Research article Received: 24 August 2014,
Revised: 6 January 2015,
Accepted: 28 February 2015
Published online in Wiley Online Library: 14 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3469
Synthesis of molecularly imprinted polypyrrole as an adsorbent for solid-phase extraction of warfarin from human plasma and urine Moazameh Peyrovi and Mohammadreza Hadjmohammadi* ABSTRACT: The aim of this work was to develop a method for the clean-up and preconcentration of warfarin from biological sample employing a new molecularly imprinted polymer (MIP) as a selective adsorbent for solid-phase extraction (SPE). This MIP was synthesized using warfarin as a template, pyrrole as a functional monomer and vinyl triethoxysilane as a crosslinker. The molar ratio of 1:4:20 (template–functional monomer–cross-linker) showed the best results. Nonimprinted polymers (NIPs) were prepared and treated with the same method, but in the absence of warfarin. The prepared polymer was characterized by Fourier transmission infrared spectrometry and scanning electron microscopy. An adsorption process (SPE) for the removal of warfarin using the fabricated MIPs and NIPs was evaluated under various conditions. Effective parameters on warfarin extraction, for example, type and volume of elution solvent, pH of sample solution, breakthrough volume and maximum loading capacity, were studied. The limits of detection were in the range of 0.0035–0.0050 μg mL 1. Linearity of the method was determined in the range of 0.0165–10.0000 μg mL 1 for plasma and 0.0115–10.0000 μg mL 1 for urine with coefficients of determination (R2) ranging from 0.9975 to 0.9985. The recoveries for plasma and urine samples were >95%. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: molecularly imprinted polymer; solid-phase extraction; warfarin; HPLC
Introduction
Biomed. Chromatogr. 2015; 29: 1623–1628
* Correspondence to: M. Hadjmohammadi, Department of Chemistry, University of Mazandran, Niroo-Havayii Boulevard, 47416-95447 Babolsar, Iran. Email: [email protected] Department of Chemistry, University of Mazandran, Niroo-Havayii Boulevard 47416-95447 Babolsar, Iran Abbreviations used: FT-IR, Fourier transmission infrared spectrometry; INR, international normalized ratio; MIP, molecularly imprinted polymer; NIP, nonimprinted polymer; PPy, polypyrrole; PT, prothrombin time; SEM, scanning electron microscopy; SPE, solid-phase extraction; VTEOS, vinyl triethoxysilane.
Copyright © 2015 John Wiley & Sons, Ltd.
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Warfarin [3-(α-acetonyl benzyl)-4-hydroxy coumarin] is an anticoagulant drug normally used in the prevention of thrombosis and thromboembolism, the formation of blood clots in the blood vessels and their migration elsewhere in the body, respectively (Holbrook et al., 2005). In order to ensure the effectiveness and safety of warfarin, the dosage must be adjusted accurately and frequently, which is now critically dependent on maintaining the prothrombin time (PT), expressed as the international normalized ratio (INR), within the therapeutic range. However, the INR has its limitations in detecting factors such as patient compliance, resistance to anticoagulants, drug interaction and food variety. Knowledge of the plasma concentration of warfarin is valuable for clinical decisions and allows for effective treatment of severe intoxication. Therefore, many studies have been performed to measure the plasma concentrations of warfarin and its correlation with INR (Sun et al., 2006). It has been reported that the correlation of warfarin dosage or concentration with INR is very poor, although concentration monitoring has a role in research/development and may show value clinically (Huang et al., 2008). Several methods, such as high-performance liquid chromatography (HPLC) using ultraviolet or fluorescence detection (Sun et al., 2006; Osman et al., 2005; Locatelli et al., 2005; Ring and Bostick, 2000; Boppana et al., 2002; Chua et al., 2012), liquid chromatography–tandem mass spectrometry (Zuo et al., 2010; Naidong et al., 2001; Zhang et al., 2001), micellar electrokinetic chromatography–electrospray ionization mass spectrometry (Hou et al., 2007), supercritical fluid chromatography–tandem mass spectrometry (Coe et al., 2006), capillary zone electrophoresis (Yau and Chan, 2002; Gareil et al., 1993; Balchen et al., 2007) and
square-wave adsorptive cathodic stripping voltammetry (Ghoneim and Tawfik, 2004), have been reported for the determination of warfarin in biological samples. Owing to the complex matrix of the real samples and the low concentration of warfarin, making efforts to develop a simple and reliable method for preconcentration and determination of the warfarin is the main challenge and a very important step for the analysis of it. SPE is frequently used for enrichment and clean-up of biological samples owing to its benefits of simplicity, rapidity and low consumption of organic solvents. Nevertheless, the traditional SPE sorbents (C8, C18, SCX, PCX, HLB, etc.) lack special selectivity, which commonly leads to the co-extraction of impurities from the sample matrix. Therefore, the development of selective sorbent material for SPE is desired. A relatively new development in the area of SPE is the use of molecularly imprinted polymers (MIPs) for the sample clean-up (Sellergren, 2001; Caro et al., 2006; Hu et al., 2005; Masque et al., 2001). MIPs are synthetic polymers possessing specific cavities designed for a target molecule.
