Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine.

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Journal of Separation Science

Water-compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine Mojgan Pourfarziba,c, Rasoul Dinarvandc, Behrouz Akbari-adergania, Ali Mehramizib, Hossein Rastegar a , Maryam Shekarchia*

a

Water Safety Research Center, Food and Drug Laboratory Research Center, Food and Drug Control Laboratories, MOH ME, Tehran, Iran. b Tehran Chemie Pharmaceutical Co., Tehran, Iran. c Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Mojgan pourfarzib: [email protected] Rasoul Dinarvand: [email protected] Behrouz Akbari-adergani: [email protected] Ali Mehramizi: [email protected] Hossein Rastegar: [email protected]

Corresponding author*: Maryam Shekarchi No 31, Imam Khomeini St., Food and drug Control Laboratories, Tehran, Iran. P.O. Box: 1113615911 Fax: +98-21-66404330 Email: [email protected] Tel: +98-912-2613640

Received: 30-Dec-2014; Revised: 26-Feb-2015; Accepted: 28-Feb-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201401492. This article is protected by copyright. All rights reserved.

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Abstract A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid-phase extraction before highperformance liquid chromatography with UV analysis. The imprinted polymers were prepared in bulk polymerization by non-covalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross-linker and chloroform as porogen. Molecular recognition properties, binding capacity and selectivity of the molecularly imprinted polymers were evaluated and the results represented that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high-performance liquid chromatography showed a linear calibration curve in the range of 20–100 µg/L with excellent precisions (2.5 and 2.8% for 50 µg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 µg/L), and urine (5.45 and 16 µg/L). The recoveries for serum and urine samples were found to be 88.2–93.5% and 84.3–90.2%, respectively. Keywords: Adefovir; Human serum; Molecularly imprinted polymers; Urine

This article is protected by copyright. All rights reserved.

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1 Introduction Adefovir dipivoxil (ADV) belongs to the group of dideoxynucleoside reverse transcriptase inhibitors licensed for the treatment of viral infection chronic hepatitis B (HBV), which has been used with higher oral bioavailability against both negative and positive hepatitis B antigen. All nucleoside analogs are competitive inhibitors of the viral polymerase as they compete with the incorporation of the natural endogenous intracellular nucleotides in nascent viral DNA [1, 2]. The acidic condition of gastrointestinal tract hydrolyzed ADV to adefovir following transportation into cells and conversion to active adefovir diphosphate (12–36 h intracellular half-life)[3,4]. After oral administration of single dose (10 mg) of ADV to patients with HBV, the mean maximum concentration (Cmax) of drug in plasma was 18.4±6.26 ng/mL, and the steady urinary recovery was approximately 45.3% [5]. Since ADV is widely used in the antiviral therapy, it is important to develop and validate analytical methods for its determination in biological fluids. Adefovir (Fig.1) has been measured by different methods such as UV/vis spectrophotometry [6], ion-pair LC with fluorescence detection [7], HPLC with MS/MS or UV detection [5,8–10] and voltammetry [11]. Some of these methods involve many derivatization and pretreatment procedures such as plasma protein precipitation or LLE before final analysis which are very time consuming and the demand of expensive, sophisticated instrumentation and highly skilled personnel restrict their use in routine analysis. SPE is nowadays routinely established as clean up techniques for the target enrichment and clarification to assist analytical quantification. Compared to LLE, SPE can decrease the time required, simplify the automation, can handle little samples and low solvent consumption. Due to high load, high recovery, better reproducibility and wide spectrum of stationary phases SPE is the most popular sample pretreatment techniques today [12–14]. A relatively new improvement in the area of SPE is


