691
J. Sep. Sci. 2014, 37, 691–695
Homayon Ahmad Panahi1 Ali Mehramizi2 Somayeh Ghassemi1 Elham Moniri3 1 Department
of Chemistry, Islamic Azad University, Central Tehran Branch, Iran 2 TAM Pouya pharmaceutical research Co., Tehran, Iran 3 Department of Chemistry, Islamic Azad University, Varamin (Pishva) Branch, Iran Received October 27, 2013 Revised December 19, 2013 Accepted December 23, 2013
Research Article
Selective extraction of clonazepam from human plasma and urine samples by molecularly imprinted polymeric beads A molecularly imprinted polymer (MIP) based on free-radical polymerization was prepared with 1-(N,N-biscarboxymethyl)amino-3-allylglycerol and N,N-dimethylacrylamide as functional monomers, N,N-methylene diacrylamide as the cross-linker, copper ion-clonazepam as the template and 2,2-azobis(2-methylbutyronitrile) as the initiator. The imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermogravimetric analysis, and SEM. The MIP of agglomerated microparticles with multipores was used for SPE. The imprinted polymer sorbent was selective for clonazepam. The optimum pH and sorption capacity were 5 and 0.18 mg/g at 20⬚C, respectively. The profile of the drug uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. The MIP-SPE was the most feasible technique for the extraction of clonazepam with a high recovery from human plasma and urine samples. Keywords: Clonazepam / Human plasma / Human urine / Molecularly imprinted polymers / SPE DOI 10.1002/jssc.201301144
1 Introduction Clonazepam is a well-known prescription medication. Clonazepam, 5-(2-chlorphenyl)-1,3-dihydro-7-nitro-2H-1,4benzodiazepin-2-one, a benzodiazepine derivative, is an anticonvulsant agent primarily used in the treatment of epilepsy for both adults and children. A single oral administration of 2 mg of clonazepam leads to an average plasma concentration of 17 ng/L (7–24 ng/L) [1]. Previously published methods revealed that the analysis of clonazepam in biological and pharmaceutical samples is vitally important [2]. Thus, several methods have been reported for the determination of this drug, including liquid–liquid extraction and SPE techniques [3–5], GC [6, 7], HPLC [8–10] with MS [11] and GC– MS [12–14], and electrochemiluminescence techniques [15]. The molecular imprinting technique involving the formation of cavities in a synthetic polymer for a template molecule is useful for selective extraction. This technique is a rapidly developing method for the preparation polymeric materials that are capable of high molecular recognition [16–19]. This method usually involves cross-linking of the functional monomers in the presence of template structures by radical polymerization, then removing the target. Molecularly Correspondence: Dr. Ahmad Panahi, Department of Chemistry, Islamic Azad University, Central Tehran Branch, No. 4, Riyahi Alley, Barzegar Alley, Vali Asr street, Tehran, Iran E-mail: [email protected] Fax: +98-21-44164539
Abbreviations: AGE, allyl glycidyl ether; AIBN, 2,2 -azobis (2methylbutyronitrile); DMAA, N,N -dimethylacrylamide; IDA, iminodiacetic acid; MIP, molecularly imprinted polymer; NIP, nonimprinted polymer; TGA, thermogravimetric analysis
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
imprinting polymer preparation has generally been based on hydrogen bonding interactions for small molecules. Researchers always try to prepare imprinted polymers for high affinity toward the template molecules. For achieving this purpose, the type of monomers and polymerization were designed for better selectivity. The method presented in this paper employs a new synthetic monomer for the preparation of a clonazepam-imprinted polymer based on free-radical polymerization in the presence of copper ions. The imprinted polymer in batch-wise mode as the solid-phase extractant was used for the pretreatment of trace clonazepam in human plasma and urine samples prior to the determination of the concentration of clonazepam by HPLC.