M. Peyrovi and M. Hadjmohammadi Polypyrrole (PPy) was the first conducting polymer to show relatively high conductivity. Until now, there has been little information about using PPy for the chemical synthesis of MIPs (Mehdinia et al., 2013; Miah et al., 2012, 2014). Polymerization occurs readily in the presence of different oxidants, such as FeCl3 (Ansari et al., 2008) and K2S2O8 (Park et al., 2003 ). In this work, we synthesized a warfarin MIP based on polypyrrole and applied it as special sorbent of SPE for extraction of warfarin from plasma and urine samples.
Experimental Materials and methods Sodium warfarin (≥98%) was purchased from Sigma–Aldrich (St Louis, MO, USA). Methanol (HPLC-grade,( sodium hydroxide, hydrochloric acid and phosphoric acid were obtained from Merck (Darmstadt, Germany). Pyrrole and vinyl triethoxysilane (VTEOS), were obtained from Merck. The water used was double-distilled deionized.
1
Stock solution of warfarin (500.0 μg mL ) was prepared in methanol and stored in the dark at 4°C. The working solutions were prepared daily by an appropriate dilution of the stock solution with double distilled deionized water.
Instrumentation Chromatographic measurements were carried out using a series 10 liquid chromatography pump (Perkin-Elmer, Norwalk, CT, USA) system equipped with a series 10 LC pump, UV detector model LC-95 set at 320 nm and model 7125i manual injector with a 20 μL sample loop (Perkin-Elmer, Norwalk, CT, USA). The column used was a C18 (250 × 4 mm, 5 μm particle size) from Dr Maisch (Ammerbuch, Germany). A mixture of methanol and 1 phosphoric acid 0.5% (80:20, v/v) at a flow rate of 1.0 mL min and room temperature was used as mobile phase. Measurement of solution pH was done by a 3030 Jenway pH meter (Leeds, UK).
Preparation of biological samples Blank plasma and urine samples were provided by healthy donors. In order to eliminate the protein binding of the drug in plasma (>99%), the pretreatment, as outlined in the work of Tahmasebi et al. (2009), was performed. For this purpose, 3 mL methanol was added to 2 mL of the plasma and the resulting mixture was strongly vortexed for 10 min. The mixture was placed for 10 min in an ice bath, followed by 10 min at ambient temperature and then centrifuged at 3500 rpm for 10 min. The supernatant was evaporated and diluted with double-distilled deionized water. The pH of the final solution was adjusted to 3 using an appropriate amount of HCl solution and the extraction procedure was performed under the optimized conditions. The urine sample was filtered through a 0.45 μm filter from Millipore (Bedford, MA). The filtrate was collected in a glass container. Then, 10 mL of the urine sample was spiked with mixed standard solution to obtain the desired concentration and diluted to 20 mL with deionized water. Next ,an appropriate amount of HCl solution was added to achieve a pH value of 3. These samples were subsequently submitted to the MIP-SPE procedure.
