe to syringe dispersive liquid phase microextraction-solidified floating
organic drop combined with high performance liquid chromatography

Mohammad Asadi, Shayessteh Dadfarnia, Ali Mohammad Haji Shabani

PII: S0003-2670(16)30625-0
DOI: 10.1016/j.aca.2016.05.014
Reference: ACA 234580

To appear in: Analytica Chimica Acta

Received Date: 1 March 2016


Revised Date: 7 May 2016
Accepted Date: 9 May 2016

Please cite this article as: M. Asadi, S. Dadfarnia, A.M.H. Shabani, Simultaneous extraction and
determination of albendazole and triclabendazole by a novel syringe to syringe dispersive liquid phase
microextraction-solidified floating organic drop combined with high performance liquid chromatography,
Analytica Chimica Acta (2016), doi: 10.1016/j.aca.2016.05.014.

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ACCEPTED MANUSCRIPT

Simultaneous extraction and determination of albendazole and triclabendazole using two

syringes assisted emulsification microextraction based on solidification of floating organic

drop combined with high performance liquid chromatography and fluorescence detection

Mohammad Asadi, Shayessteh Dadfarnia1, Ali Mohammad Haji Shabani

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Department of Chemistry, Faculty of Science, Yazd University, Yazd, 89195-741, Iran

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Corresponding author. Tel.: +983531232667; fax: +98 3538210644.
E-mail Address: sdadfarnia@yazd.ac.ir (S. Dadfarnia).
ACCEPTED MANUSCRIPT

1 Simultaneous extraction and determination of albendazole and triclabendazole by a novel


2 syringe to syringe dispersive liquid phase microextraction-solidified floating organic drop
3 combined with high performance liquid chromatography
4
5 Mohammad Asadi, Shayessteh Dadfarnia*, Ali Mohammad Haji Shabani

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6 Department of Chemistry, Faculty of Science, Yazd University, Yazd, 89195-741, Iran
7

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8 Abstract
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10 A syringe to syringe dispersive liquid phase microextraction-solidified floating organic drop was

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11 introduced and used for the simultaneous extraction of trace amounts of albendazole and

12 triclabendazole from different matrices. The extracted analytes were determined by high

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13 performance liquid chromatography along with fluorescence detection. The analytical parameters

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affecting the microextraction efficiency including the nature and volume of the extraction

solvent, sample volume, sample pH, ionic strength and the cycles of extraction were optimized.
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16 The calibration curves were linear in the range of 0.1-30.0 µg L-1 and 0.2-30.0 µg L-1 with
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17 determination coefficients of 0.9999 and 0.9998 for albendazole and triclabendazole respectively.
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18 The detection limits defined as three folds of the signal to noise ratio were found to be 0.02 µg L-
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19 for albendazole and 0.06 µg L-1 for triclabendazole. The inter-day and intra-day precision
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20 (RSD%) for both analytes at three concentration levels (0.5, 2.0 and 10.0 µg L-1) were in the

21 range of 6.3-10.1% and 5.0-7.5% respectively. The developed method was successfully applied
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22 to determine albendazole and triclabendazole in water, cow milk, honey, and urine samples.
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23 Keywords: Syringe to syringe dispersive liquid phase microextraction-solidified floating organic

24 drop; High performance liquid chromatography; Albendazole; Triclabendazole

25

*
Corresponding author. Tel.: +983531232667; fax: +98 3538210644.
E-mail Address: sdadfarnia@yazd.ac.ir (S. Dadfarnia).

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26 1. Introduction

27 Albendazole (ALB) and triclabendazole (TCB) are two kinds of benzimidazole

28 anthelmintic drugs (BADs). These drugs are widely used for the control and treatment of

29 parasitic infections in animals and agriculture. However, long-term use of BADs can cause

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30 several toxic effects including congenic malformations, diarrhea, polyploidy, necrotic

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31 lymphoadenopathy, and pulmonary edemas [1]. The widespread and improper use of BADs can

32 also result in contamination of dairy products and water by their residue, which is a potential

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33 threat to human consumers [2]. So, the government and corresponding organizations have set a

34 maximum concentration level (MCL) or maximum residue limits (MRLs) to control the amount

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35 of BADs in drinking water and food samples. For example, Codex [3] and the Australian

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Government has recommended an MRL of <100 µg kg-1 for benzimidazole anthelmintics in milk

[4], the European Water Framework Directive has set an MCL of 0.1 mg L-1 for most
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38 benzimidazole compounds in natural waters [5], and the European Community legislation has set
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39 an MRL of 10 and 100 µg Kg-1 for TCB and ALB in milk respectively [6]. Therefore,
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40 development of accurate analytical techniques to monitor and determine these drugs in food and

41 environmental samples is a challenging task [7].


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42 Various analytical techniques such as spectrophotometry [8], spectrofluorimetry [9],

43 capillary zone electrophoresis (CZE) [10], and high performance liquid chromatography (HPLC)
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44 [11] have been used to determine these drugs. Among the mentioned techniques, high
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45 performance liquid chromatography combined with mass-spectrometry [12], ultraviolet [13], or

46 fluorescence detection system [14] is widely applied to determine ABDs in different real

47 samples. However, due to the low concentration of the drugs in real samples and the complexity

48 of sample matrices, direct drug determination by HPLC is difficult or impossible. Thus, a

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49 separation and preconcentration step is required prior to drug analysis by HPLC. In this regard,

50 several sample preparation methods including liquid-liquid extraction (LLE) [15], solid-phase

51 extraction (SPE) [16], solid phase microextraction (SPME) [11] and liquid phase microextraction

52 (LPME) [17] have been used. However, traditional sample pretreatment methods (i.e. LLE and

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53 SPE) are time-consuming as well as labor-intensive, and LLE needs large volumes of highly

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54 pure and toxic organic solvents. To overcome these limitations, sample pretreatment has moved

55 toward miniaturization of traditional extraction methods, which has resulted in development of

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56 SPME and LPME.

57 Liquid phase microextraction (LPME) is a miniaturized mode of LLE that greatly reduces

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58 the consumption of organic phases [18]. LPME has the advantages of low cost, simple operation,

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low toxicity and a high enrichment factor. Different modes of LPME that have been developed

include single drop microextraction (SDME) [19], hollow fiber liquid phase microextraction
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61 (HF-LPME) [20], dispersive liquid phase microextraction (DLPME) [21,22], and solidified
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62 floating organic drop microextraction (SFODME) [23,24]. Among these modes, DLPME has
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63 been widely used for the extraction of different analytes from various matrices [25]. In this mode

64 of LPME, the extraction solvent with a density higher or lower than that of water is mixed with a
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65 polar solvent such as methanol or acetonitrile (as a dispersive solvent) and is rapidly injected into

66 the aqueous sample containing the analyte. In this state, a cloudy solution containing fine
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67 droplets of the extraction solvent is formed in the aqueous phase which results in rapid extraction
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68 of the target analyte. DLPME has the advantages of rapidity, simplicity, low cost, use of low

69 amounts of organic solvents, possibility of obtaining a high enrichment factor as well as no need

70 for a large sample volume and a specific extraction vessel. However, this method is a ternary

71 component solvent system, and the dispersive solvent may cause an increase in the solubility of

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72 the extraction solvent in the aqueous phase as well as a decrease in the partition coefficients of

73 analytes between the aqueous phase and the extraction solvent. Furthermore, separation and

74 recovery of the extraction solvent is difficult. To resolve these drawbacks, ultrasonic-assisted

75 emulsification microextraction (UAEME) [26], vortex assisted emulsification microextraction

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76 (VAEME) [27,28], and DLPME based on solidified floating organic drop (DLPME-SFOD)

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77 [29,30] have been developed. In VAEME and UAEME, the dispersive solvent is eliminated and

78 the extraction solvent is dispersed in the aqueous sample by the use of vortex energy and

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79 ultrasound radiation respectively. However, these methods require longer extraction time than

80 the traditional DLPME as the dispersion of the solvent takes longer time, and the ultrasonic

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81 energy may cause the degradation of analytes and complexes. In DLPME-SFOD mode, a low-

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toxin and low-density organic solvent, such as 1-dodecanol, with a melting point near the room

temperature is used as the extraction solvent. This simplifies the solvent recovery at the end of
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84 extraction as the solvent solidifies and floats on the top of the sample solution. However, the
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85 method is still a three-phase system and does not alleviate the need for a dispersive solvent.
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86 In this research, a new mode of DLPME called ‘syringe to syringe dispersive liquid phase

87 microextraction-solidified floating organic drop (SS-DLPME-SFOD) is designed. In this


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88 method, two disposal syringes are connected to each other through one needle and used as an

89 extraction flask. The sample solution along with the extraction solvent is put in syringe 1, and
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90 then syringe 2 is connected to it. The solvent is simply dispersed in the aqueous phase through
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91 the injection and back injection of the mixture of the sample and the extraction solvent into the

92 extraction vessel. Finally, the mixture is transferred to a centrifuge tube, and the phases are

93 separated and easily recovered by centrifugation and solidification of the extraction solvent in an

94 ice bath. This technique does not require a dispersive solvent, vortex mixer or ultrasound bath. In

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95 addition, as the extraction is performed in a closed system, it prevents the loss of the extraction

96 solvent through evaporation and alleviates the harmful effects of hazardous analytes and toxic

97 organic solvents on the operator and the environment. The designed method is applied for

98 simultaneous separation and preconcentration of trace amounts of ALB and TCB from various

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99 samples before their determination by high performance liquid chromatography.

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101 2. Experimental

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102 2.1. Chemicals and reagents

103 Albendazole and triclabendazole of analytical reagent grades were supplied from Sigma

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104 Aldrich (St. Louis, MO, USA). HPLC grade water and methanol, dipotassium hydrogen phosphate,

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hydrochloric acid, sodium chloride, 1-dodecanol, 1-decanol, and 2-undecanol (of analytical grade)

were provided from Merck (Darmstadt, Germany). The stock standard solutions of ALB and
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107 TCB were prepared at the concentration of 100 mg L-1 by dissolving an appropriate amount of
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108 each drug in methanol. The solutions were then kept in a refrigerator at -4 °C. Working standard
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109 solutions were prepared on a daily basis by diluting the stock standard solution adequately with

110 high purity water (HPLC grade). All the prepared standard and solutions were kept in clean
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111 polypropylene bottles (Nalgene, Lima, OH, USA).

112 A phosphate buffer solution (2.0 mol L-1, pH = 8.0) was prepared by dissolving 358 g of
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113 dipotassium hydrogen phosphate in 900 mL of deionized water, adjusting its pH to 8.0 with
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114 hydrochloric acid and diluting the solution to 1000 mL with deionized water.

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116 2.2. Instrumentation

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117 A Knauer HPLC system (Berlin, Germany) composed of LC-pump 1000, 20 µL sample

118 loop and a fluorescence detector RF-10AXL (Shimadzu, Japan) was used for chromatographic

119 analysis. The detector excitation and emission wavelengths were set at 290 and 330 nm

120 respectively. Separation of the analytes was carried out on a Nucleosil-C18 column (250 mm ×

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121 4.6 mm, 5 µm). The mobile phase consisted of a mixture of methanol and water (80: 20). The

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122 flow rate and the column temperature were set at 0.9 mL min-1 and 40 °C respectively.

123

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124 2.3. SS-DLPME-SFOD procedure

125 In the design of the extraction system, two medical syringes were connecting to each other. For

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126 this purpose, the needle of one of the medical syringe was removed, the needle of the second one

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was cut and shortened to 5 mm and was inserted into the head of the first syringe. Finally, the

connection was insulated with PVC glue so that no leak was observed during the injection and
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129 back injection process. The pH of ten milliliters of the sample or the standard solution was
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130 adjusted to 8.0 (using a 0.5 mL phosphate buffer 2.0 mol L-1, pH = 8.0), the solution was drawn
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131 into syringe 1, and 30 µL of 1-dodecanol was added to it (Fig. 1). Then, syringe 1 was connected

132 to syringe 2, and the mixture (i.e. the sample solution and the organic solvent) in syringe 1 was
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133 rapidly injected into syringe 2 followed by back injection of the mixture in syringe 2 to syringe

134 1. This procedure was repeated four times until the solvent was completely dispersed in the
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135 aqueous phase and the equilibrium was achieved. The resulting dispersed mixture was
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136 transferred to a closed conical centrifuged tube and centrifuged at 5000 rpm for four minutes.

137 Finally, the sample tube was transferred into an ice bath where 1-dodecanol was solidified after

138 about four minutes, and the solidified organic drop was transferred into a conical vial in room

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139 temperature where it melted immediately. 20 µL of the extract containing ALB and TCB was

140 injected into HPLC for quantification.

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142 2.4. Sample preparation

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143 2.4.1. Water samples

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144 A tap water sample was collected freshly from Bijan Chemistry laboratory (Mashhad,

145 Iran), and wastewater was taken from the effluent of a wastewater treatment plant (Mashhad,

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146 Iran) and poured in polypropylene bottles. The water samples were filtered through a Millipore

147 0.45 µm pore-size filter, the pH was adjusted to 8.0 by a phosphate buffer, and the samples were

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148 treated according to the given procedure.

149 2.4.2. Urine sample


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A urine sample was obtained from a volunteer working in the laboratory, and then it was
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151 kept frozen. It was melted at room temperature just before the extraction, centrifuged for 15
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152 minutes at 5000 rpm and filtered. The matrix effect was reduced through dilution of the sample
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153 for five times and, subsequently, the sample was subjected to SS-DLPME-SFOD procedure and

154 HPLC measurement.


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155 2.4.3. Milk sample

156 Pasteurized and homogenized cow milk was obtained from a local market (Mashhad,
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157 Iran). The milk sample was prepared according to the given procedure [31]. Thus, 5.0 mL of the
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158 sample was mixed with 4.0 mL of 3.0% (w/v) trichloroacetic acid in a 15 mL vial and vortexed

159 for 30 seconds. The mixture was left on its own for 15 minutes to allow precipitation of the

160 proteins. Afterwards, it was centrifuged at 4000 rpm for 10 minutes. The supernatant solution

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161 was filtered, and the pH was adjusted to 8.0 by a phosphate buffer. Finally, the volume was

162 adjusted to 10.0 mL with HPLC grade water and treated according to the extraction procedure.

163 2.4.4. Honey sample

164 A honey sample was purchased from a local market (Mashhad, Iran). One gram of the

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165 sample was diluted to 10 mL with 0.5 mL of a phosphate buffer (pH = 8.0) and HPLC grade

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166 water and vortexed for four seconds. Then, the mixture was filtered and extracted according to

167 the given procedure [32].

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169 3. Results and discussion

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170 In the preliminary study, an extraction system was designed, and albendazole (ALB) and

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triclabendazole (TCB), as two kinds of benzimidazole anthelmintic drugs, were selected as

model compounds. Then, the experimental parameters influencing the extraction efficiency of
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173 the analytes, such as the type and volume of the organic phase, sample pH, ionic strength,
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174 number of dispersion cycles of the extraction and sample volume, were studied and optimized.
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175 Each experiment was performed for three times, and the corresponding average and relative

176 standard deviation were calculated.


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178 3.1. Effect of sample pH


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179 The pH of a sample solution is an important factor that determines the ionic state of
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180 analytes as well as their distribution between aqueous and organic phases. Thus, pH must be

181 adjusted to the value at which analytes are mainly in their neutral forms so that their affinity for

182 the extraction solvent can become high. According to the pKa values of ALB (pKa1 = 5.54, pKa2 =

183 13.11) and TCB (pKa1 = 5.31, pKa2 = 12.91), these drugs should be mainly in their neutral forms

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184 at pHs within their two pKa values [7]. So, several experiments were performed by varying the

185 sample pH in the range of 4.0-11.0 while keeping the other parameters constant. The results of

186 the study (Fig. 2) revealed that the peak areas of ALB and TCB increase by an increase of pH

187 from 4.0 to 8.0. However, further increase in the sample pH causes a slight decrease in the

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188 extraction efficiency. Thus, the sample pH was adjusted to 8.0 by a phosphate buffer for further

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189 experiments.

190

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191 3.2. Effect of nature and volume of extraction solvent

192 In SS-DLPME-SFOD, like in other LPME methods, the nature of extraction solvent is an

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193 important parameter affecting the efficiency of extraction. The extraction solvent should have

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several characteristics such as high affinity for analytes, low solubility in sample solutions, low

density, and a melting point close to an ambient temperature. Therefore, the effect of several
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196 organic solvents including 1-dodecanol, 1-undecanol, 1,10-dichlorodecane and 1-decanol on the
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197 extraction of ALB and TCB by SS-DLPME-SFOD method was studied. The results (Fig. 3)
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198 showed that, with 1-dodecanol, the peak area and, therefore, the extraction efficiencies for ALB

199 and TCB were higher than those with the other solvents. This observation can be related to the
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200 lower polarity and, consequently, the higher affinity of 1-dodecanol (dielectric constant = 5.82)

201 for analytes as compared to 1-undecanol (dielectric constant = 5.98), 1,10-dichlorodecane


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202 (dielectric constant = 6.68) and 1-decanol (dielectric constant = 7.93). Therefore, 1-dodecanol
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203 was selected as the extracting solvent.

204 To study the effect of the volume of extraction solvent on the extraction efficiency of the

205 developed method, some experiments were carried out using various volumes of 1-dodecanol

206 (30, 40, 50 and 60 µL). The results revealed that the analytical signals of ALB and TCB

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207 decreased proportionally to the increase in the extraction solvent volume, indicating that the

208 extraction was constant within this range of 1-dodecanol volume. An extraction solvent volume

209 less than 30 µL was not taken into account due to the difficulty of collecting the solvent after

210 extraction. So, in order to obtain a high enrichment factor, 30 µL of 1-dodecanol was selected as

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211 the optimum volume of the extraction solvent.

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213 3.3. Effect of ionic strength

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214 In liquid phase extraction, adding salt to the aqueous phase usually improves the

215 extraction efficiency of the analytes through the salting out effect. However, in LPME in some

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216 cases, a decrease in the extraction efficiency at a high concentration of salt has been observed

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[33], which can be attributed to the increase in the sample viscosity and the restriction of the
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218 transfer of the analyte to the organic phase. Therefore, in order to investigate the effect of salt on

219 the SS-DLPME-SFOD performance, several experiments were carried out with different NaCl
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220 concentrations (0.0–6.0%) while keeping the other experimental parameters constant. The results
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221 indicated that addition of salt had no significant effect on the analytical responses of ALB and

222 TCB. Thus, the method is suitable for the extraction of analytes from saline samples.
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224 3.4. Effect of the number of injections


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225 SS-DLPME-SFOD is a close system in which the organic phase is dispersed in the
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226 aqueous phase through successive injections and back injections of the sample solution and the

227 extraction solvent using two syringes connected to each other with a gauge needle. In order to

228 perform SS-DLPME-SFOD with high efficiency in a short time, the number of injections must

229 be optimized. The effect of the number of injections and back injections was examined through

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230 1 to 9 injections under other constant experimental conditions. The results (Fig. 4) showed that

231 the analytical responses of ALB and TCB reached a maximum and remained constant after eight

232 injections, indicating that the system had reached a state of equilibrium. Therefore, eight-time

233 injection was selected as the optimum practice for extraction.

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234

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235 3.5. Effect of sample volume

236 The sample volume influences the efficiency of convection during the extraction

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237 procedure, the LPME efficiency as well as the enrichment factor. Therefore, to investigate the

238 effect of sample volume on the efficiency of the developed method, several experiments were

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239 performed using different sample volumes (6-20 mL) containing a fixed amount of analytes (0.1

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µg ALB and 0.2 µg TCB). According to the results (Fig. 5), the analytical response was

maximum and constant up to 10 mL, but further increase in the sample volume caused a decrease
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242 in the analyte response. This observation can be attributed to the incomplete dispersion of the
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243 extraction solvent within a fixed number of injections. Thus, 10 mL was chosen as the maximum
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244 sample volume for the extraction.

245
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246 3.6. Figures of merit

247 The recommendations of the Food and Drug Administration (FDA) [34] were used to
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248 investigate the figures of merit of the developed SS-DLPME-SFOD technique. The regression
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249 equation, linear dynamic range (LDR), coefficient of determination (R2), limit of detection

250 (LOD), enhancement factor, and extraction recovery were determined under optimal conditions.

251 The results are summarized in Table 1. The inter-day (n = 3) and intra-day (n = 5) precision

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252 (RSD%) of the SS-DLPME-SFOD method (Table 2) were evaluated by analyzing the samples

253 spiked at three concentration levels (i.e. 0.5, 2.0, 10.0 µg L-1).

254 The enhancement factor was defined as the ratio of the slope of the calibration curve after

255 the SS-DLPME-SFOD method to the slope of the calibration curve without extraction. The ratio

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256 was found to be 281 and 311 for ALB and TCB respectively. The extraction recovery (ER) was

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257 calculated using the following equation:

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ER% = EF × ( ) × 100 (Eq. 1)

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258 where Va and Vo are the volumes of the aqueous sample and the organic phase respectively

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259 [28,35].

260 AN
261 3.7. Application of SS-DLPME -SFOD
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262 The applicability of the developed SS-DLPME-SFOD-HPLC/FLD method in the analysis

of real samples was evaluated through extraction and determination of ALB and TCB in water,
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264 wastewater, milk and urine samples. The accuracy of the method was determined through
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265 recovery experiments by spiking the samples at two different concentrations of ALB and TCB.
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266 The recovery (R%), and accuracy (Error%) were calculated according to the following equations

267 respectively:
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Cf -Ci
R% = ( ) × 100 (Eq. 2)
Ca
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Error% = R% - 100 (Eq. 3)

268 where Ci, Ca and Cf are the initial concentrations of the analyte in the sample, the concentration

269 of the analyte in the sample after spiking by a certain amount of the standard, and the

270 concentration of the analyte determined in the spiked sample by the proposed method

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271 respectively. The results of this study (Table 3) show that the recoveries and the errors are in the

272 range of 96.0 to 104.3% and + 4.3 to - 4.0% respectively, suggesting that the present method

273 provides acceptable rates of recovery and accuracy for the determination of ALB and TCB in

274 real samples, as examined in the study.

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275 The chromatograms of the standard along with the milk sample (spiked and unspiked)

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276 after extraction by the developed method (Fig. 6) show that the peaks are symmetrical, and the

277 retention times of the analytes are constant in the standard and the real sample. Furthermore, the

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278 unspiked milk chromatogram shows a lack of any interference peak at the retention time of the

279 analytes. Thus, the method can be considered accurate for the determination of ALB and TCB.

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3.8. Comparison of the developed method with other recent microextraction methods combined

with HPLC for determination of ALB and TCB


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283 Table 4 presents a comparison of the developed SS-DLPME-SFOD-HPLC/FLD with


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284 other microextraction techniques [36,37] combined with HPLC for the extraction and
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285 determination of ALB and TCB. As it is observed, the developed SS-DLPME-SFOD-

286 HPLC/FLD method provides a higher enhancement factor and, consequently, lower LODs
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287 values than the other techniques do. The precision of the method is also comparable to that of

288 other methods, and the method can be applied to a wider range of matrices.
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289
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290 4. Conclusion

291 In this study, a syringe to syringe dispersive liquid phase microextraction-solidified

292 floating organic drop (SS-DLPME-SFOD) was introduced and used for the simultaneous

293 extraction of trace amounts of albendazole and triclabendazole from different matrices. The

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294 designed microextraction technique was combined with high performance liquid

295 chromatography-fluorescence detection and successfully applied to simultaneous

296 separation/preconcentration and determination of trace amounts of ALB and TCB in water,

297 urine, milk and honey samples. The SS-DLPME-SFOD method is privileged for its high

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298 enhancement factor, low detection limit, good accuracy and precision. It also has the advantages

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299 of rapidity, simplicity, low cost and being environment-friendly. Moreover, it does not need any

300 dispersing agent (e.g. a polar solvent or surfactant) or specific instrument (e.g. a vortex mixer,

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301 magnetic stirrer or ultrasonic bath), and as the extraction is performed in a close system, the

302 possibility of loss of the organic solvent during the extraction is minimized.

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304 References

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308 [2] D. Chen, Y. Tao, Z. Liu, Z. Liu, L. Huang, Y. Wang, Y. Pan, D. Peng, M. Dai, Z. Yuan,

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311 [3] CAC F. WHO, Maximum residue limits for veterinary drugs in foods, Proceedings of the

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318 [6] Council Regulation of the European Union No. 2377/90 and Commission Regulation Nos.

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326 [9] S. Küçükkolbaşı, B. Gündüz, E. Kılıç, Development of a spectrofluorimetric method for

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330 capillary zone electrophoresis, Talanta 89 (2012) 335-341.

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425 Determination of benzimidazole anthelmintics using HPLC after vortex-assisted mixed

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floating organic droplet procedure, J. Food Compos. Anal. 37 (2015) 30-37.


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430 Figure captions

431 Fig. 1. SS-DLPME procedure

432 Fig. 2. Effect of the sample pH on the microextraction. Conditions: ALB and TCB

433 concentrations are10 and 20 µg L−1, respectively; sample volume, 10 mL; extraction solvent, 1-

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434 dodecanol (30 µL); number of injections, eight times.

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435 Fig. 3. Effect of the nature of extraction solvent on the microextraction. Conditions: sample pH,

436 8;ALB and TCB concentrations are 10 and 20 µg L−1, respectively; sample volume, 10 mL;

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437 volume of extraction solvent, 30 µL; number of injections, eight times.

438 Fig. 4. Effect of the number of injections on the microextraction. Conditions: sample pH, 8; ALB

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439 and TCB concentrations are 10 and 20 µg L−1, respectively; sample volume, 10 mL; extraction

440 solvent, 1-dodecanol (30 µL).


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Fig. 5. Effect of the sample volume on the microextraction. Conditions: sample pH, 8; amounts
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442 of ALB and TCB are 0.1and 0.2 µg, respectively; extraction solvent, 1-dodecanol (30 µL);
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443 number injections, eight times.


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444 Fig. 6. Chromatograms of the spiked milk sample with 20 µg L−1 of ALB and TCB (a), unspiked

445 milk samples (b) and standard with 20 µg L−1 of ALB and TCB (c) after the SS-DLPME-SFOD
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10.0

8.0

Peak area × 100000


ALB

6.0

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4.0

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3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0

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Sample pH

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10.0

8.0
Peak area × 100000

6.0 ALB

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4.0

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1-Dodecanol 1-Undecanol 1,10-Dichlorodecane 1-Decanol

Nature of extraction solvent

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8.0

6.0
Peak area × 100000
ALB
4.0

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1 2 3 4 5 6 7 8 9

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Number of injections
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Table 1 Analytical performance of the SS-DLPME-SFOD-HPLC/FLD.

Analyte Linearity (µg L-1) Linear equation R2 LOD (µg L-1) EF ER (%)

ALB 0.1-30.0 y = 69137x + 1217 0.9999 0.02 281 84

TCB 0.2-30.0 y = 26935x + 1243 0.9998 0.06 311 93

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R2: Coefficient of determination; LOD: limit of detection; EF: enhancement factor; ER: Extraction recovery.

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Table 2

Precision of the developed method for the determination of ALB and TCB in blank real samples.

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Matrix Precision (RSD%)

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ALB (µg L-1) TCB (µg L-1)

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Inter-assay (n = 3) Intra-assay (n = 5) Inter-assay (n = 3) Intra-assay (n = 5)

0.5 2 10 0.5 2 10 0.5 2 10 0.5 2 10

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Tap water 7.9 7.3 6.9 6.6 5.3 5.0 7.7 6.5 6.3 6.1 5.3 5.5

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Waste water 8.9 6.8 7.7 6.9 6.0 5.7 9.2 7.0 7.4 7.2 6.6 6.3

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Cow milk 9.7 9.4 8.0 7.1 6.4 6.0 9.5 8.7 6.0 6.8 7.0 5.9

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Honey 9.1 6.5 7.2 6.1 6.5 5.6 7.8 7.3 6.8 7.0 6.6 6.5

Urine 10.1 7.7 8.5 7.1


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Table 3 Determination of ALB and TCB in different real matrices (n = 3).


Sample ALB TCB
Added Found Recovery (%) Error % Added Found Recovery (%) Error %
(µg L-1) (µg L-1) (µg L-1) (µg L-1)

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Tap water - - - - - -
1 1.01 ± 0.06 101.0 +1.0 1 0.98 ± 0.07 98.0 -2.0

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10 9.83 ± 0.52 98.3 -1.7 10 10.15 ± 0.64 101.5 +1.5
Waste water - 5.60 - - - - - -

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1 6.58 ± 0.28 98.0 -2.0 1 0.99 ± 0.06 99.0 -1.0
10 15.63 ± 0.74 103.0 +3.0 10 10.12 ± 0.48 101.2 +1.2
Honey - - - - - - - -

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1 1.01 ± 0.09 101.0 +1.0 1 1.02 ± 0.08 102.0 +2.0

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10 9.90 ± 0.61 99.0 -0.1 10 9.76 ± 0.51 97.6 -2.4
Cow milk - - - - - - - -
1 0.99 ± 0.05 99.0 -1.0 1 0.97 ± 0.04 97.0 -3.0

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10 10.08 ± 0.49 100.8 +0.8 10 10.43 ± 0.65 104.3 +4.3
Urine - 14.36 - - - - - -
1 15.39 ± 0.78 103.0 +3.0 1 0.96 ± 0.08 96.0 -4.0

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Table 4 Comparison of the SS-DLPME-SFOD with other recent microextraction methods combined with HPLC.

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Analyte Analytical technique Sample RSD (%) LOD EF Ref.

Inter-day Intra-day (µg L-1)

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ALB (TBZ, OFZ, MEB and SPME–HPLC/DAD Milk and honey 2.3 8.5 0.21 52 [11]

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FBZ)

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ALB (OFZ, MBZ and FBZ) UAEME-HPLC/PDA Egg <11.3 <9.0 12.5 - [13]

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ALB (TBZ, OFZ and UASEME –HPLC/DAD Milk 3.4 6.7 2.9 60 (PF) [17]

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MEB)

ALB (FBZ and MBZ) SSEME-HPLC/PDA Milk <8.8 <8.8 2.6 38 (PF) [36]

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ALB (TBZ, MBZ and FBZ) VASEME -HPLC/PDA TE Liver and kidney <8.0% <8.0% 0.5 60 (PF) [37]

ALB SS-DLPME-SFOD- Water, wastewater, milk, 6.5 5.0 0.02 281 This
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TCB HPLC/FLD honey and urine 6.0 5.3 0.06 311 work
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RSD: relative standard deviation; LOD: limit of detection; EF: enhancement factor: PF: preconcentration factor UASEME: ultrasound-assisted
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surfactant-enhanced emulsification microextraction; SSEME: surfactant-solvent-based quaternary component emulsification microextraction;

VASEME: Vortex-assisted surfactant-enhanced emulsification microextraction; UAEME: ultrasound assisted emulsification microextraction;

TBZ: thiabendazole; OFZ: oxfendazole; MEB: mebendazole; FBZ: fenbendazole.

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Highlights

- A novel dispersive liquid phase microextraction was developed for ALB and TCB.
- The analytes are extracted in a closed system using of two connected syringes.
- The dispersion is done by injection & back injection of mixture of solvent/ sample.

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- The dispersion is done without the use of dispersing agent, ultrasonic bath or vortex mixer.
- The combination of method with HPLC-FLD allows trace determination of ALB and TCB.
- The method is simple, rapid, green and accurate.

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