Accepted Manuscript Solid phase microextraction of diclofenac using molecularly imprinted polymer sorbent in hollow fiber combined with fiber optic-linear array spectrophotometry Arezou Amiri Pebdani, Ali Mohammad Haji Shabani, Shayessteh Dadfarnia, Saeid Khodadoust PII: DOI: Reference:

S1386-1425(15)00353-4 http://dx.doi.org/10.1016/j.saa.2015.03.057 SAA 13471

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

20 November 2014 5 February 2015 2 March 2015

Please cite this article as: A.A. Pebdani, A.M.H. Shabani, S. Dadfarnia, S. Khodadoust, Solid phase microextraction of diclofenac using molecularly imprinted polymer sorbent in hollow fiber combined with fiber optic-linear array spectrophotometry, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http:// dx.doi.org/10.1016/j.saa.2015.03.057

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Solid phase microextraction of diclofenac using molecularly imprinted polymer sorbent in hollow fiber combined with fiber optic-linear array spectrophotometry Arezou Amiri Pebdani a, Ali Mohammad Haji Shabani a, Shayessteh Dadfarnia* a, Saeid Khodadoust b a b

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

Department of Chemistry, Behbahan Khatam Alanbia University of Technology, Behbahan, Iran * Corresponding author, Email: [email protected] (S. Dadfarnia)

Abstract A simple solid phase microextraction method based on molecularly imprinted polymer sorbent in the hollow fiber (MIP-HF-SPME) combined with fiber optic-linear array spectrophotometer has been applied for the extraction and determination of diclofenac in environmental and biological samples. The effects of different parameters such as pH, times of extraction, type and volume of the organic solvent, stirring rate and donor phase volume on the extraction efficiency of the diclofenac were investigated and optimized. Under the optimal conditions, the calibration graph was linear (r2=0.998) in the range of 3.0-85.0 µg L-1 with a detection limit of 0.7 µg L-1 for preconcentration of 25.0 mL of the sample and the relative standard deviation (n = 6) less than 5%. This method was applied successfully for the extraction and determination of diclofenac in different matrices (water, urine and plasma) and accuracy was examined through the recovery experiments.

Keywords: Molecularly imprinted polymer; Hollow fiber; Solid phase microextraction; Diclofenac; Spectrophotometry

1

Introduction Non-steroid anti-inflammatory drugs (NSAIDs) are a new class of emerging environmental pollutants that are widely used by human and animals all over the world [1]. One of the most consumed drug of NSAIDs world-wide is diclofenac (DFC), (2-[2,6-dichlorophenyl] amino-benzene acetic acid monosodium salt). It is used for the treatment of rheumatoid arthritis, ankyllosing spondylitis, osteoarthritis, and sport injuries [2, 3]. Due to various functions of DFC, there is a great challenge to develop a sensitive, simple and high accuracy method for its extraction and determination from real samples with complicated matrices. Thus, monitoring and evaluation of trace levels of this compound is imperative. However, due to the complexity of the matrix of biological and environmental samples and the low level of DFC in real samples, a sample preparation technique with the aim of separation and preconcentration of analyte is commonly an essential step prior to its determination by analytical instruments. Numerous sample preparation techniques such as liquid–liquid extraction (LLE) [4] and solid phase extraction (SPE) [5] had been used for the preconcentration and clean up of the drugs from different samples. These traditional techniques suffer from the drawbacks including timeconsuming, high cost, and consumption of large volumes of samples and toxic organic solvents [6, 7]. In order to cope with these limitations, solid phase microextraction (SPME) was developed [6]. This technique is fast, time-efficient, easy to use and has been applied for pretreatment of various samples [7]. However, SPME also suffers from some drawbacks including; fiber fragility, limited lifetime of the fiber, relatively high price and also sample carryover [8]. To overcome these problems, recently Eshaghi and coworkers introduced a new version of SPME named hollow fiber solid phase microextraction (HF-SPME). In this method, a silica-based, organic–inorganic polymer containing carbon nanotubes, was prepared by sol-gel technique and was injected into a piece of polypropylene hollow fibre (HF) where the process of in situ gelation occurred and give a suitable sorbent for the HF-SPME procedure [12, 13]. The molecularly imprinted polymer (MIP) is a synthetic polymer, which has selective molecular binding sites for the recognition of a particular chemical substance with its complementary cavity. The synthesis of MIP materials typically consists of polymerizing functional and cross-linking monomers around a template molecule [14, 15]. Most investigations for the synthesis of imprinted polymers were carried out using entirely organic polymeric material [16–19]. However, these types of polymers may shrink or swell when exposed to 2