MIP and NIP preparation with bulk polymerization Figure 1. Scanning electron microscopy image of warfarin imprinted polymer.
For preparation of the warfarin-imprinted polymer, 4 mmol pyrrole (as a monomer), 20 mmol VTEOS (as a cross-linker) and 40 mL HCl 10% were stirred for 30 min. Afterwards, 1 mmol warfarin (template), 10 mL ACN
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Figure 2. Fourier transmission infrared spectrometry (FT-IR) spectra of (a) nonimprinted polymers (NIP), (b) unleached molecularly imprinted polymer (MIP), (c) leached MIP and (d) warfarin.
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1623–1628
Molecularly imprinted polypyrrole for warfarin extraction .
(progen solvent) and, finally, 2.4 g FeCl36H2O were added to the mixed solution. The final solution was kept in refrigerator for 24 h. The obtained hard polymers were dried and crushed. The polymer particles were washed with methanol three times and with distilled water twice. The complete removal of template was followed by HPLC-UV. In order to verify that retention of template was due to the molecular recognition and not to nonspecific binding, a control, nonimprinted polymer (NIP) was prepared as the same procedure, includingwashing, but withthe omission of the target molecule, warfarin. Moreover, the ratios of template, monomer and cross-linker were investigated from 1:4:8 to 1:4:30 to obtain MIPs with satisfactory mechanical strength and affinity to warfarin. As a consequence, the MIPs prepared at the molar ratio of 1:4:20 were suitable. The morphology of the MIPs evaluated by scanning electron microscope is shown in Fig. 1.
NIP were recorded in the range of 400–4000 cm 1 by the KBr pellet method (Fig. 2). The similarity in these IR spectra showed that these polymers have similar backbones. No band was present in leached warfarin imprinted polymer in the region of 1680 cm 1, indicating the absence of the carbonyl group of template. This confirmed the complete removal of the template. The FT-IR spectrum obtained for MIP-PPy shows the presence of a characteristic absorption band at 1570.7 cm 1 (C = C stretching of pyrrole ring) and 1360.7 cm 1(C–N stretching vibration in the ring). In order to determine the most appropriate elution solvent, five different solvents, n-hexane, ethylacetate, methanol, dichloromethane and acetonitrile, were investigated. It was found that methanol gave the best recovery among these solvents (Fig. 3a).
SPE procedure The MIPcartridge was conditioned with 2 mL methanoland 2 mL ultrapure water. Then, an appropriate volume of aqueous standard solution of warfarin was passed through the cartridge at an optimized flow rate by a vacuum pump. Then, the cartridge was washed with 3 mL of ultrapure water to remove the co-adsorbed matrix materials from the cartridge. Subsequently, warfarin retained on the cartridge was eluted with an optimum volume of methanol. The eluate was dried by evaporation in vacuum. Finally, the residue was reconstituted in 200 μL of methanol and 20 μL of it was injected onto the HPLC.
Results and discussion Characterization The Fourier transmission infrared spectrometry (FT-IR) spectra of leached and unleached warfarin imprinted polymers (MIPs) and Figure 4. Effect of sample pH on the extraction for MIP cartridge, 3 mL of methanol. Other conditions as in Fig. 3.
Biomed. Chromatogr. 2015; 29: 1623–1628
Figure 5. (a) Breakthrough volume of warfarin for MIP cartridge. (b) Maximum loading capacity of MIP cartridge for adsorption of warfarin with a concentration of 20 ppm. Other conditions as in Fig. 3.
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 3. (a) Selection of appropriate elution solvent for MIP cartridge; (b) selection of suitable volume of elution solvent for MIPcartridg. Conditions: sample loaded, 20 mL of 0.1 ppm of warfarin standard solution; elution solvent volume, 4 mL; HPLC mobile phase, methanol–phosphoric acid 0.5% 1 (80:20, v/v); flow rate, 1.0 mL min ; flow rate, 1 mL/min; column, C18 (250 × 4.6 mm, 10 mm); λ = 320 nm; room temperature.
M. Peyrovi and M. Hadjmohammadi For determination of a suitable volume of elution solvent, different volumes (1, 2, 3, 4, 5 and 6 mL) of methanol were used for elution of retained warfarin from the cartridge. The appropriate volume of methanol was 3 mL (Fig. 3b).
pH of sample solution The pH of a sample plays an important role in the SPE procedure because the pH of the solution determines the state of the analyte in solution as ionic or molecular form, and thus, determines the extraction recovery of target analytes. In this work, a wide range of sample pHs from 2 to 8 was evaluated. According to the results shown in Fig. 4, the best recovery was obtained at pH 3.
Determination of breakthrough volume and maximum loading capacity of solid-phase cartridge for warfarin The breakthrough volume of the cartridges was determined by Hennion’s (2000) procedure. Thus, 5 μg of a warfarin standard was dissolved in 50–1500 mL of double-distilled deionized water (adjusted at pH 3 with concentrated HCl) and passed through the cartridge. The maximum loading capacity of solid-phase cartridge for adsorption of warfarin was determined by passing different volumes (10–100 mL of 2 μg mL 1 of each aqueous standard solution adjusted at pH 3 with concentrated HCl) through the cartridge. In both cases, the retained analyte was eluted with 3 mL methanol and dried by evaporation in vacuum. The residue was reconstituted in 1 mL methanol and injected into the HPLC system. Figure 5(a) shows that the breakthrough volume was 750 mL. According to Fig. 5(b), the maximum loading capacity for warfarin was 160 μg. Study of MIP selectivity Acenucoumarul, dicoumarol and 7-hydroxy-4-methyl coumarin as structural analogues of warfarin were selected to investigate the selectivity of the MIP. Their molecular structures are shown in Fig. 6. Solutions of all compounds were prepared individually with the concentration of 0.5 μg mL 1. Extraction solvent was 3 mL methanol. The extraction yields of the selected compounds with the MIP and NIP are shown in Fig. 7. Obviously, this demonstrated that warfarin based-MIP possessed better affinity to the structural analogs of the template molecule.
Figure 7. Extractions of warfarin, dicoumarol, acenocumarol and 7hydroxy-methylcoumarin with MIP and NIP.
Analytical performance of the MIP-SPE-HPLC For determination of warfarin under optimum condition, figures of merit of the proposed method, consisting of linear range, determination coefficient (r2), limit of detection, limit of quantification, extraction recovery and preconcentration factor were studied in blank plasma and urine samples. Limits of detection and quantification of the method for each matrix were determined by spiking samples with standard warfarin at low concentrations, extracted by the described MIP-SPE method and calculated as the concentration giving peaks for which the signal-to-noise ratio was 3 and 10, respectively. The preconcentration factor was defined as the ratio of the concentrations of analyte in the final extraction phase (concentration after preconcentration) and in the initial aqueous sample solution (concentration before dilution). As shown in Table 1, the intra- and inter-day precisions of the method in different spiked real samples were determined as relative standard deviation (RSD, %). Intra-day precision was assessed by five determinations per concentration in 1 day, while inter-day precision was evaluated by five determinations at each of the three levels on three different days in a week. For evaluating the performance of the MIP-SPE-HPLC method for the extraction of warfarin from biological samples, calibration curves were drawn by spiking the standards directly into urine and plasma and water samples (Table 2). Figure 8 shows the chromatograms obtained from plasma and urine samples by MIP-SPE-HPLC-UV. Table 1. Inter- and intra-day precision and recovery of distilled water, plasma and urine spiked with warfarin after MIP-SPE-HPLC (n = 5) Sample
Concentration added (μg mL 1)
Plasma
Urine
0.05 0.15 1.00 0.05 0.15 1.00
Inter-day
Intra-day
ER (%) ± preci- ER (%) ± precision sion (RSD, %) (RSD, %) 95.0 ± 7.3 96.1 ± 7.0 96.5 ± 6.8 96.8 ± 6.8 97.0 ± 6.5 97.5 ± 6.3
96.5 ± 4.5 97.0 ± 4.2 97.9 ± 4.0 97.5 ± 4.2 98.5 ± 3.9 98.8 ± 3.7
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ER, Extraction recovery. Figure 6. Chemical structure of investigated organic compounds and drugs.
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Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1623–1628
Molecularly imprinted polypyrrole for warfarin extraction Table 2. Analytical performance of MIP-SPE-HPLC for determination of warfarin in biological samples Sample
Sample volume (mL)
Plasma Urine
LOD (μg mL 1)
LOQ (μg mL 1)
LR (μg mL 1)
0.0050 0.0035
0.0165 0.0115
0.0165–10.0000 0.0115–10.0000
2 10
R2 0.9985 0.9975
ER ± RSD (%) 98.8 ± 4.5 99.2 ± 3.7
Calibration equation y = 3662.8x + 166.95 y = 3609.1x + 213.36
PF 10 51
MIP, Molecularly imprinted polymer; SPE, solid-phase extraction; LOD, limit of detection; LOQ, limit of quantitation; LR, linear range; PF, preconcentration factor.
Figure 8. Representative chromatograms of (a) blank plasma sample, (b) spiked plasma sample, (c) blank urine sample and (d) spiked urine sample.
Table 3. Comparison of the present method with other reported methods for the determination of warfarin Method
LOD (μg mL 1)
Linear range (μg mL 1)
HF-LPME-HPLC-UVa
0.0050
0.0150–0.5500
LD-DLLME HPLC-UVb
0.0050
0.0050–3.0000
SPE-MEKC-ESI-MSc LLE-HPLC-ESI-MSd LLE-HPLC-UVe Stripping voltammetry
0.100 — — 0.0003
MIP-HPLC-UVf
0.0050
RSD (%) (intra-day)
RSD (%) (inter-day)
Real sample
Reference
4.2
11.1
Plasma
2.8
6.5
Plasma
0.2500–5.0000 0.0005–0.10000 0.1200–3.0000 0.0150–0.1230
—
Revised: 6 January 2015,
Accepted: 28 February 2015
Published online in Wiley Online Library: 14 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3469
Synthesis of molecularly imprinted polypyrrole as an adsorbent for solid-phase extraction of warfarin from human plasma and urine Moazameh Peyrovi and Mohammadreza Hadjmohammadi* ABSTRACT: The aim of this work was to develop a method for the clean-up and preconcentration of warfarin from biological sample employing a new molecularly imprinted polymer (MIP) as a selective adsorbent for solid-phase extraction (SPE). This MIP was synthesized using warfarin as a template, pyrrole as a functional monomer and vinyl triethoxysilane as a crosslinker. The molar ratio of 1:4:20 (template–functional monomer–cross-linker) showed the best results. Nonimprinted polymers (NIPs) were prepared and treated with the same method, but in the absence of warfarin. The prepared polymer was characterized by Fourier transmission infrared spectrometry and scanning electron microscopy. An adsorption process (SPE) for the removal of warfarin using the fabricated MIPs and NIPs was evaluated under various conditions. Effective parameters on warfarin extraction, for example, type and volume of elution solvent, pH of sample solution, breakthrough volume and maximum loading capacity, were studied. The limits of detection were in the range of 0.0035–0.0050 μg mL 1. Linearity of the method was determined in the range of 0.0165–10.0000 μg mL 1 for plasma and 0.0115–10.0000 μg mL 1 for urine with coefficients of determination (R2) ranging from 0.9975 to 0.9985. The recoveries for plasma and urine samples were >95%. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: molecularly imprinted polymer; solid-phase extraction; warfarin; HPLC
Introduction
Biomed. Chromatogr. 2015; 29: 1623–1628
* Correspondence to: M. Hadjmohammadi, Department of Chemistry, University of Mazandran, Niroo-Havayii Boulevard, 47416-95447 Babolsar, Iran. Email: [email protected] Department of Chemistry, University of Mazandran, Niroo-Havayii Boulevard 47416-95447 Babolsar, Iran Abbreviations used: FT-IR, Fourier transmission infrared spectrometry; INR, international normalized ratio; MIP, molecularly imprinted polymer; NIP, nonimprinted polymer; PPy, polypyrrole; PT, prothrombin time; SEM, scanning electron microscopy; SPE, solid-phase extraction; VTEOS, vinyl triethoxysilane.
Copyright © 2015 John Wiley & Sons, Ltd.
1623
Warfarin [3-(α-acetonyl benzyl)-4-hydroxy coumarin] is an anticoagulant drug normally used in the prevention of thrombosis and thromboembolism, the formation of blood clots in the blood vessels and their migration elsewhere in the body, respectively (Holbrook et al., 2005). In order to ensure the effectiveness and safety of warfarin, the dosage must be adjusted accurately and frequently, which is now critically dependent on maintaining the prothrombin time (PT), expressed as the international normalized ratio (INR), within the therapeutic range. However, the INR has its limitations in detecting factors such as patient compliance, resistance to anticoagulants, drug interaction and food variety. Knowledge of the plasma concentration of warfarin is valuable for clinical decisions and allows for effective treatment of severe intoxication. Therefore, many studies have been performed to measure the plasma concentrations of warfarin and its correlation with INR (Sun et al., 2006). It has been reported that the correlation of warfarin dosage or concentration with INR is very poor, although concentration monitoring has a role in research/development and may show value clinically (Huang et al., 2008). Several methods, such as high-performance liquid chromatography (HPLC) using ultraviolet or fluorescence detection (Sun et al., 2006; Osman et al., 2005; Locatelli et al., 2005; Ring and Bostick, 2000; Boppana et al., 2002; Chua et al., 2012), liquid chromatography–tandem mass spectrometry (Zuo et al., 2010; Naidong et al., 2001; Zhang et al., 2001), micellar electrokinetic chromatography–electrospray ionization mass spectrometry (Hou et al., 2007), supercritical fluid chromatography–tandem mass spectrometry (Coe et al., 2006), capillary zone electrophoresis (Yau and Chan, 2002; Gareil et al., 1993; Balchen et al., 2007) and
square-wave adsorptive cathodic stripping voltammetry (Ghoneim and Tawfik, 2004), have been reported for the determination of warfarin in biological samples. Owing to the complex matrix of the real samples and the low concentration of warfarin, making efforts to develop a simple and reliable method for preconcentration and determination of the warfarin is the main challenge and a very important step for the analysis of it. SPE is frequently used for enrichment and clean-up of biological samples owing to its benefits of simplicity, rapidity and low consumption of organic solvents. Nevertheless, the traditional SPE sorbents (C8, C18, SCX, PCX, HLB, etc.) lack special selectivity, which commonly leads to the co-extraction of impurities from the sample matrix. Therefore, the development of selective sorbent material for SPE is desired. A relatively new development in the area of SPE is the use of molecularly imprinted polymers (MIPs) for the sample clean-up (Sellergren, 2001; Caro et al., 2006; Hu et al., 2005; Masque et al., 2001). MIPs are synthetic polymers possessing specific cavities designed for a target molecule.
M. Peyrovi and M. Hadjmohammadi Polypyrrole (PPy) was the first conducting polymer to show relatively high conductivity. Until now, there has been little information about using PPy for the chemical synthesis of MIPs (Mehdinia et al., 2013; Miah et al., 2012, 2014). Polymerization occurs readily in the presence of different oxidants, such as FeCl3 (Ansari et al., 2008) and K2S2O8 (Park et al., 2003 ). In this work, we synthesized a warfarin MIP based on polypyrrole and applied it as special sorbent of SPE for extraction of warfarin from plasma and urine samples.
Experimental Materials and methods Sodium warfarin (≥98%) was purchased from Sigma–Aldrich (St Louis, MO, USA). Methanol (HPLC-grade,( sodium hydroxide, hydrochloric acid and phosphoric acid were obtained from Merck (Darmstadt, Germany). Pyrrole and vinyl triethoxysilane (VTEOS), were obtained from Merck. The water used was double-distilled deionized.
1
Stock solution of warfarin (500.0 μg mL ) was prepared in methanol and stored in the dark at 4°C. The working solutions were prepared daily by an appropriate dilution of the stock solution with double distilled deionized water.
Instrumentation Chromatographic measurements were carried out using a series 10 liquid chromatography pump (Perkin-Elmer, Norwalk, CT, USA) system equipped with a series 10 LC pump, UV detector model LC-95 set at 320 nm and model 7125i manual injector with a 20 μL sample loop (Perkin-Elmer, Norwalk, CT, USA). The column used was a C18 (250 × 4 mm, 5 μm particle size) from Dr Maisch (Ammerbuch, Germany). A mixture of methanol and 1 phosphoric acid 0.5% (80:20, v/v) at a flow rate of 1.0 mL min and room temperature was used as mobile phase. Measurement of solution pH was done by a 3030 Jenway pH meter (Leeds, UK).
Preparation of biological samples Blank plasma and urine samples were provided by healthy donors. In order to eliminate the protein binding of the drug in plasma (>99%), the pretreatment, as outlined in the work of Tahmasebi et al. (2009), was performed. For this purpose, 3 mL methanol was added to 2 mL of the plasma and the resulting mixture was strongly vortexed for 10 min. The mixture was placed for 10 min in an ice bath, followed by 10 min at ambient temperature and then centrifuged at 3500 rpm for 10 min. The supernatant was evaporated and diluted with double-distilled deionized water. The pH of the final solution was adjusted to 3 using an appropriate amount of HCl solution and the extraction procedure was performed under the optimized conditions. The urine sample was filtered through a 0.45 μm filter from Millipore (Bedford, MA). The filtrate was collected in a glass container. Then, 10 mL of the urine sample was spiked with mixed standard solution to obtain the desired concentration and diluted to 20 mL with deionized water. Next ,an appropriate amount of HCl solution was added to achieve a pH value of 3. These samples were subsequently submitted to the MIP-SPE procedure.
MIP and NIP preparation with bulk polymerization Figure 1. Scanning electron microscopy image of warfarin imprinted polymer.
For preparation of the warfarin-imprinted polymer, 4 mmol pyrrole (as a monomer), 20 mmol VTEOS (as a cross-linker) and 40 mL HCl 10% were stirred for 30 min. Afterwards, 1 mmol warfarin (template), 10 mL ACN
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Figure 2. Fourier transmission infrared spectrometry (FT-IR) spectra of (a) nonimprinted polymers (NIP), (b) unleached molecularly imprinted polymer (MIP), (c) leached MIP and (d) warfarin.
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1623–1628
Molecularly imprinted polypyrrole for warfarin extraction .
(progen solvent) and, finally, 2.4 g FeCl36H2O were added to the mixed solution. The final solution was kept in refrigerator for 24 h. The obtained hard polymers were dried and crushed. The polymer particles were washed with methanol three times and with distilled water twice. The complete removal of template was followed by HPLC-UV. In order to verify that retention of template was due to the molecular recognition and not to nonspecific binding, a control, nonimprinted polymer (NIP) was prepared as the same procedure, includingwashing, but withthe omission of the target molecule, warfarin. Moreover, the ratios of template, monomer and cross-linker were investigated from 1:4:8 to 1:4:30 to obtain MIPs with satisfactory mechanical strength and affinity to warfarin. As a consequence, the MIPs prepared at the molar ratio of 1:4:20 were suitable. The morphology of the MIPs evaluated by scanning electron microscope is shown in Fig. 1.
NIP were recorded in the range of 400–4000 cm 1 by the KBr pellet method (Fig. 2). The similarity in these IR spectra showed that these polymers have similar backbones. No band was present in leached warfarin imprinted polymer in the region of 1680 cm 1, indicating the absence of the carbonyl group of template. This confirmed the complete removal of the template. The FT-IR spectrum obtained for MIP-PPy shows the presence of a characteristic absorption band at 1570.7 cm 1 (C = C stretching of pyrrole ring) and 1360.7 cm 1(C–N stretching vibration in the ring). In order to determine the most appropriate elution solvent, five different solvents, n-hexane, ethylacetate, methanol, dichloromethane and acetonitrile, were investigated. It was found that methanol gave the best recovery among these solvents (Fig. 3a).
SPE procedure The MIPcartridge was conditioned with 2 mL methanoland 2 mL ultrapure water. Then, an appropriate volume of aqueous standard solution of warfarin was passed through the cartridge at an optimized flow rate by a vacuum pump. Then, the cartridge was washed with 3 mL of ultrapure water to remove the co-adsorbed matrix materials from the cartridge. Subsequently, warfarin retained on the cartridge was eluted with an optimum volume of methanol. The eluate was dried by evaporation in vacuum. Finally, the residue was reconstituted in 200 μL of methanol and 20 μL of it was injected onto the HPLC.
Results and discussion Characterization The Fourier transmission infrared spectrometry (FT-IR) spectra of leached and unleached warfarin imprinted polymers (MIPs) and Figure 4. Effect of sample pH on the extraction for MIP cartridge, 3 mL of methanol. Other conditions as in Fig. 3.
Biomed. Chromatogr. 2015; 29: 1623–1628
Figure 5. (a) Breakthrough volume of warfarin for MIP cartridge. (b) Maximum loading capacity of MIP cartridge for adsorption of warfarin with a concentration of 20 ppm. Other conditions as in Fig. 3.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1625
Figure 3. (a) Selection of appropriate elution solvent for MIP cartridge; (b) selection of suitable volume of elution solvent for MIPcartridg. Conditions: sample loaded, 20 mL of 0.1 ppm of warfarin standard solution; elution solvent volume, 4 mL; HPLC mobile phase, methanol–phosphoric acid 0.5% 1 (80:20, v/v); flow rate, 1.0 mL min ; flow rate, 1 mL/min; column, C18 (250 × 4.6 mm, 10 mm); λ = 320 nm; room temperature.
M. Peyrovi and M. Hadjmohammadi For determination of a suitable volume of elution solvent, different volumes (1, 2, 3, 4, 5 and 6 mL) of methanol were used for elution of retained warfarin from the cartridge. The appropriate volume of methanol was 3 mL (Fig. 3b).
pH of sample solution The pH of a sample plays an important role in the SPE procedure because the pH of the solution determines the state of the analyte in solution as ionic or molecular form, and thus, determines the extraction recovery of target analytes. In this work, a wide range of sample pHs from 2 to 8 was evaluated. According to the results shown in Fig. 4, the best recovery was obtained at pH 3.
Determination of breakthrough volume and maximum loading capacity of solid-phase cartridge for warfarin The breakthrough volume of the cartridges was determined by Hennion’s (2000) procedure. Thus, 5 μg of a warfarin standard was dissolved in 50–1500 mL of double-distilled deionized water (adjusted at pH 3 with concentrated HCl) and passed through the cartridge. The maximum loading capacity of solid-phase cartridge for adsorption of warfarin was determined by passing different volumes (10–100 mL of 2 μg mL 1 of each aqueous standard solution adjusted at pH 3 with concentrated HCl) through the cartridge. In both cases, the retained analyte was eluted with 3 mL methanol and dried by evaporation in vacuum. The residue was reconstituted in 1 mL methanol and injected into the HPLC system. Figure 5(a) shows that the breakthrough volume was 750 mL. According to Fig. 5(b), the maximum loading capacity for warfarin was 160 μg. Study of MIP selectivity Acenucoumarul, dicoumarol and 7-hydroxy-4-methyl coumarin as structural analogues of warfarin were selected to investigate the selectivity of the MIP. Their molecular structures are shown in Fig. 6. