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Molecularly Imprinted Polymers for Selective Solid-Phase Extraction of Verapamil from Biological Fluids and Human Urine Mehran Javanbakht,1,* Narges Shaabani,2 Majid Abdouss,1 Mohammad R. Ganjali,3 Ali Mohammadi,4 and Parviz Norouzi3 1
Department of Chemistry, Amirkabir University of Technology, Tehran, Iran
2
Department of Chemistry, Young Researchers Club, Islamic Azad University, Shahre-ray Branch, Shahre-ray, Iran
3
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran
4
Department of Drug and Food Control, School of Pharmacy, Medical Sciences, University of Tehran, Tehran, Iran Abstract: In this work, water-compatible molecularly imprinted polymers (MIPs) were prepared for fast, accurate and selective solid-phase extraction of verapamil (VPM) from complex matrices such as biological fluids and human urine followed by its UV spectroscopic determination at 278 nm. The effective factors influencing the precipitation polymerization have been studied. Molecular recognition properties, binding capability and selectivity of the MIPs were evaluated and the results revealed that the obtained MIPs have high affinity for VPM in aqueous media. Equilibrium binding experiments were done to assess the performance of the MIP relative to non imprinted polymer (NIP). After optimization of molecularly imprinted solid-phase extraction (MISPE) method with 2 mL water plus 2 mL acetone as washing solvents and 5 mL of methanol and acetic acid (10:1, v/v) as elution solvent, successful imprinting was confirmed by comparison of the recoveries between NIP (4%) and MIP (97%) polymers. The binding capacity of the MIP for VPM was determined to be 196 mg g-1 (400 μmol g-1). Accuracy and precision were checked by the HPLC technique and the results did not present significant difference at 95% confidence levels according to the t-test.
Keywords: Molecularly imprinted polymer, Solid-phase extraction, Verapamil, Biological fluids, Human urine. INTRODUCTION Molecularly Imprinted Polymers (MIPs) are able to bind to a particular compound of interest, present in a complex mixture. This aspect is directly related to selectivity and, for analytical purposes, it is often highly desirable to develop a selective procedure to extract a compound from very complex matrices [1]. The imprinting technique involves the formation of a complex between a template molecule and the functional monomers in an appropriate solvent. The procedure for synthesizing an MIP is based on the chemical polymerization of a functional monomer and a cross-linking agent in the presence of a molecule used as a template. After removing the imprinted molecule, an imprinted polymer is obtained. This polymer contains sites with a high affinity for the template molecule, due to their shapes and the arrangement of the functional groups of the monomer units. These MIPs have been employed in the fields in which a certain degree of selectivity is required such as sensors [2], chromatography [3], and catalysis [4]. The imprinted polymers are used as selective sorbents for the SPE, owing to their longterm stability, cheap, rapid preparation, high thermal and chemical stability, and insolubility in water and most organic solvents. However, these days their use in solid-phase
*Address correspondence to this author at the Department of Chemistry, Amirkabir University of Technology, Tehran, Iran; Tel: +98-21-64543295; Fax: +98-21-64543296; E-mail:
[email protected]
1573-4129/09 $55.00+.00
extraction, so-called molecularly imprinted solid-phase extraction (MISPE), is undoubtedly the most advanced technical application of MIPs. The use of MIPs as selective sorbent materials allows performing a customized sample treatment step prior to the final determination. This is of special interest, when the sample is complex and the presence of interferences could prevent final quantification by typical chromatographic techniques coupled to common detectors. Due to the inherent selectivity provided by MIPs, past years have seen a growing interest in this area and it has been extensively reviewed [5-8]. Recently, we applied MIPs as new sensing material in potentiometric detection of hydroxyzine [9] and cetirizine [10]. In this work, we developed a sorbent based on MIPs for selective solid phase extraction of (±)VPM and its spectrophotometric determination at 278 nm. W. M. Mullett and coworkers [11] were developed a method for the synthesis and multidimensional on-line sample preparation of verapamil and its metabolites by the MIP coupled to liquid- chromatography–mass spectrometry. In mentioned work, the time of verapamil loading in acetonitrile as solvent is 24 h. Moreover, the extraction of verapamil from the MIP was continued for 24 h. Although, the verapamil detection in that work is selective and sensitive, but our scheme allows a fast, simple, sensitive and inexpensive extraction and detection method for the analyte without using additional reagents or instruments. In the present study, the elution and loading was done only in a few minutes.
© 2009 Bentham Science Publishers Ltd.
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O O
OCH3 OH N
H3CO
CH3
H3C
O
O
HO
N CH3
HO
CH3
OCH3
HO
H3CO
CH3Cl
CN
OCH3
HO H3CO
O
OCH3
H3CO
HO
CN H3C
O
CH3
EGDMA
O HO
O
O
HO HO
HO OCH3
O
Remove
HO
HO
O
HO
O
O
HO H3CO
O
Rebind H3CO
O
OCH3
HO
O
HO
N O
HO
CH3
H3C
CN HO CH3
O
Verapamil Recognition Site
Fig. (1). Schematic representation of the MIP synthesis.
Verapamil as a chiral molecule, [(+)-5-[N-(3,4-dimethoxyphenethyl)-N-methylamino]-2,3-(dimethoxyphenyl)-2iso-propyl-valeronitrile], is a calcium channel blocker (CCB) and is classified as a class IV anti-arrhythmic agent. By antagonizing L-type calcium channels in the myocardium, VPM has negative inotropic and chronotropic effects, and overdoses of this drug can lead to decreased cardiac output, hypotension, and shock [12]. It is used to control supra ventricular tachyarrhythmia, and in the management of classical and variant angina pectoris. It is also used in the treatment of hypertension [13]. The drug and its formulations are officially listed in British Pharmacopoeia [14], which suggests potentiometric titration method; The United States Pharmacopoeia [15] recommends a gas chromatographic method. Different analytical methods that are reported for its determination include high performance liquid chromatography [16], high performance thin layer [17], liquid chromatography [18], spectrofluorometry [19], resonance Rayleigh scattering [20], stripping voltammetry [21] and atomic emission spectrometry [22]. The more common approach of using silica based SPE [23] materials such as C18 or C8, is also limited by awkward solvent wetting requirements, poor extraction efficiency and for polar drugs, undesirable interaction between residual silanols and basic analytes and time and labor consumption [24].
EXPERIMENTAL SECTION Materials and Reagents Methacrylic acid (MAA) from Merck (Darmstadt, Germany) was distilled in vacuum prior to use in order to remove the stabilizers. Ethylene glycol dimethacrylate (EGDMA) and 2, 2’-azobis isobutyronitrile (AIBN) from Merck (Darmstadt, Germany), were of reagent grade and used without any further purification. The phosphate buffer solutions with a pH value of 8.5 were prepared in de-ionized water and used. All the other chemicals were of analytical reagent grade and the solutions were prepared with distilled water. Drug free human serum was obtained from the Iranian blood transfusion service (Tehran, Iran) and stored at -20°C until use after gentle thawing. Urine was also collected from healthy volunteers. MIP and NIP Preparation with Precipitation Polymerization The schematic representation of the imprinting and the removal of VPM from the imprinted polymer are shown in Fig. (1). The MIP and NIP preparation with different template:monomer:cross-linker molar ratio, were completed. As reported previously [13], the monomer MAA (240 L, 2.82 mmol), VPM (as hydrochloride salt) print molecule
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Fig. (2). TGA plots of the leached NIP, unleached and leached MIP particles.
(107 mg, 0.217 mmol) and 30 mL of chloroform were placed in a glass sample vial. Then, cross-linker EGDMA (2.6 mL, 13.77 mmol) was added. The mixture was uniformly dispersed by sonication (sonic bath model Ultrasonic UTD35Falc, Italy). After sonication, it was purged with nitrogen for 10 min and the glass tube was sealed under this atmosphere. Then the reaction initiator AIBN (57 mg, 0.347 mmol) was added. The polymerization was carried out for 24 hrs in a water bath at 60°C. After the polymerization procedure and drying, the polymer particles were washed with methanol and acetic-acid (10:1, v/v, of 98 % methanol and pure acetic acid) for three times and with distilled water for two times. The complete removal of template was followed by UV spectrophotometer, and after three times washing, spectra of VPM weren’t seen. In order to verify that retention of template was due to molecular recognition and not because of non-specific binding, a control (non-imprinted) polymer was prepared following the same procedure, including washing, but with the omission of the target molecule, VPM. INSTRUMENTATION Apparatus pH of solutions were adjusted using a model 630 digital Metrohm pH meter equipped with a combined glass–calomel electrode. IR spectra of grounded polymer were recorded on a Shimadzu IR-460 spectrometer (Kyoto, Japan) using KBr pellets in the range of 400–4000 cm1. The thermal analysis of polymer was carried out on a model PL-STA-1500 thermogravimetric analysis (TGA)–differential thermal analysis (DTA) instrument from Polymer Laboratories’ Company (Church Stretton, Shropshire, UK). 5.00 mg of the grounded polymer was heated at a heating rate of 5°C min-1 from ambient temperature up to 600 C under nitrogen atmosphere (flow rate = 20 mL min1) and the corresponding TG curves were obtained.
Infrared Spectroscopy As a result of hydrogen binding with the -COOH group of the methacrylic acid, the O-H and C=O stretching vibrations at 3581 cm1 and 1731 cm1 in the leached MIP materials were shifted to 3554 cm1 and 1721 cm1 in the corresponding unleached MIP, respectively. Further more, there was another distinct difference between the IR spectra of the leached and unleached MIPs. In the leached polymer, there was a broad band around 1477 cm-1. This band had a shoulder in the unleached polymer spectrum and a peak around ~1517 cm-1 was observed. Thermogravimetric Analysis Fig. (2) depicts the TGA plots of the unleached and leached MIP particles. Regarding the unleached MIP particles, TGA revealed two decomposition states: one mass loss starting at 100 °C downward, assigned to the decomposition of the free monomer and the cross-linker, and one starting at ~155 °C, related to the VPM hydrochloride decomposition, as the melting point of VPM hydrochloride is 140-143 °C [25]. All the materials were completely decomposed prior to reaching the temperature of 450 °C. PROCEDURES Batch Binding Assay Adsorption of drug in aqueous solutions was investigated in batch experiments. The general procedure for extraction of VPM by the MIP was as follows: The polymer beads were suspended in aqueous solutions and the pH was adjusted at 8.5. In all experiments, the polymer concentration was kept constant at 50 mg/25 mL. The mixtures were thermostated at 25 °C for 10 min, under continuous stirring and then were filtrated on a paper filter (flow rate= 100 ml min-1 by applied vacuum). The concentration of free VPM in the filtrate was
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Table 1. Compositions and Comparisons of the Extraction of Polymers
a
MIP
MAA (mmol)
VPM (mmol)
EGDMA (mmol)
AIBN (mmol)
Extraction(%) (Mean ± SD) a
MIP1
1.32
0.217
13.77
0.347
52 (± 2.1)
MIP2
2.82
0.217
13.77
0.347
99 (± 3.2)
MIP3
5.00
0.217
13.77
0.347
45 (± 1.7)
MIP4
7.60
0.217
13.77
0.347
37 (± 2.4)
MIP5
2.85
0.217
6.88
0.347
35 (± 2.5)
MIP6
2.85
0.217
20.6
0.347
50 (± 3.3)
MIP7
2.85
0.217
27.5
0.347
54 (± 3.6)
NIP1
1.32
----
13.77
0.347
18 (± 1.9)
NIP2
2.85
----
13.77
0.347
32 (± 3.2)
NIP3
5.00
----
13.77
0.347
20 (± 3.4)
NIP4
7.60
----
13.77
0.347
22 (± 3.5)
Average of five determinations.
determined by spectrophotometer of UV. The instrument response was periodically checked with known VPM standard solutions. Three replicate extractions and measurements were performed for each aqueous solution. Percent extraction of VPM was calculated from the following equation:
C Cf Extraction% = i 100 Ci In which, Ci and Cf are the concentrations of VPM before and after extraction in the solution. The adsorbed VPM was desorbed from the MIP by treating with 5 mL methanol and acetic-acid (10:1, v/v, of 98% methanol and pure acetic acid). The imprinted polymer containing VPM was placed in the desorption medium and stirred continuously at 600 rpm and room temperature for designated time. The final VPM concentration in the aqueous phase was determined by spectrophotometer of UV. The same procedure was followed for NIP particles. Extraction Procedure for Plasma and Urine Samples Drug free human serum was obtained from the Iranian blood transfusion service (Tehran, Iran) and stored at -20 °C until use after gentle thawing. Urine was also collected from healthy volunteers (males, around 35-years-old). The samples were centrifuged for 20 min at 8000 rpm and then filtered through a cellulose acetate filter (0.20 μm pore size, Advantec MFS Inc. CA, USA). The filtrates were collected in glass containers and stored at -20 °C until analysis was performed, with the minimum possible delay. Stock standard solutions of VPM were prepared in water. Standard solutions were prepared by adding appropriate volumes of VPM solution to a 5 mL volumetric flask and the solution was diluted to the mark with biological fluids and vortexed for 3 minute.
Then the solution was adjusted to pH=8.5 and the analysis was followed up as indicated in the general analytical procedure. RESULTS AND DISCUSSION Optimization of MIP Formulations There are several variables, such as amount of monomer or nature of cross-linker and solvent that affect the final characteristics of the obtained materials in terms of capacity, affinity, and selectivity for the target analyte. Primary experiments revealed that the imprinted polymers prepared in chloroform show better molecular recognition ability than acetonitrile (AN) in aqueous environment. Thus, in chloroform, different formulations for the obtainment of MIPs with improved molecular recognition capabilities have been used (Table 1). Generally, proper molar ratios of functional monomer to template are very important to enhance specific affinity of polymers and number of MIPs recognition sites. High ratios of functional monomer to template result in high non-specific affinity, while low ratios produce fewer complexation due to insufficient functional groups [26]. Five molar ratios of the monomer MAA to the template of 6:1, 13:1, 23:1 and 34:1 were used in the experiments. The optimum ratio of functional monomer to template for the specific rebinding of VPM was 13:1 (Table 1), which had the best specific affinity and the highest recovery of 99%, while that of the corresponding NIPs had low recovery at 32%. The specific adsorption recovery of VPM at 13:1 was 67%, while those at 6:1, 23:1 and 34:1 were 34%, 25% and 15%, respectively. For the polymers with a ratio of 23:1 and 34:1, an excess of the functional monomer with respect to the template yielded higher non-specific affinity. Therefore, the typical 1:13:63 template:monomer:cross-linker molar ratio was used for further studies.
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Fig. (4). Effect of time on sorption of VPM on imprinted polymer particles. Fig. (3). Effect of pH on sorption of VPM on imprinted polymer particles.
Effect of pH The effect of pH on the sorption of VPM was investigated by varying the solution pH from 2.0 to 10.0. Several batch experiments were performed by equilibrating 50 mg of the imprinted particles with 25 mL of solutions containing 0.1 mM of VPM under the desired range of pH. The pH dependence of extracted percentage of VPM is shown in Fig. (3). As seen, binding of VPM increased with the increasing pH and reached to maximum at pH of 8.0–9.5. Since the pKa-value of the verapamil is about 9 [27], at low pHs, the nitrogen hetero-atoms can be protonated and, therefore, negligible amounts of VPM are adsorbed to the polymer. The Effect of the Extraction Time The effect of the extraction time on the efficiency of the extraction was investigated and the results are depicted in Fig. (4). As it is seen, the time of the extraction from 5 to 60 min. has not any significant effect on extraction efficiency of the VPM. Choice of Washing and Eluent Solution Optimization of the washing procedure is critical in MISPE. The selectivity of MISPE is generally obtained by the introduction of a selective washing procedure in order to remove the compounds retained only by non-specific interactions. A VPM solution (50 mL of 0.10 mM) was loaded on MIP and NIP. For the washing step, 2 mL water plus 2 mL acetone eliminated VPM from NIP by suppressing of nonspecific interactions (Table 2). These washing solutions are able to disrupt Van der Waals interactions and, probably, a part of the hydrogen bonds thanks to their hydrogen bond donor properties. Finally, in order to choose the most effective eluent for desorbing VPM from the sorbent, a series of selected eluent solutions such as methanol, ethanol and acetonitrile (10:1, v/v, of eluent and pure acetic acid) a total of 5.0 mL of the above mentioned eluents were used for desorb-
ing the adsorbed VPM. The results showed that recovery was the best (97 %) when mixed solution of methanol and acetic acid (10:1, v/v) was used as eluent (Table 2). Indeed, MeOH and acetic acid are protic and polar solvents able to break hydrogen bonds between functional groups of VPM and carboxyl groups present in MIP cavities. Effect of Amount of MIP In order to investigate the optimum amount of MIP on the quantitative extraction of VPM, the extraction for a series of 50 mL of 0.1 mM VPM solutions was conducted by varing the amount of the MIP from 50 to 200 mg. The results showed the extraction of VPM is quantitative by using 90 mg of MIP. Adsorption Capacity The capacity of the sorbent is an important factor that determines how much sorbent is required to remove a specific amount of drug from the solution quantitatively. For investigation of adsorption of VPM, the same volumes of VPM solution (50 mL) with different concentrations of VPM were contacted with 100 mg of sorbent in the batch mode. Then, the concentration of the remaining VPM in the solution was determined by UV. The adsorption isotherm that is the number of milligram adsorbed per gram of adsorbent (N) versus the equilibrium concentration of VPM is shown in Fig. (5). According to these results, the maximum amount of VPM that can absorb by MIP was found to be 196 mg g-1 (400 μmol g-1) at pH 8.5. Taking into account templatemonomer ratio during MIP preparation, the theoretical number of imprints was 1.4 mmol per gram of polymer. However, experimental data showed strong specific retention of VPM (400 μmol per gram of MIP), which means that approximately thirty percent of the theoretical number of sites was formed. Such behavior is coherent with previous data [28, 29] with MIP capacities ranged from 50 to 500 μmol per gram. For higher VPM amounts (higher than 400 μmol g-1), a slight increase of retained VPM was observed on MIP capacity curve. As all the accessible specific cavities of the MIP are saturated, the retention of the analyte is only due to non specific interactions which can be identical for MIP and NIP polymers.
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most commonly used methods to investigate the selectivity of the imprinted materials [30]. For equilibrium batch rebinding experiments, a known mass of template in solution is added to a vial containing a fixed mass of polymer. Once the system has come to equilibrium, the concentration of free template in solution is measured and the mass of template adsorbed to the MIP is calculated [13, 31]. The initial concentrations of 50 mL of the drugs (100 μM) were extracted by 100 mg of imprinted material at a pH of 8.5 on MIP and NIP. As can be seen in Table 3, distribution ratio (KD) and selectivity coefficient of the sorbent (ksel) were obtained in these competitive experiments. The distribution ratio (mL g-1) of VPM between the MIP particles and aqueous solution was determined by the following equation:
KD = Fig. (5). Curve of capacity obtained after the loading of 5 mL aqueous solution spiked with increasing amounts of VPM onto the MIP particles.
(Ci Cf ) V Cf m
Where V is the volume of initial solution and m is the mass of MIP materials. Selectivity coefficients for VPM ion relative to foreign compounds are defined as:
Calibration Curve and Precision Under optimized conditions, calibration curves were obtained by the MISPE protocol measured at ten increasing concentrations, in a range from 1 to 1000 μM of VPM. The results showed good linearity (r=0.9983) in the dynamic range of 5.0–500 μM. The results confirmed that the SPE method based on MIP beads could be directly applied to real sample analysis. According to signal-to-noise relation rule equal to 3.0, the limit of detection for VPM was 3 μM. The precision of the method were assessed by performing replicate analyses of quality control samples at three different concentrations of VPM (10, 50 and 100 μM) in four replicates at the same day and consecutive days. The results showed that the intra and inter-assay relative standard deviations of the proposed method were lower than 3.4% and 7.2%, respectively. Study of MIP Selectivity
(2)
k sel VPM j =
K VPM D K Dj
VR
(3) j
Where K D and K D are the distribution ratios of VPM and foreign compound, respectively. The previous MISPE protocol was applied also to these drug molecules on NIP particles. The relative selectivity coefficient ( k ) was also determined by following equation:
k =
k(MIP) k(NIP)
(4)
In the case of VPM, a quantitative extraction and an excellent MIP/NIP selectivity were obtained. ANALYTICAL APPLICATIONS Vrapamil Assay in Spiked Human Serum and Urine
The MIPs are usually evaluated to check theirs recognition properties for a target analyte. Chromatographic evaluation and equilibrium batch rebinding experiments are the
The extraction procedure has to be optimized in order to eliminate low energy interactions at the surface without
Table 2. Recoveries (%) Obtained from After the Loading of 100 mg of MIP and NIP with 5 μmol of VPM Recovery %
a
No.
Fraction
MIP (Mean ± SD)a
NIP (Mean ± SD)a
1
Washing 1, 2 mL, water
Not detected
8 ± 1.1
2
Washing 2, 2 mL, acetone
4 ± 1.0
14 ± 1.4
3
Elute, 5 mL, methanol and pure acetic acid (10:1, v/v)
97 ± 2.0
10 ± 1.5
4
Ethanol and pure acetic acid (10:1, v/v)
95 ± 2.2
14 ± 1.5
5
Acetonitril and pure acetic acid (10:1, v/v)
91 ± 2.4
14 ± 2.2
Total (Nos. 1, 2 and 3)
101 ± 2.2
32 ± 2.3
Average of three determinations.
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Table 3. Distribution Ratio (KD), Selectivity Coefficient (ksel) and Relative Selectively Coefficient (k') Values of MIP and NIP Material For Different Drugs
Drug
KD(MIP) (mL g-1)
Verapamil
16167
Dextromethorphan
KD(NIP)
ksel(MIP)
ksel(NIP)
k'
81.4
---
---
---
88.2
55.5
183.2
1.46
125
Metoclopramide
171.1
92.4
94.4
0.88
73
Prometazin
125
102.4
129.3
0.79
164
Terazosin
141
125
114.6
0.65
176
Salbutamol
115.7
68.8
139.7
1.18
118
Methyldopa
55.5
40.5
291.2
2.0
146
Norverapamil
2830
76.2
5.7
1.07
5.3
(mL g-1 )
Table 4. Assay of VPM in Human Serum and Urine by Means of the Described MISPE Procedure and the HPLC Method [13]
Sample
Human Serum
Human Urine
a
Spiked Value
Proposed SPE Procedure (Recovery% ±SD)a
(μM)
HPLC (Recovery% ±SD)a
MIP
NIP
10
100.1 (± 3.5)
15.5 (± 2.1)
98.3 (± 2.9)
50
104 (± 5.3)
10.8 (± 1.1)
---
100
102 (± 4.2)
12.2 (± 1.5)
---
10
95.5 (± 4.6)
15.2 (± 1.6)
---
50
98.2 (± 4.7)
17.1 (± 1.5)
101.4 (± 3.4)
100
100.2 (± 4.1)
19.3 (± 1.9)
---
Average of four determinations.
damaging specific interactions taking place in the cavities and that are of stronger energy due to the spatial recognition. To evaluate the risk of non-specific interactions with the external surface of the MIP, the procedure of extraction has to be tested in parallel with the NIP. Therefore, the procedure should be based on the use of a solvent for the percolation step and/or the washing step that possess an elution strength sufficiently high to disrupt the interactions that can take place with residual monomers at the surface of the polymer without affecting the overall retention in the imprints. Thus, the proposed SPE procedure was successfully applied to assay of VPM in spiked human serum and urine. The obtained results (Table 4) were statistically compared with those obtained by high performance liquid chromatography using ultraviolet detection [32]. No significant difference was noticed between the two methods regarding accuracy and precision as revealed by t-value [33]. CONCLUSIONS In this paper, water-compatible molecularly imprinted polymers were synthesized via a non-covalent molecular imprinting approach in chloroform for selective extraction of VPM from biological fluids and human urine samples.
Molecular recognition properties, binding capability, and analytical applications of the MIPs were evaluated and the results revealed that the obtained MIPs have high affinity for VPM in aqueous environment without any relevant pretreatment of the sample. The development of a fast, accurate and selective analytical method in this study provides a strategy the for development of drug MIPs and related analytical means for further drug determination in biological samples. REFERENCES [1] [2]
[3]
[4] [5]
Komiyama, M.; Takeuchi, T.; Mukawa, T.; Asanuma, H.; Molecular imprinting: from fundamentals to applications, John Wiley & Sons, 2003. Ye, L.; Haupt, K. Molecularly imprinted polymers as antibody and receptor mimics for assays, sensors and drug discovery. Anal. Bioanal, Chem., 2004, 378(8), 1887-1897. Turiel, E.; Martin-Esteban, A. Molecularly imprinted polymers: Towards highly selective stationary phases in liquid chromatography and capillary electrophoresis. Anal. Bioanal. Chem., 2004, 378, 1876-1886. Alexander, C.; Davidson, L.; Hayes, W. Imprinted polymers: Artificial molecular recognition materials with applications in synthesis and catalysis. Tetrahedron, 2003, 59, 2025-2057. Martin-Esteban, A.; Fresenius, J. New molecular recognition materials for selective solid-phase extraction of organic compounds. Anal. Chem., 2001, 370, 795-802.
276 Current Pharmaceutical Analysis, 2009, Vol. 5, No. 3 [6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14] [15] [16]
[17]
[18]
[19]
Javanbakht et al.
Caro, E.; Marce, R.M.; Borrull, F.; Cormack, P.A.G.; Sherrington, D.C. Application of molecularly imprinted polymers to solid-phase extraction of compounds from environmental and biological samples. Trends. Anal. Chem., 2006, 25, 143-145. Pichon, V.; Haupt, K. Affinity Separations on molecularly imprinted polymers with special emphasis on solid-phase extraction J. Liq. Chromatogr. Relat. Technol., 2006, 29, 989-1023. Tamayo, F.G.; Turiel, E.; Martin-Esteban, A. Molecularly imprinted polymers for solid-phase extraction and solid-phase microextraction: Recent developments and future trends. J. Chromatogr. A, 2007, 1152, 32-40. Javanbakht, M.; Eynollahi Fard, S.; Mohammadi, A.; Abdouss, M.; Ganjali, M.R.; Norouzi, P.; Safaraliee, L. Molecularly imprinted polymer based potentiometric sensor for the determination of hydroxyzine in tablets and biological fluids. Anal. Chim. Acta, 2008, 612, 56-74. Javanbakht, M.; Eynollahi Fard, S.; Abdouss, M.; Mohammadi, A.; Ganjali, M.R.; Norouzi, P.; Safaraliee, L. A biomimetic potentiometric sensor using molecularly imprinted polymer for the cetirizine assay in tablets and biological fluids. Electroanalysis, 2008, 20, 2023-2030. Mullett, W.M.; Walles, M.; Levsen, K.; Borlak, J.; Pawliszyn, J. Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry. J. Chromatogr. B, 2004, 801, 297-306. Patel, N.P.; Pugh, M.E.; Goldberg, S.; Eiger, G. Hyperinsulinemic euglycemia therapy for verapamil poisoning: Case Report. Am. J. Crit. Care., 2007, 16, 498-503. Eisenberg, M.J.; Brox, A.; Bestawros, A.N. Major side effects and safety of calcium channel blockers. Am. J. Med., 2004, 116, 35-43. British Pharmacopoeia, H.M. Stationery Office, London, 1998, Vol. I, pp. 1353-1354. The United States Pharmacopoeia, National Formulary 18, The United States Pharmacopoeial Convention, XXIII ed, USA, Rockville, MD, 1995, pp. 1623-1626. Ozkan, Y.; Yilmaz, N.; Ozkan, S.A.; Biryol, I. High performance liquid chromatographic assay and drug dissolution studies of fluoxetine hydrochloride in capsule formulations. J. Liq. Chromatogr. Relat. Technol., 2000, 23, 1699-1710. ElGhany, M.F.A.; Moustafa, A.A.; ElZeany, B.E.; Stewart, J.T. High performance thin-layer chromatographic analysis of verapamil hydrochloride in drug substance and dosage forms. J. Planar Chromatogr. Mod. TLC, 1996, 9, 388-390. Gumieniczek, A.; Hopkala, A. Development and validation of a liquid choromatographic method for the determination of trandolapril. J. Liq. Chromatogr. Relat. Technol., 2001, 24, 393-400. Walash, M.; Belal, F.; El-Enany, N.; Abdelsalam, A. Spectrofluorometric determination of verapamil hydrochloride in pharmaceutical preparations and human plasma using organized media: application to stability studies. J. AOAC Int., 2006, 89, 1565-1572.
Received: 30 September, 2008
[20]
[21]
[22]
[23]
[24]
[25] [26] [27]
[28]
[29]
[30]
[31] [32]
[33]
Xu, D.; Liu, S.; Liu, Z.; Hu, X. Determination of verapamil hydrochloride with 12-tungstophosphoric acid by resonance Rayleigh scattering method coupled to flow injection system. Anal. Chim. Acta, 2007, 588, 10-15. Kasim, E.A.; Ghandour, M.A.; El-Haty, M.T., Ahmed, M.M. Determination of verapamil by adsorptive stripping voltammetry in urine and pharmaceutical formulations. J. Pharm. Biomed. Anal., 2002, 30, 921-929. Khalil, S.; Kelzieh, A. Determination of verapamil in pharmaceutical formulations using atomic emission spectrometry. J. Pharm. Biomed. Anal., 2002, 27, 123-131. Asafu-Adjaye, E. B.; Shiu, G. K. Solid-phase extraction-highperformance liquid chromatography determination of verapamil and norverapamil enantiomers in urine. J. Chromatogr. B., 1998, 707, 161-167. Cheng, Y.F.; Neue, U.D. Bean, L. Straightforward solid-phase extraction method for the determination of verapamil and its metabolite in plasma in a 96-well extraction plate. J. Chromatogr. A, 1998, 828, 273-281. Anthony C.M. Clarke,s Analysis of drugs and poisons in pharmaceuticals, Third ed., Pharmaceutical Press, 2004, Vol. 2, p. 1659. Rachkov, A.; Minour, N. Molecular imprinting for the recognition of N-terminal histidine peptides in aqueous solution. J. Chromatogr. A, 2000, 889, 111-118. Walles, M.; Mullett, W.M.; Levsen, K.; Borlak, J.; Wünsch, G.; Pawliszyn, J. Verapamil drug metabolism studies by automated intube solid phase microextraction. J. Pharm. Biomed. Anal. 2002, 30, 307-319. Zi-Hui, M.; Qi, L. Determination of degradation products of nerve agents in human serum by solid phase extraction using molecularly imprinted polymer. Anal. Chim. Acta, 2001, 435, 121-127. Cacho, C.; Turiel, E.; Matrin-Esteban, A.; Perez-Conde, C.; Camara, C. Characterisation and quality assessment of binding sites on a propazine-imprinted polymer prepared by precipitation polymerization. J. Chromatogr. B, 2004, 802, 347-353. Martin, P.; Jones, G.R.; Stringer, F.; Wilson, I.D. Comparison of normal and reversed-phase solid phase extraction methods for extraction of -blockers from plasma using molecularly imprinted polymers. Analyst, 2003, 128, 345-350. Shea, K.J.; Spivak, D.A.; Sellergre, B. Influence of polymer morphology on the ability of imprinted network polymers to resolve enantiomers. J. Am. Chem. Soc., 1993, 115, 3368-3369. Garcia, M.A.; Aramayona, J.J.; Bregante, M.A.; Fraile. L.J.; Solans, C. Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection. J. Chromatogr. B, 1997, 693(2), 377382. Miller, J. N.; Miller, J. C. Statistics and Chemometrics for Analytical Chemistry, Prentice Hall, England, 2000, Fourth Edition, 40.
Revised: 30 October, 2008
Accepted: 01 December, 2008