different samples and may noticeably change the morphology of the polymer network [20–24]. Nowadays, organic–inorganic hybrid MIPs have been found to be highly advantageous as their remarkable structural rigidity, stability, low manufacturing cost, low density, long shelf-life with excellent mechanical properties [25-28]. A synthetic method based on sol-gel process, as one of the fastest emerging fields of material chemistry, is a convenient one for material preparation from organic precursors under mild conditions at low temperature [29]. The aim of the present study is to prepare a molecularly imprinted polymer (MIP) sorbent in presence of MWCNT for DFC, reinforce it in hollow fiber and use it in the hollow fiber solid phase microextraction (MIPHF-SPME) for the preconcentration of DFC prior to its determination by fiber optic-linear array spectrophotometry. This extraction process was successfully used for the determination of DFC in various samples.

Experimental Chemicals and Apparatus Methacrylic acid (MAA), vinyl triethoxysilane (VTEOS), 2,2-azobisisobutyronitrile (AIBN), tetraethyl orthosilicate (TEOS), acetonitrile, methanol, ethanol, chloroform, dichloromethane, hydrochloric acid and sodium hydroxide were all purchased from Merck (Schuchardt, Germany). Diclofenac (DFC) sodium salt was purchased from Sigma-Aldrich (St, Louis, MO, USA). The neutral form of diclofenac was obtained by extracting its sodium salt from an acidic media into the chloroform [30]. Stock standard solution of diclofenac (1000 mg L−1) was prepared in methanol and was stored at -4 ºC. Working solutions were prepared daily from the stock solution by serial dilutions with double distilled water. The hollow fiber polypropylene membranes, Q3/2 Accurel PP (200 µm thick wall, 600 µm inner diameter and 0.2 µm average pore size) were purchased from Membrana (Wuppertal, Germany). The multiwalled carbon nanotubes (MWCNTs) were obtained from the Research Institute of the Petroleum Industry (Tehran, Iran). The mean diameter of the MWCNTs was 10–15 nm, the length was 50– 100 nm and purity >98%. An Avantes Photodiode Array Spectrophotometer model AvaSpec-2048 equipped with a source model of Ava Light-DH-S-BAL was used for recording absorbance spectra. All measurements were made against a reagent blank solution. The pH measurements were carried

3

out with a Metrohm pH meter (model 827, Switzerland) using a combined glass calomel electrode.

Preparation of MIP Preparation of MIP The synthesis procedure of MIP-HF-SPME was done based on the previous reported method [31]. Briefly, the solutions of functional monomer (20 mmol MAA), cross linker (18.5 mmol VTEOS), and initializer (0.5 mmol AIBN) were mixed with template solution. The template solution was prepared by mixing 2.7 mL TEOS, 2.7 mL ethanol (stirred at 50 ◦C), 0.4 mmol of DFC and final addition of 0.8 mL of HCl 12.5% (v/v). Then, 20 mg (equivalent to 2 mg MWCNT per milliliter of solution) of MWCNT which had been acid functionalized [32] was added gradually to the mixture and then it was put in the ultrasonic bath (for 2 h) until the black homogeneous gelatin solution was formed. This process caused the MWCNT to merge into the MIP structure and increase the rate of mass transfer of DFC from aqueous sample into the sorbent.

Fiber treatment The polypropylene hollow fiber was cut into 2.5 cm segments. Then 15.0 µL of the homogeneous solution was injected into the fiber using a Hamilton micro-syringe and the ends of the fiber were closed by a nylon string to prevent the leakage of the solution. The fiber was left for 72 h till the gel network was formed in situ in the HF. Then the prepared sorbent in HF was put in a glass vial and subjected to methanol for removal of the trapped template DFC. Nonimprinted polymer (NIP) control fibers were prepared and treated in the same manner but in the absence of DFC.

Preparation of real samples To evaluate the accuracy and applicability of the proposed method, the extraction and determination of DFC was performed on different samples including tap water, mineral water, human urine and plasma samples. Water samples were filtered through 0.45 µm Millipore filter, the pH was adjusted to ~ 4.0 and was treated according to given procedure. Urine and plasma samples were provided by healthy volunteer in our lab which were not exposed to any drug for at 4

least 4 months and were kept frozen at -20 ˚C before analysis [33, 34]. The frozen urine and plasma samples were thawed at room temperature and centrifuged for 10 min at 5000 rpm. In this step the white lipid solid was sediment in the bottom of the conical test tube which may be attributed to the co-sedimentation of the urine and plasma matrices ingredients. Then, the supernatants were decanted into clean glass tube and filtered through a 0.45 µm filter. The matrix effect was lowered by 10 times dilution of urine and plasma sample and subsequently the samples were subjected to MIP-HF-SPME procedure and measurement.

MIP-HF-SPME procedure MIP-HF-SPME procedure The prepared HF was submerged into 25.0 mL of the samples containing not more than 0.5 µg DFC in a round bottom flask and stirred at 800 rpm for 30 min. The preconcentrated DFC into HF was desorbed into 0.25 mL of methanol under sonical agitation for 8 min and was determined by measuring its absorption at 283 nm. All the experiments were performed in triplicate and averages were reported.

Results and discussion Effect of pH on the extraction The pH of the aqueous sample play an important role in the sorption of DFC into MIPHF-SPME, because it can affect the interaction between the MIP surface and the target analyte. Therefore, the effect of pH on the DFC sorption was investigated by varying the pH of the aqueous samples in the range of 1.0 to 6.0. The pH was adjusted by using either diluted nitric acid or ammonia solution. As it is depicted in Fig. 1, the maximum extraction recoveries of DFC were found in the pH range of 2.0-4.0 which is corresponding to the neutral form of DFC.

Effect of extraction time HF-SPME is an equilibrium process, so the extraction time is an important factor that influences the extraction yield. This is because a long solid-liquid contact time guarantee that the system has reached equilibrium and the maximum amount of analyte is extracted. As can be seen from Fig. 2, the extraction efficiency increases with the increasing of contact time from 5 to 30

5

min, and then becomes constant with the further increase in contact time. Thus, accordingly, 30 min was selected as the optimal extraction time for the subsequent experiments. The rate of stirring can reduce the time required to reach thermodynamic equilibrium by enhancing the mass transfer rate of the DFC from the aqueous phase to solid phase and reducing the thickness of boundary layer at the outer membrane surface [32]. The effect of stirring rate on the extraction was investigated by varying the stirring rates between 300 rpm to 900 rpm. It was found that a stirring rate of 800 rpm to be adequate for obtaining the maximum extraction recovery.

Effect of elution condition The nature of eluting agents has significant effect on the desorbing process of retained analyte from the solid phase and the performance of the preconcentration method. In order to choose the most effective eluent several organic solvents such as methanol, acetonitrile, ethanol and dichloromethane were investigated. The recoveries with these eluent were as follows: methanol (98.6%), ethanol (25.3%), dichloromethane (33.4%) and acetonitrile (42.5%). According to the results, methanol was chosen as the best desorbing solvent and was used in the subsequent work. The methanol volume is also an important factor that affects the efficiency of the elution. In order to study this factor, four different volumes of methanol in the range of 0.150.5 mL were tested and was found that 0.25 mL to be sufficient for complete desorption of DFC. To ensure that desorption of DFC from the fiber was complete; the elution time was investigated in the 1 to 10 min. It was observed that the complete elution was achieved after 8 minutes (Fig. 3).

Effect of sample volume In order to obtain a higher enhancement factor, the possibility of extraction of DFC from the large sample volume was examined. For this purpose, different volumes (15-50 mL) of sample containing 0.5 µg of DFC were subjected to the extraction procedure while the volume of acceptor phase was kept constant at 0.25 mL. It was found that up to 35.0 mL of aqueous phase the recovery was quantitative (> 98%) and then gradually decreased with the further increase in sample volume. Thus, a sample volume up to 35 mL can be subjected to the extraction

6

procedure. However, in the optimization and analysis of real samples 25 mL of sample was used as it was more convenient.

Interference study The selectivity of the MIP-HF-SPME for DFC was investigated by application of the method to mefenamic acid (MFA) and carbamazephine (CBZ) with similar molecular structure. The experiments were performed using the standard solution of 85.0 µg L-1 of each compound individually. As can be seen from Fig. 4, the extraction efficiency of other drugs was less than 10% in compare to DFC. In addition, comparison of extraction efficiency of MIP-HF-SPME and NIP-HF-SPME for DFC revealed that the MIP-HF-SPME has much more affinity for DFC than NIP-HF-SPME. This indicates that the proposed MIP-HF-SPME method exhibited a much higher selectivity for diclofenac.

Analytical performance Under the optimal conditions, a series of experiments (n=8) were performed to consider the linear range, correlation coefficient (r2), precision, limit of detection (LOD), and limit of quantification (LOQ) of the proposed method. The calibration graphs for DFC were linear over the concentration ranges of 3.0-85.0 µg L-1 and 2.0-70.0 µg L-1 with correlation coefficients (r2) of 0.998 and 0.997 for preconcentrating the analyte from 25.0 and 35.0 mL of aqueous solution, respectively. The equations of calibration curves were A = 7.103 C + 0.002 and A = 10.12 C + 0.016 (where A is the absorbance and C is the concentration of diclofenac in mg L-1) for 25.0 and 35.0 mL sample, respectively. The ratio of the slopes of two calibration curves (1.42) is very close to the ratio of the preconcentrated sample volumes (1.40), indicating the constant sorption and desorption of the analyte. The enhancement factor (EF) defined as the slope ratio of the calibration curve with and without preconcentration was found to be 96.0 and 136.7 with sampling volume of 25.0 and 35.0 mL, respectively. The closeness of EF (96.0) to preconcentration factor (100.0) indicates the extraction process is quantitative (96%). The limits of detection (LOD) and quantification (LOQ) defined as 3 and 10 times of the standard deviation of the blank to the slope of calibration curve were found to be 0.7 and 2.3 µg L-1 for 25.0 mL and 0.48 and 1.6 µg L-1 for 35.0 mL of sample, respectively. The relative

7

standard deviation (RSD) for six replicate measurements of the calibration solution of 0.5 µg of DFC in 25.0 and 35.0 mL sampling volume were 3.4 and 4.1%, respectively.

Application of the method to real samples In order to found the validity of the proposed method, the optimum procedure has been applied to the determine DFC concentration in various samples with different matrices, such as water, human urine and plasma. The accuracy of the method was evaluated by recovery experiment. For urine or plasma sample, 2.5 mL of it was diluted to 25.0 mL prior to analysis, whereas the water samples were directly subjected to the preconcentration procedure under the optimum conditions. The results of this analysis are summarized in Table 1 and indicate that the recoveries at the two spiked levels are good (95.0-104.2). The figures of merit for the determination of DFC by the proposed MIP-HF-SPME were compared with those of the previously published methods (Table 2). As it can be seen, the proposed method has a good precision with a suitable dynamic linear range and with one exception [1] it provides a higher preconcentration factor and lower detection limit than the other reported methods.

Conclusion In present study, molecularly imprinted polymer (MIP) sorbent in presence of MWCNT was synthesized and was reinforced in the hollow fiber (HF) for solid phase microextraction (MIP-HF-SPME) of DFC. The combination of molecular imprinted sorbent and HF-SPME technique provides a powerful sample preparation tool in terms of selectivity, simplicity, and high extraction recovery. The various factors affecting extraction efficiency were investigated and optimized. The linear response (r2 > 0.998) was achieved in the range of 3.0-85.0 µg L−1 with a detection limit of 0.7 µg L−1 for 25.0 mL of sample solution. The capability of the MIPHF-SPME method for extraction of DFC in different samples including water, human urine and plasma was successfully demonstrated.

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[24] F. Qin, C. Xie, Z. Yu, L. Kong, M. Ye, H. Zou, J. Sep. Sci. 29 (2006) 1332-1343. [25] Z.S. Liu, C. Zheng, C. Yan, R.Y. Gao, Electrophoresis 28 (2007) 127-136. [26] C. I. Lin, A. K. Joseph, C. K. Chang, Y. C. Wang, Y. D. Lee, Anal. Chim. Acta 481 (2003) 175-180. [27] K.J. Shea, D.A. Loy, Chem. Mater. 13 (2001) 3306-3319. [28] J.P. Corriu, Angew. Chem. Int. Ed. Engl. 39 (2000) 1376-1398. [29] L. Hench, J. West, Chem. Rev. 90 (1990) 33-72. [30] Z. Sun, W. Schüssler, M. Sengl, R. Niessner, D. Knopp, Anal. Chim. Acta 620 (2008) 7381. [31] M. Ahmadi Golsefidi, Z. Eshaghi, A. Sarafraz-Yazdi, J. Chromatogr. A 1229 (2012) 24-29. [32] Z. Eshaghi, M.A. Golsefidi, A. Saify, A. A. Tanha, Z. Rezaeifar, Z. Alian-Nezhadi, J. Chromatogr. A 1217(2010) 2768-2775. [33] S. Khodadoust, M. Ghaedi, J. Sep. Sci. 36 (2013) 1734-1742. [34] M. Rezaee, Y. Yamini, M. Faraji, J. Chromatogr. A 1217 (2010) 2342-2357. [35] Zh.O. Kormosh, I.P. Hunka, Ya. R. Bazel, J. Chin. Chem. Soc. 55 (2008) 356-361. [36] L. Vera-Candioti, M.D. GilGarcía, M. Martínez Galera, H.C. Goicoechea, J. Chromatogr. A 1211 (2008) 22-32. [37] A. Azzouz, B. Souhail, E. Ballesteros, Talanta 84 (2011) 820-828.

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Figure captions

Fig. 1. Effect of sample pH on the extraction of DFC. Conditions: initial DFC concentration 20 µg L−1, sample volume 25.0 mL, extraction time 30 min, desorption time 8 min, and eluent 0.25 mL. Fig. 2. Effect of time on the extraction of DFC. Conditions: sample pH ~ 4.0, sample volume 25.0 mL, initial DFC concentration 20 µg L−1, desorption time 8 min, and eluent 0.25 mL. Fig. 3. Effect of desorption time on the extraction of DFC. Conditions: sample pH ~ 3.0, sample volume 25.0 mL, initial DFC concentration 20 µg L−1, extraction time 30 min, and eluent 0.25 mL. Fig. 4. Selective recognition capability of MIP and NIP-HF-SPME. Conditions: sample pH~ 3.0, initial concentration 85 µg L-1, sample volume 25.0 mL, and extraction time 30 min.

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Recovery (%)

100

90

80

70

60 0

1

2

3

pH

Figure 1

12

4

5

6

100

Recovery (%)

80

60

40

20 0

5

10

15

20

25

30

Extraction time (min)

Figure 2

13

35

40

45

100

Recovery (%)

80

60

40

20 0

1

2

3

4

5

6

7

Desorption time (min)

Figure 3

14

8

9

10

11

Figure 4

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Table 1 Recovery of DFC in a spiked water, plasma and urine samples (n = 3). Sample

Tap water

Well water

Mineral water

Plasma

Urine

Added (µg L-1)

Found (µg L-1)

Recovery (%)

-

-

-

10

9.62 ± 0.17

96.2

20

19.10 ± 0.56

95.5

-

-

-

10

9.66±0.33

96.6

20

19.25 ± 0.63

96.3

-

-

-

10

10.15 ± 0.49

101.5

20

20.67 ± 1.09

103.3

-

-

-

10

9.57 ± 0.46

95.7

20

19.02 ± 0.18

95.1

-

-

-

10

10.42 ± 0.46

104.2

20

20.60 ± 0.71

103.0

16

Table 2 Comparison of the proposed method with other methods of extraction and determination of the DFC. Analysis method

LOD

DLR

RSD

(µg L-1)

(µg L-1)

(%)

Pharmaceuticals

860

900-11000

SPME-LC-DAD

Water

1.5

SPE-HPLC(UV)

Animal tissue/

Ion associates-

Matrices

PF

Ref.

-

-

[35]

4-50

5.9

-

[36]

1.2

5-80

5.1

-

[37]

Water

0.4

10-2560

2.8

184

[1]

Water

3500

600–10000

-

-

[2]

0.7

3.0-85.0

3.4

100

This

spectrophotometer

plasma CNTs-HF-SLPMEHPLC (UV) Ion associatesspectrophotometer MIP-HF-SPME-

Water/urine/plasma

spectrophotometer

work

17

18

Highlights


Preparation of a sorbent for DFC by synthesis of MIP in presence of MWCNT and its reinforcement in HF.
Development of a MIP-HF-SPME method for preconcentration of DFC.
Combination of MIP-HF-SPME with fiber optic-linear array spectrophotometry.
Development of a simple, sensitive & precise method for determination of DFC in various samples.

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