2 Materials and methods 2.1 Instruments Elemental analysis was carried out on a Thermo-Finnigan (Milan, Italy) model Flash EA elemental analyzer. In addition, thermogravimetric analysis (TGA) was carried out using a TGA-50H (Shimadzu Corporation, Kyoto, Japan). Infrared spectra were recorded on a Jasco Fourier transform infrared spectrometer (FTIR-410, Jasco, Easton, Maryland). The SEM micrographs were obtained with a SEM-PHILIPS XL30 scanning electron microscope. 2.2 Reagents and solutions Allyl glycidyl ether (AGE) was purchased from Fluka Chemica (Buchs Switzerland). N,N-Dimethylacrylamide (DMAA) was
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J. Sep. Sci. 2014, 37, 691–695
acquired from Aldrich (Steinheim, Germany). 2,2 -Azobis(2methylbutyronitrile) (AIBN) was purchased from Across (New Jersey, USA). Divinylbenzene, N,N-methylene diacrylamide, iminodiacetic acid (IDA), tetrahydrofuran, sodium heptasulfonate, hydrogen disodium phosphate, dihydrogen sodium phosphate, phosphoric acid, potassium hydroxide, copper(II) chloride, nitric acid, TFA, acetic acid, methanol, and ethanol were products of Merck (Darmstadt, Germany). The stock solution (100 mg/L) of clonazepam was prepared by dissolving appropriate amounts of clonazepam in methanol. To adjust the pH of the solution, 0.01 M acetic acid/acetate buffer (pH 3–6.5) or 0.01 M phosphate buffer (pH 6.5–9) were used wherever suitable.
2.3 Synthesis of poly(AGE/IDA-co-DMAA) 2.3.1 Synthesis of the functional monomer 1-(N,N-biscarboxymethyl)amino-3-allylglycerol (AGE/IDA) Details of the preparation and characterization of the poly[1-(N,N-biscarboxymethyl)amino-3-allylglycerolcodimethylacrylamide] have been reported in our previous work [20]. The IDA was neutralized with a potassium hydroxide solution to keep carboxylic acid from reacting with the epoxy ring of AGE. Then, the dipotassium salt solution (1 M, 50 mL) was added dropwise to AGE at a 1:1 molar ratio. The mixture was kept at 65⬚C for 1 h with stirring. At this point, the oil/water mixture changed to a transparent water phase. Consequently, the yellowish liquid monomer was purified by being poured into acetone and being dissolved in distilled water. 2.3.2 Preparation of clonazepam-imprinted polymer (MIP) The MIP was prepared by the noncovalent approach with the free radical copolymerization of AGE/IDA and DMAA. In a polymerization reactor, 53 mg of clonazepam, as the template, was dissolved in 20 mL methanol; and then, 8 mg copper chloride, 100 mg AIBN as the initiator, and 15 mL AGE/IDA and 1 mL DMAA as the functional monomers were added. The mixture was sparged with nitrogen for 5 min and it was placed in a thermostatted water bath at 65⬚C for 120 min with vigorous stirring under a nitrogen atmosphere. Then the degassed mixture of N,N-methylene diacrylamide or divinylbenzene, as cross-linker, and 0.1 g of AIBN in 10 mL of methanol was added to the polymerization reactor. After complete polymerization, the blue-greenish polymer was filtered immediately and washed with 100 mL methanol and then was dried in the oven at 45⬚C. The resultant bulk polymers were ground in a mechanical mortar and sieved through a 100 m sieve and washed again with methanol containing acetic acid (0.3%) and TFA (0.1%) for 24 h. The second washing step was performed with 100 mL of 0.1 M nitric acid while C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Schematic presentation of synthesis process of poly(AGE/IDA-co-DMAA).
stirring for 1 day to remove the Cu(II). So, the color of the polymer turned white. The product was recovered by filtration and washed with ultrapure water until the filtrate pH was no longer acidic. Finally, the sorbent was dried under vacuum, ground in a mechanical mortar, sieved through a 100 m sieve, and stored at 4⬚C. The methodology used to synthesize MIP is summarized in Fig. 1. The MIP was characterized by FTIR spectroscopy, elemental analysis, SEM, and TGA. FTIR (NaCl): 3386 (OH), 1651 (C = O), 2936 bending (CH2 ), 1208 (C–O), 1114 (CN) cm−1 . The elemental analysis for poly DMAA-AGE/IDA was performed and the percentage of carbon, hydrogen, and www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2014, 37, 691–695
693
nitrogen was found (C: 49.56; H: 7.85; N: 13.73%). The nonimprinted polymer (NIP) was prepared in the same way as described above but with no template molecule added.
2.4 HPLC System Chromatographic separations were carried out on an Agilent HPLC, 1200 series, equipped with a UV/Vis detector. Separations were carried out on a Zorbax Extend C18 column (15 cm × 4.6 mm, with 3 mm particle size) from Agilent (Wilmington, DE, USA). The mixture of 48 mL solution of 6.6 g/L of ammonium hydrogen phosphate, pH 8.0, 10 mL tetrahydrofuran and 42 mL methanol at a flow rate of 1 mL/min was used as a mobile phase in isocratic elution mode. The injection volume was 20 L for all the samples and the detection was performed at a wavelength of 254 nm.
2.5 Clonazepam adsorption/desorption method A set of solutions (the volume of each 1 mL) containing 1– 30 g/mL of clonazepam was taken in a microtube and their pH were adjusted to 5 with 0.01M acetate buffer solutions. Poly(AGE/IDA-co-DMAA) (0.01 g) was added to each solution and the mixture was shaken for 2 min. The sorbent was filtered and the adsorbed clonazepam were eluted with 0.3% acetic acid and 0.1% of TFA in methanol. The concentration of clonazepam in the eluate was determined by HPLC.
2.6 Sample treatment Human blood was collected from thoroughly controlled voluntary blood donors. Each unit was separately controlled and found negative for HBS antigen and HIV I, II, and hepatitis C antibodies. Human blood was collected into test tubes containing EDTA, and red blood cells were separated from plasma by centrifugation at 4000 ×g for 30 min at room temperature, then filtered (3 m Sartorius filter) and frozen at −20⬚C. Before use, the plasma was thawed for 1 h at 37⬚C.
Figure 2. Effect of pH sorption of clonazepam onto imprinted poly(AGE/IDA-co-DMAA). Volume of each 1 mL containing 20 g/mL of clonazepam. pH values were adjusted with buffer solutions. 0.01 g of imprinted poly(AGE/IDA-co-DMAA) was added to each solution and the mixture was shaken for 20 min.
metal ions. Therefore, it seems that this method can be useful for other separations with other templates.
3.1 Characterization of clonazepam-imprinted poly(AGE/IDA-co-DMAA) FTIR spectroscopy confirmed the presence of hydroxyl and carbonyl groups in the polymer. The presence of nitrogen in the polymer by elemental analysis indicates that DMAA or AGE/IDA molecule is present in the repeat unit of the polymer. The TGA of the polymer showed a weight loss up to 150⬚C due to the desorption of water molecules from the surface, and the weight loss at 200–600⬚C was due to the decomposition of the polymeric matrix. Briefly, FTIR, TGA, and elemental analysis consistently confirmed the structure of the polymer as presented in Fig. 1. SEM was used to observe the external surface of the polymer. The surface of the polymer has a smooth and highly homogeneous appearance. The specific surface area of poly(AGE/IDA-co-DMAA) was calculated by using the Brunauer–Emmett–Teller method using nitrogen isotherm adsorption data. The surface area of the imprinted polymer was 8.41 m2 g−1 .
3 Results and discussion 3.2 Sorption conditions In this method, intermediate metal ion (Cu(II)) was used for the sorption of clonazepam in human plasma and urine. The intermediate metal ion can coordinate nitrogen, oxygen, and sulfur. The principle of separation is based on affinity between Cu(II) with nitrogen and oxygen atoms in the polymer and/or in the clonazepam molecules. So the intermediate metal ion can play role of a bridge between the sorbent polymer and the drug. Moreover, the cavities in the molecularly imprinted polymer (MIP) are better formed in presence of Cu(II). Perhaps the drug template transfers easier into the cavities due to the bigger size of cavities in the presence of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The imprinted polymers were doped with Cu(II) and then subjected to clonazepam sorption in different pH levels (Fig. 2). The maximum adsorption of the drug was achieved at pH 5. The extraction recovery was defined as the percentage of the total clonazepam, which was extracted into the solid phase and subsequently into eluent. The sorption, as a function of contact time for clonazepam, was studied at different times and shown in Fig. 3. Just 1 min was sufficient for maximal sorption. The profile of clonazepam uptake by the MIP reflects good accessibility of the active sites in the www.jss-journal.com
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Table 1. Effect of eluting agent on clonazepam recovery
Figure 3. Kinetics of clonazepam sorption on imprinted poly(AGE/IDA-co-DMAA). Volume of each 1 mL containing 20 g/mL of clonazepam. A total of 0.01 g of imprinted poly(AGE/IDA-co-DMAA) was added to each solution and the mixture was shaken for different length of time at pH 5.
Eluent
Recovery (%)
TFA 0.1% in methanol Acetic acid 0.3% + TFA 0.1% in methanol Acetic acid 0.5% + TFA 0.1% in methanol Acetic acid 1% + TFA 0.1% in methanol Acetic acid 2% + TFA 0.1% in methanol Acetic acid 10% + TFA 0.1% in methanol Acetic acid 15% + TFA 0.1% in methanol Acetic acid 20% + TFA 0.1% in methanol Acetic acid 30% + TFA 0.1% in methanol
88.1 97.6 97.1 82.9 76.4 76.2 76.1 74.3 73.2
use of the imprinted poly(AGE/IDA-co-DMAA) is feasible. After loading with samples, the imprinted polymer can be readily regenerated with methanol containing 0.1% of TFA and 0.3% of acetic acid.
3.4 Selectivity of the imprinted sorbent The sorption comparison of clonazepam on NIP and MIP was investigated. A recognition coefficient (␣), an important parameter that is used to evaluate the binding performance of the molecular imprinting polymers, can be defined as: [24] ␣ = Q(imprinted) /Q(nonimprinted) Figure 4. Effect of initial concentration of clonazepam in the solution on sorption capacity of clonazepam onto imprinted poly(AGE/IDA-co-DMAA).
imprinted polymer. The sorption capacity of the MIP sorbent for clonazepam was ascertained from the difference between concentrations in solution before and after the sorption. The sorption capacity was around 0.18 mg/g at pH 5 and 20⬚C (Fig. 4). The affinity to target compound by the polymer in the presence and absence of Cu(II) was compared. The binding capacity of polymer in the presence of Cu(II) is more than the other one, so Cu2+ can fit the drug into polymer cavity. The degree of drug desorption with different eluting agents after clonazepam adsorption at pH 5, was determined by a batchwise method (Table 1). The eluents generally used are methanol and a small percentage of acid such as TFA and acetic acid, which were added for a more efficient elution of the more strongly bound analytes [21–23]. Methanol, containing 0.1% of TFA and 0.3% of acetic acid, provided the best recovery. The clonazepam was sorbed and desorbed on the imprinted polymer several times. It was found that the sorption capacity of imprinted polymer after six cycles of its equilibration with clonazepam, changes
J. Sep. Sci. 2014, 37, 691–695
Homayon Ahmad Panahi1 Ali Mehramizi2 Somayeh Ghassemi1 Elham Moniri3 1 Department
of Chemistry, Islamic Azad University, Central Tehran Branch, Iran 2 TAM Pouya pharmaceutical research Co., Tehran, Iran 3 Department of Chemistry, Islamic Azad University, Varamin (Pishva) Branch, Iran Received October 27, 2013 Revised December 19, 2013 Accepted December 23, 2013
Research Article
Selective extraction of clonazepam from human plasma and urine samples by molecularly imprinted polymeric beads A molecularly imprinted polymer (MIP) based on free-radical polymerization was prepared with 1-(N,N-biscarboxymethyl)amino-3-allylglycerol and N,N-dimethylacrylamide as functional monomers, N,N-methylene diacrylamide as the cross-linker, copper ion-clonazepam as the template and 2,2-azobis(2-methylbutyronitrile) as the initiator. The imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermogravimetric analysis, and SEM. The MIP of agglomerated microparticles with multipores was used for SPE. The imprinted polymer sorbent was selective for clonazepam. The optimum pH and sorption capacity were 5 and 0.18 mg/g at 20⬚C, respectively. The profile of the drug uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. The MIP-SPE was the most feasible technique for the extraction of clonazepam with a high recovery from human plasma and urine samples. Keywords: Clonazepam / Human plasma / Human urine / Molecularly imprinted polymers / SPE DOI 10.1002/jssc.201301144
1 Introduction Clonazepam is a well-known prescription medication. Clonazepam, 5-(2-chlorphenyl)-1,3-dihydro-7-nitro-2H-1,4benzodiazepin-2-one, a benzodiazepine derivative, is an anticonvulsant agent primarily used in the treatment of epilepsy for both adults and children. A single oral administration of 2 mg of clonazepam leads to an average plasma concentration of 17 ng/L (7–24 ng/L) [1]. Previously published methods revealed that the analysis of clonazepam in biological and pharmaceutical samples is vitally important [2]. Thus, several methods have been reported for the determination of this drug, including liquid–liquid extraction and SPE techniques [3–5], GC [6, 7], HPLC [8–10] with MS [11] and GC– MS [12–14], and electrochemiluminescence techniques [15]. The molecular imprinting technique involving the formation of cavities in a synthetic polymer for a template molecule is useful for selective extraction. This technique is a rapidly developing method for the preparation polymeric materials that are capable of high molecular recognition [16–19]. This method usually involves cross-linking of the functional monomers in the presence of template structures by radical polymerization, then removing the target. Molecularly Correspondence: Dr. Ahmad Panahi, Department of Chemistry, Islamic Azad University, Central Tehran Branch, No. 4, Riyahi Alley, Barzegar Alley, Vali Asr street, Tehran, Iran E-mail: [email protected] Fax: +98-21-44164539
Abbreviations: AGE, allyl glycidyl ether; AIBN, 2,2 -azobis (2methylbutyronitrile); DMAA, N,N -dimethylacrylamide; IDA, iminodiacetic acid; MIP, molecularly imprinted polymer; NIP, nonimprinted polymer; TGA, thermogravimetric analysis
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
imprinting polymer preparation has generally been based on hydrogen bonding interactions for small molecules. Researchers always try to prepare imprinted polymers for high affinity toward the template molecules. For achieving this purpose, the type of monomers and polymerization were designed for better selectivity. The method presented in this paper employs a new synthetic monomer for the preparation of a clonazepam-imprinted polymer based on free-radical polymerization in the presence of copper ions. The imprinted polymer in batch-wise mode as the solid-phase extractant was used for the pretreatment of trace clonazepam in human plasma and urine samples prior to the determination of the concentration of clonazepam by HPLC.
2 Materials and methods 2.1 Instruments Elemental analysis was carried out on a Thermo-Finnigan (Milan, Italy) model Flash EA elemental analyzer. In addition, thermogravimetric analysis (TGA) was carried out using a TGA-50H (Shimadzu Corporation, Kyoto, Japan). Infrared spectra were recorded on a Jasco Fourier transform infrared spectrometer (FTIR-410, Jasco, Easton, Maryland). The SEM micrographs were obtained with a SEM-PHILIPS XL30 scanning electron microscope. 2.2 Reagents and solutions Allyl glycidyl ether (AGE) was purchased from Fluka Chemica (Buchs Switzerland). N,N-Dimethylacrylamide (DMAA) was
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H. A. Panahi et al.
J. Sep. Sci. 2014, 37, 691–695
acquired from Aldrich (Steinheim, Germany). 2,2 -Azobis(2methylbutyronitrile) (AIBN) was purchased from Across (New Jersey, USA). Divinylbenzene, N,N-methylene diacrylamide, iminodiacetic acid (IDA), tetrahydrofuran, sodium heptasulfonate, hydrogen disodium phosphate, dihydrogen sodium phosphate, phosphoric acid, potassium hydroxide, copper(II) chloride, nitric acid, TFA, acetic acid, methanol, and ethanol were products of Merck (Darmstadt, Germany). The stock solution (100 mg/L) of clonazepam was prepared by dissolving appropriate amounts of clonazepam in methanol. To adjust the pH of the solution, 0.01 M acetic acid/acetate buffer (pH 3–6.5) or 0.01 M phosphate buffer (pH 6.5–9) were used wherever suitable.
2.3 Synthesis of poly(AGE/IDA-co-DMAA) 2.3.1 Synthesis of the functional monomer 1-(N,N-biscarboxymethyl)amino-3-allylglycerol (AGE/IDA) Details of the preparation and characterization of the poly[1-(N,N-biscarboxymethyl)amino-3-allylglycerolcodimethylacrylamide] have been reported in our previous work [20]. The IDA was neutralized with a potassium hydroxide solution to keep carboxylic acid from reacting with the epoxy ring of AGE. Then, the dipotassium salt solution (1 M, 50 mL) was added dropwise to AGE at a 1:1 molar ratio. The mixture was kept at 65⬚C for 1 h with stirring. At this point, the oil/water mixture changed to a transparent water phase. Consequently, the yellowish liquid monomer was purified by being poured into acetone and being dissolved in distilled water. 2.3.2 Preparation of clonazepam-imprinted polymer (MIP) The MIP was prepared by the noncovalent approach with the free radical copolymerization of AGE/IDA and DMAA. In a polymerization reactor, 53 mg of clonazepam, as the template, was dissolved in 20 mL methanol; and then, 8 mg copper chloride, 100 mg AIBN as the initiator, and 15 mL AGE/IDA and 1 mL DMAA as the functional monomers were added. The mixture was sparged with nitrogen for 5 min and it was placed in a thermostatted water bath at 65⬚C for 120 min with vigorous stirring under a nitrogen atmosphere. Then the degassed mixture of N,N-methylene diacrylamide or divinylbenzene, as cross-linker, and 0.1 g of AIBN in 10 mL of methanol was added to the polymerization reactor. After complete polymerization, the blue-greenish polymer was filtered immediately and washed with 100 mL methanol and then was dried in the oven at 45⬚C. The resultant bulk polymers were ground in a mechanical mortar and sieved through a 100 m sieve and washed again with methanol containing acetic acid (0.3%) and TFA (0.1%) for 24 h. The second washing step was performed with 100 mL of 0.1 M nitric acid while C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Schematic presentation of synthesis process of poly(AGE/IDA-co-DMAA).
stirring for 1 day to remove the Cu(II). So, the color of the polymer turned white. The product was recovered by filtration and washed with ultrapure water until the filtrate pH was no longer acidic. Finally, the sorbent was dried under vacuum, ground in a mechanical mortar, sieved through a 100 m sieve, and stored at 4⬚C. The methodology used to synthesize MIP is summarized in Fig. 1. The MIP was characterized by FTIR spectroscopy, elemental analysis, SEM, and TGA. FTIR (NaCl): 3386 (OH), 1651 (C = O), 2936 bending (CH2 ), 1208 (C–O), 1114 (CN) cm−1 . The elemental analysis for poly DMAA-AGE/IDA was performed and the percentage of carbon, hydrogen, and www.jss-journal.com
Sample Preparation
J. Sep. Sci. 2014, 37, 691–695
693
nitrogen was found (C: 49.56; H: 7.85; N: 13.73%). The nonimprinted polymer (NIP) was prepared in the same way as described above but with no template molecule added.
2.4 HPLC System Chromatographic separations were carried out on an Agilent HPLC, 1200 series, equipped with a UV/Vis detector. Separations were carried out on a Zorbax Extend C18 column (15 cm × 4.6 mm, with 3 mm particle size) from Agilent (Wilmington, DE, USA). The mixture of 48 mL solution of 6.6 g/L of ammonium hydrogen phosphate, pH 8.0, 10 mL tetrahydrofuran and 42 mL methanol at a flow rate of 1 mL/min was used as a mobile phase in isocratic elution mode. The injection volume was 20 L for all the samples and the detection was performed at a wavelength of 254 nm.
2.5 Clonazepam adsorption/desorption method A set of solutions (the volume of each 1 mL) containing 1– 30 g/mL of clonazepam was taken in a microtube and their pH were adjusted to 5 with 0.01M acetate buffer solutions. Poly(AGE/IDA-co-DMAA) (0.01 g) was added to each solution and the mixture was shaken for 2 min. The sorbent was filtered and the adsorbed clonazepam were eluted with 0.3% acetic acid and 0.1% of TFA in methanol. The concentration of clonazepam in the eluate was determined by HPLC.
2.6 Sample treatment Human blood was collected from thoroughly controlled voluntary blood donors. Each unit was separately controlled and found negative for HBS antigen and HIV I, II, and hepatitis C antibodies. Human blood was collected into test tubes containing EDTA, and red blood cells were separated from plasma by centrifugation at 4000 ×g for 30 min at room temperature, then filtered (3 m Sartorius filter) and frozen at −20⬚C. Before use, the plasma was thawed for 1 h at 37⬚C.
Figure 2. Effect of pH sorption of clonazepam onto imprinted poly(AGE/IDA-co-DMAA). Volume of each 1 mL containing 20 g/mL of clonazepam. pH values were adjusted with buffer solutions. 0.01 g of imprinted poly(AGE/IDA-co-DMAA) was added to each solution and the mixture was shaken for 20 min.
metal ions. Therefore, it seems that this method can be useful for other separations with other templates.
3.1 Characterization of clonazepam-imprinted poly(AGE/IDA-co-DMAA) FTIR spectroscopy confirmed the presence of hydroxyl and carbonyl groups in the polymer. The presence of nitrogen in the polymer by elemental analysis indicates that DMAA or AGE/IDA molecule is present in the repeat unit of the polymer. The TGA of the polymer showed a weight loss up to 150⬚C due to the desorption of water molecules from the surface, and the weight loss at 200–600⬚C was due to the decomposition of the polymeric matrix. Briefly, FTIR, TGA, and elemental analysis consistently confirmed the structure of the polymer as presented in Fig. 1. SEM was used to observe the external surface of the polymer. The surface of the polymer has a smooth and highly homogeneous appearance. The specific surface area of poly(AGE/IDA-co-DMAA) was calculated by using the Brunauer–Emmett–Teller method using nitrogen isotherm adsorption data. The surface area of the imprinted polymer was 8.41 m2 g−1 .
3 Results and discussion 3.2 Sorption conditions In this method, intermediate metal ion (Cu(II)) was used for the sorption of clonazepam in human plasma and urine. The intermediate metal ion can coordinate nitrogen, oxygen, and sulfur. The principle of separation is based on affinity between Cu(II) with nitrogen and oxygen atoms in the polymer and/or in the clonazepam molecules. So the intermediate metal ion can play role of a bridge between the sorbent polymer and the drug. Moreover, the cavities in the molecularly imprinted polymer (MIP) are better formed in presence of Cu(II). Perhaps the drug template transfers easier into the cavities due to the bigger size of cavities in the presence of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The imprinted polymers were doped with Cu(II) and then subjected to clonazepam sorption in different pH levels (Fig. 2). The maximum adsorption of the drug was achieved at pH 5. The extraction recovery was defined as the percentage of the total clonazepam, which was extracted into the solid phase and subsequently into eluent. The sorption, as a function of contact time for clonazepam, was studied at different times and shown in Fig. 3. Just 1 min was sufficient for maximal sorption. The profile of clonazepam uptake by the MIP reflects good accessibility of the active sites in the www.jss-journal.com
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Table 1. Effect of eluting agent on clonazepam recovery
Figure 3. Kinetics of clonazepam sorption on imprinted poly(AGE/IDA-co-DMAA). Volume of each 1 mL containing 20 g/mL of clonazepam. A total of 0.01 g of imprinted poly(AGE/IDA-co-DMAA) was added to each solution and the mixture was shaken for different length of time at pH 5.
Eluent
Recovery (%)
TFA 0.1% in methanol Acetic acid 0.3% + TFA 0.1% in methanol Acetic acid 0.5% + TFA 0.1% in methanol Acetic acid 1% + TFA 0.1% in methanol Acetic acid 2% + TFA 0.1% in methanol Acetic acid 10% + TFA 0.1% in methanol Acetic acid 15% + TFA 0.1% in methanol Acetic acid 20% + TFA 0.1% in methanol Acetic acid 30% + TFA 0.1% in methanol
88.1 97.6 97.1 82.9 76.4 76.2 76.1 74.3 73.2
use of the imprinted poly(AGE/IDA-co-DMAA) is feasible. After loading with samples, the imprinted polymer can be readily regenerated with methanol containing 0.1% of TFA and 0.3% of acetic acid.
3.4 Selectivity of the imprinted sorbent The sorption comparison of clonazepam on NIP and MIP was investigated. A recognition coefficient (␣), an important parameter that is used to evaluate the binding performance of the molecular imprinting polymers, can be defined as: [24] ␣ = Q(imprinted) /Q(nonimprinted) Figure 4. Effect of initial concentration of clonazepam in the solution on sorption capacity of clonazepam onto imprinted poly(AGE/IDA-co-DMAA).
imprinted polymer. The sorption capacity of the MIP sorbent for clonazepam was ascertained from the difference between concentrations in solution before and after the sorption. The sorption capacity was around 0.18 mg/g at pH 5 and 20⬚C (Fig. 4). The affinity to target compound by the polymer in the presence and absence of Cu(II) was compared. The binding capacity of polymer in the presence of Cu(II) is more than the other one, so Cu2+ can fit the drug into polymer cavity. The degree of drug desorption with different eluting agents after clonazepam adsorption at pH 5, was determined by a batchwise method (Table 1). The eluents generally used are methanol and a small percentage of acid such as TFA and acetic acid, which were added for a more efficient elution of the more strongly bound analytes [21–23]. Methanol, containing 0.1% of TFA and 0.3% of acetic acid, provided the best recovery. The clonazepam was sorbed and desorbed on the imprinted polymer several times. It was found that the sorption capacity of imprinted polymer after six cycles of its equilibration with clonazepam, changes