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the use of molecularly imprinted polymers (MIPs) for the sample clean up [15–17]. MIPs are synthetic polymers having specific cavities planned for a template molecule which are prepared by copolymerization of a functional monomer with a crosslinker in the presence of target analyte to produce a three-dimensional polymer network. In the most common preparation procedure, monomers make a complex with a template throughout covalent or non-covalent interactions. The imprinted polymers based on non-covalent interactions between print molecules and suitable monomers have been widely applied. An ideal MIP adsorbent should have the following characteristics: high binding affinity, specificity and sample load capacity, physical robustness, fast association and dissociation kinetics, broad solvent compatibility, long-term stability to extremes of pH, organic solvents and heating which permit for more flexibility in the analytical methods [18]. The use of MIPs for SPE can include different forms, with conventional SPE where the MIP is filled into columns or cartridges [19, 20] and batch mode SPE where the MIP is equilibrated with the sample [21]. In our previously published papers, the technique of molecular imprinting has been applied with success to the preparation of high affinity SPE for bromhexine [22], metoclopramide [23], tramadol [24], dextromethorphan [25], penicillin G [26], carbamazepine [27], dipyridamole [28], lamivudine[29], and efavirenz [30]. Herein we present a simple and straightforward method for the performance evaluation of adefovir based MIPs as selective sorbents for efficient sample clean-up and further determination of adefovir from biological matrices by HPLC. This scheme as molecularly imprinted polymers permits the sensitive, uncomplicated and inexpensive separation and determination of the analyte in human serum and urine samples which can be adopted by pharmaceutical laboratories for industrial QC.


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2 Materials and methods 2.1 Chemicals and reagents

Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) from Merck Darmstadt, Germany) were distilled in vacuum before use to remove the stabilizers. 2,2Azobisisobutyronitrile (AIBN) was purchased from Merck (Darmstadt, Germany) and recrystallized from ethanol. Water was obtained from a Milli-Q purification system (Purelab UHQ Elga). All solvents used in chromatography were HPLC grade and obtained from Merck (Darmstadt, Germany). Adefovir, acyclovir, lamivudine and tenofovir reference standards were supplied from Hetero drug limited (India, Hyderabad). 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 samples were collected from healthy volunteers. The adefovir stock solution as standard solution (10 mg/L) was prepared in acetonitrile monthly and stored at 4°C. Intermediate standard solution (1 mg/L) was prepared weekly by dilution of stock solutions. Working standard of different concentrations (20–80 µg/L) for serum and urine were prepared daily by diluting the intermediate standard solution with phosphate buffer pH=7. 2.2 Instrumentation

FTIR spectra of the ground polymer were recorded on a Bomem FTIR MB 155S spectrometer (Canada) using KBr pellets in the range of 400–4000 cm−1. In all solutions the pH was adjusted by digital Metrohm pH meter (model 744) equipped with a combined glass– calomel electrode. The HPLC experiment was performed using a Waters Alliance system equipped with a vacuum degasser, quaternary detector. The UV spectra were collected across the range of 200–900 nm, extracting 270 nm for chromatograms. Empower software was utilized for instrument control, data collection and data processing. The column was Lichrospher 5, C18 (4.6 mm × 100 mm). The mobile phase was an isocratic mode of


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acetonitrile: phosphate buffer pH 5.2 (6:94) at a flow rate of 1.5 mL/min. The injection volume for all samples and standards was 100 µL [9]. Particle size analysis of polymers performed by photon correlation spectroscopy (Malvern Zetasizer ZS, Malvern, UK). Particles suspended in deionized water and sonicated for 20 min until no aggregated particles observed. The colloid samples measured at a fixed scattering angle of 90° at 25°C. Morphology of polymers characterized by SEM (Philips XL30 scanning microscope, Philips, Netherlands). Samples were sputter coated with gold before the SEM measurement. 2.3 Preparation of the imprinted polymer

The monomer MAA (340µl, 4 mmol), adefovir print molecule (136.6 mg, 0.5 mmol) and 10mL of chloroform were placed in a screw-capped glass vial and shake for 2 h. Then crosslinker EGDMA (3.016 mL, 16 mmol) was added. The mixture was uniformly dispersed by sonication and the reaction initiator AIBN (57 mg, 0.347 mmol) was added. The mixture was purged with a stream of nitrogen for 10 min and the glass vial was sealed under this atmosphere. The polymerization was performed for 24 h under a UV wavelength 366 nm and then for 24 h in water bath 40°C. After the polymerization procedure, the hard polymers, poly (MAA-co-EGDMA) were crushed and dried. By repeated sedimentation in acetone, the overly small particles were discarded. To remove the template and nonreactive residues, the polymer was moved to a Soxhlet apparatus and refluxed over the mixture of methanol and acetic acid (9:1 v/v, of 98% methanol and glacial acetic acid) for 24 h until the absorbance of analyte had been no longer detected in the elution by UV spectrophotometer. Non-imprinted polymer (NIP) was prepared simultaneously as the same procedure without target molecule (adefovir). The mean MIP particle size that was measured by Zetasizer was about 356 nm.


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2.4 Rebinding experiments

Batch adsorption experiments were used to estimate the binding capacity of the imprinted polymer as reported before [31, 32]. A small amount (40 mg) of MIP particles was thoroughly mixed with 5 mL adefovir (200 mg/L) solution in phosphate buffer pH=7 and thermostatted at 25°C overnight under continuous stirring. After the binding process was completed, the mixture was centrifuged for 20 min at 9000 rpm and used the clear supernatant. The free concentration of adefovir after rebinding was determined by HPLC–UV at 260 nm. Three replicate extractions and measurements were performed for each concentration. The amount of adefovir bound to the polymers (MIP and NIP) was calculated by subtracting the concentration of free adefovir from the initial concentration. After washing with the mixture of acetonitrile/acetone (4:1, v/v), the rebound adefovir was eluted from the MIP with 5 mL of methanol/acetic acid (9:1, v/v) under 20 min stirring at 600 rpm in room temperature. 2.5 Isotherm studies

Isotherm studies were performed by adding a fixed amount of sorbent (0.02 g) to a series of test tubes filled with 3 mL diluted solutions of adefovir (10–100 µg/mL). The test tubes were then sealed and shaken for 60 min at optimum pH and 25°C. The test tubes were then centrifuged, and the final concentration of adefovir in the supernatant was measured by HPLC. The amount of adefovir at equilibrium qe (mg/g) was calculated from the following equation (1): qe = (Ci –Ce)V/W (1) where Ci and Ce (mg/L) are initial and equilibrium concentrations of adefovir, respectively; V (L) is the volume of the solution and W (g) is the mass of the adsorbent used.


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2.6 Extraction procedure for serum and urine samples

Adefovir standard stock solution (10 mg/L) was prepared in acetonitrile. Standard solutions were prepared by adding proper volumes of adefovir solution to 10 mL volumetric flasks and the solutions were diluted to the mark with biological fluids and shaken for 10 min. The spiked serum and urine samples (2 mL) were diluted with buffer pH 7 (4 mL), centrifuged for 30 min at 9000 rpm and then filtered through a cellulose acetate filter (0.2 µm pore size, Advantec MFS, CA, USA). Finally 5 mL of the filtrates (20, 50, 80 µg/L) were directly loaded on to the MIP and NIP. 3 Results and discussion 3.1 Characterization studies 3.1.1 IR spectra

The IR spectra of the imprinted poly (MAA-co-EGDMA) after and before eluting template (Fig. 2) with similar typical peaks, indicated the characteristics in the backbone structure of the different polymers. The formation of hydrogen binding decreased the electric cloud density of OH and C=O and resulted in the decrease in frequency of vibration. As a result of this fact the C=O stretching, the O–H stretching and the bending vibrations at 1728 and 3456 cm⁻¹ in the leached MIP materials were shifted to 1720 and 3448 cm⁻¹ in the related unleached MIP, respectively. In addition, there were two other separate differences between the IR spectra of the MIPs before and after elution. In the unleached polymer, there were one sharp band with low relative intensity at 1458 cm⁻¹ and one band with high relative intensity at 2954 cm⁻¹ that was presented at 1465 and 2939cm⁻¹ in the same leached MIP, respectively. Other absorption peaks go with both those of MIP: 1636 cm⁻¹ (stretching vibration of residual vinylic C–C bonds), 1257, 1157 cm⁻¹ (symmetric and asymmetric ester C–O stretch bands) and 948 cm⁻¹ (out-of-plane bending vibration of vinylic C–H bond).


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3.1.2 SEM analysis

Analysis of the SEM images indicated that MIP and NIP exhibited no detectable differences in appearance. As it is clear the coagulated small polymer particles have been formed. On the basis of SEM microphotographs polymer particles in the case of both MIP and NIP demonstrated irregular shapes with heterogeneous surfaces. A porous surface could be manifestly observed for the leached MIP due to template elution from cavities. 3.2 Optimization of MIP formulations

There are several variables, such as type and amount of monomer or nature of cross-linker and solvent that affects the final characteristics of the obtained polymers in terms of capacity and selectivity for the target analyte. The structure of adefovir (Fig 1) has both amino and carbonyl groups which theoretically make it an ideal compound to interact with both basic (4vinylpyridine, VP and acrylamide, AA) and acidic (methacrylic acid, MAA) monomers. The MIP using methacrylic acid as monomer showed higher recognition ability to the target molecule than the MIPs prepared using acrylamide and 4-vinylpyridine because methacrylic acid has stronger electrostatic and hydrophobic interactions with the target in a polar environment. Due to the effect of polymerization media polarity on the strength and number of interactions and the recognition of obtained MIPs, the influence of the porogen used in the polymerization was also investigated. The main experiments revealed that the imprinted polymers prepared in chloroform rather than dimethylformamide, acetonitrile, tetrahydrofurane and dichloromethane show better molecular recognition in aqueous environment. Generally, correct molar ratios of functional monomer to template are very significant to improve specific affinity of polymers and number of MIPs recognition sites [33]. High ratios of functional monomer to template result in high non-specific affinity, while low ratios create less complexation due to insufficient functional groups. The rebinding (extraction) values and ratio range of MIPs were estimated (Table 1). For the highest specific rebinding of adefovir, the optimum molar ratio of the functional monomer to the template was 4: 0.5 (Table 1), which had the best specific affinity and the highest retention and recovery about 90.5%, while the other molar ratios at 4:0, 4:1 and 4:1.5 were 10.5, 56 and 43%, respectively. Therefore, a typical 0.5:4:16 template/monomer/cross-linker molar ratio was used for more studies.


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3.3 Effect of pH

The pH of the solution is one of the most important variables affecting retention behavior and selectivity of the MIPs as a consequence of hydrophobic interactions. Especially, the washing and elution conditions need to be carefully optimized in terms of pH, ionic strength and solvent composition to fully promote the MIPs ability to recognize adefovir. Several batch experiments were carried out by equilibrating 40 mg of the imprinted and non-imprinted polymers with 5 mL of solutions containing 200 mg/L of adefovir under the preferred range of pH (3.0–9.0). According to the results, adefovir undergoes complete rebinding at pH 7.0. The lower reactions observed at lower and higher pH, may be produced by the protonation of the amine group of adefovir and deprotonation of carboxyl groups of the polymer, respectively. 3.4 Isotherms studies 3.4.1 Langmuir Isotherm

The Langmuir isotherm assumes monolayer adsorption on a uniform surface with a limited number of adsorption sites. Once a site is in use, no further sorption can take place at that site. As such the surface will finally reach a saturation point where the maximum adsorption of the surface will be achieved. The isotherm equation further supposes that adsorption takes places at specific homogeneous sites within the adsorbent. Moreover, the Langmuir equation is based on the assumption of an adsorbent of homogeneous structure where all adsorption sites are identical and energetically equivalent. The experimental isotherm data (qe and Ce) of adefovir on MIP at 25°C were successfully fitted to the Langmuir isotherm to evaluate the qmax and KL values. Isotherm parameters calculated from following equation (2) [34]: Ce/qe = (1/qmax . KL) + (Ce/qmax) (2) Where qmax is the maximum adsorption capacity corresponding to complete monolayer coverage on the surface (mg/g), KL is the Langmuir constant (L/mg). The Langmuir This article is protected by copyright. All rights reserved.

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adsorption isotherm is one of the most famous and widely-used adsorption isotherms. Langmuir isotherms were used for the determination of sorbent capacity defined as the amount of adefovir adsorbed by one gram of sorbent. A plot of Ce/qe versus Ce showed a linear relationship (Fig. 3A), and Langmuir constants qmax and KL (it is a measure of the energy of sorption) can be calculated from the slope and intercept of the plot. The maximum monolayer capacity, qmax, and the Langmuir constant, KL (L/mg) were calculated from the Langmuir equation as 8.5178 mg/g and 0.02816 L/mg, respectively. The essential characteristics of the Langmuir isotherm parameters can be used to predict the affinity between the sorbate and sorbent using separation factor or dimensionless equilibrium parameter, RL expressed as in the following equation (3): RL =1/ (1+KL .C0) (3) Where KL is the Langmuir constant and C0 is the initial concentration of adefovir. The value of separation parameter RL provides important information about the nature of adsorption. The value of RL indicated the type of Langmuir isotherm to be irreversible (RL = 0), favorable (0 1). The RL was found to be 0.7635–0.2621 for concentration of 10–100 mg/L of adefovir. 3.4.2 Freundlich Isotherm

The Freundlich isotherm model is the earliest relationship to describe the adsorption process. This model applies to adsorption on heterogeneous surfaces with the interaction between adsorbed molecules and the application of the Freundlich equation also suggests that sorption energy exponentially decreases on completion of the sorption centers of an adsorbent. It describes reversible adsorption and is not restricted to the formation of a monolayer. This isotherm is an empirical equation and can be employed to describe heterogeneous systems and is expressed as follows in linear form [35]: ln (qe) = ln(KF)+ 1/n ln (Ce) (4)


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where KF is the Freundlich constant related to the bonding energy. 1/n is the heterogeneity factor and n (g/L) is a measure of the deviation from linearity of adsorption. The Freundlich adsorption isotherm represents the relationship between the amount of drug adsorbed per unit mass of adsorbent (qe) and the concentration of the adefovir in solution at equilibrium (Ce). Freundlich equilibrium constants were determined from the plot of ln qe versus ln Ce, (Fig. 3B) on the basis of the linear of Freundlich equation. The n value indicates the degree of nonlinearity between solution concentration and adsorption as follows: if n = 1, then adsorption is linear; if n < 1, then adsorption is a chemical process; if n > 1, then adsorption is a physical process. The n value and Freundlich constant in Freundlich equation was found to be 1.6171 and 0.446 mg/g for adefovir. The values of regression coefficients R2 are regarded as a measure of goodness of fit of the experimental data to the isotherm models. 3.5 Study of MIP selectivity

Chromatographic analysis and equilibrium batch rebinding experiments are the methods most commonly used to consider the selectivity of the imprinted materials [36]. For equilibrium batch rebinding experiments, a known amount of template in solution is added in 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 amount of template adsorbed to the MIP determined. The first concentrations of drugs (100 mg/L) were extracted by 50 mg of imprinted material at pH of 7 on MIP and NIP. The distribution ratio (ml /g) of adefovir between the MIP particles and aqueous solution was evaluated by following equation (5): KD = (Ci− Cf)V /Cf m (5) where V is the volume of initial solution, m is the mass of MIP materials, Ci is the initial concentration in solution and Cf is the free concentration of drug in supernatant after the rebinding experiment. Selectivity coefficients for adefovir relative to foreign compounds are defined as


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ksel Adefovir = KAdefovirD /KjD (6) where KAdefovirD and KjD are the distribution ratios of adefovir and foreign compound, respectively. The relative selectivity coefficient (k') was also determined by following equation (7): k' = ksel(MIP) /ksel(NIP) (7) The selectivity tests of MIP were performed using tenofovir, lamivudine and acyclovir (Fig. 1). Distribution ratio (KD), selectivity coefficient (k sel) and relative selectivity coefficient (k') values of MIP and NIP materials for these different drugs are listed in Table 2. According to the data shown in Table 2, MIP had higher k' values (5.1) for acyclovir than lamivudine (2.63) and tenofovir (2.85). For adefovir, this specific selectivity was essentially due to the predicted hydrogen bonding interactions and the molecular size recognition. For other antiviral drugs, the selectivity was caused by their similar molecular size and identical hydroxyl, amino groups with the template to form the hydrogen bonding interactions with the carboxylic groups at the expected position in the MIP. 3.6 Adefovir assay in human serum and urine samples

To display the potential of MIP for the selective clean up of analyte, the MIP was appropriated to the purification of spiked adefovir in human serum and urine (20, 50, 80 µg/L). Aqueous media with pH=7 was used for the loading solution and the wash process was evaluated for getting maximum recovery of adefovir using acetonitrile/acetone (4:1, v/v). The extracted fractions were analyzed by RP-HPLC and the chromatograms were recorded (Fig. 4A, 4B). As shown in these figures, almost all the interferences existed in human serum and urine were removed after being treated with MIP therefore it can be used for the sample clean up. The analyses result demonstrated that the MIP extraction of adefovir from serum and urine samples is linear in the ranges 20–100 µg/L with good precision (2.53% and 2.8% for 50 µg/L, respectively). The repeatability for 5 mL of spiked serum and urine (50 µg/L), expressed as %RSD (n=6), was lower than 4%. The recoveries of serum and urine were (88.2–93.5%) and (84.3–90.2%), respectively (Table 3). The LOD and LOQ for adefovir were 7.62 and 15.1 µg/L in serum samples and 5.45 and 16 µg/L in urine samples, respectively.


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4 Conclusions In this paper, water-compatible molecularly imprinted polymers were synthesized by a noncovalent molecular imprinting approach in chloroform for selective extraction and separation of adefovir from serum and urine. This proficient method provided cleaner extracts and removed interfering peaks from the complex biological matrices. The method was appropriated to the trace adefovir determination at three levels, and the recoveries for the spiked human serum and urine samples were 88.2–93.5% and 84.3–90.2% at 20–80 µg/L, respectively. Based on these results, the extraction recoveries of the analytes from the real samples were satisfactory and consequently, the proposed coupled system of MIP–HPLC can be easily employed for the analysis of adefovir in biological samples with great potential in developing selective extraction. The results presented in this study could be precious for future research toward improvement of new systems in clean-up procedures and provide a strategy for expansion of MIPs in product recovery from biological fluids.


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[23] Javanbakht, M., Shaabani, N., Adergani, B.A., J. Chromatogr. B 2009, 877, 2537–2544 [24] Javanbakht, M., Attaran, A.M., Namjumanesh, M.H., Manesh, M.E., Adergani, B.A., J. Chromatogr. B 2010, 878, 1700–1706. [25] Moein, M.M., Javanbakht, M., Adergani, B.A., J. Chromatogr. B 2011, 879, 777–782. [26] Javanbakht, M., Pishro, K.A., Nasab, A.H., Adergani, B.A., Mat. Sci. Eng. C 2012, 32, 2367–2373. [27] Manesh, M.E., Javanbakht, M., Atyabi, F., Dinarvand, R., J. Mater. Sci: Mater. Med. 2012, 23, 963–972. [28]Javanbakht, M., Atyabi, F., Mohammadi, A., Mohammadi, S., Akbari, B., Manesh, M Dinarvand, R., Mat. Sci. Eng. C 2011,8, 1692 –1699. [29]Shekarchi, M., Pourfarzib, M., Akbari-Adergani, B., Mehramizi, A., Javanbakht, M., Dinarvand, R., Journal of Chromatography B, 2013, 931, 50– 55. [30] Pourfarzib, M., Shekarchi, M., Rastegar, H., Akbari-Adergani, B., Mehramizi, A., Dinarvand, R., J. Chromatogr. B, 2015, 974, 1–8. [31] Zhai, Y.H., Liu, Y.W., Chang, X.J., Chen, S., Huang, X.J., Chen, S.B., Huang, X.P., Anal. Chim. Acta 2007, 593, 123–128. [32] Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H., Molecular Imprinting: From Fundamentals to Applications, Wiley, Weinheim 2003. [33 ]Won, J. C., Song, H. Y., Faiz, A., J. Sep. Sci. 2013, 36, 609–628. [34]Langmuir, L., J. Am. Chem. Soc. 1918, 40, 1361–1403. [35] Freundlich, H., Hellen, W., J. Am. Chem. Soc. 1993, 61, 2228–2230 [36] Mullett, W.M. , Walles, M., Levsen, K., Borlak, J., Pawliszyn, J., J. Chromatogr. B: Biomed. Anal. 2004, 801, 297–306.


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Fig 1. Structures of the drugs used in this study.



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Fig 2. IR spectra of the unleached (B) and leached (A) MIP particle.



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Fig 3. A) Langmuir isotherm for adefovir adsorption onto imprinted polymer at 25°C. B) Freundlich isotherm for adefovir adsorption onto imprinted polymer at 25°C.


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Fig 4. A)HPLC chromatogram obtained after clean up a 50 µg/L solution of adefovir in serum samples with (A) MIP and (B) NIP monitored at 260 nm; conditions: column C18, 5 µm, 4.6×100 mm, eluent: phosphate buffer pH 5.2/acetonitrile (94:6) at flow rate 1.5 mL/min. B) HPLC chromatogram obtained after clean up a 50 µg/L solution of adefovir in urine samples with (A) MIP and (B) NIP.


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Table 1. Compositions and comparison of the extraction of adefovir from adefovir standard solution ( 5 ml, 200 mg/L) using 40 mg of various polymers as sorbents at pH 7, eluted by 5 ml methanol : acetic acid (9: 1, v/v) . Polymer MAAb adefovir EGDMAb AIBNb Extraction (%)a NIP

4.0

0.0

16

0.34

10.2%±2.3

MIP1

4.0

0.5

16

0.34

90.5%±3.2

MIP2

4.0

1.0

16

0.34

56%±2.5

MIP3

4.0

1.5

16

0.34

43%±2.7

a

represent average of three determinations as maen±SD, brepresent as mmol, MAA=methacrylic acid, EGDMA = ethylene glycol dimethacrylate, AIBN= 2,2Azobisisobutyronitrile


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Table 2. Distribution ratio (KD), selectivity coefficient (Ksel) and relative selectively coefficient (K′) values of MIP and NIP material for different drugs. Polymer

KD mip

KD nip

Ksel (mip)

Ksel (nip)

K′

Adefovir

495.4

64.3

-

-

-

Acyclovir

60.4

40

8.2

1.61

5.1

Lamivudine

51.7

19.1

9.58

3.36

2.85

Tenofovir

159.7

54.2

3.1

1.18

2.63


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Table 3. Assay of Adefovir in human serum and urine by means of the described method. Spiked value

(Recovery%±SD)a

Sample

Human serum

Human urine

a

(ng/ml)

MIP

NIP

20

88.2±2.83

22.8±2.52

50

93.5±2.53

12.9±2.19

80

90.3±3.32

18.6 ±3.54

20

86.7±2.80

24.5±3.71

50

90.2±2.80

15.5±3.06

80

84.3±3.16

23.6 ±2.55

Average of three determinations.


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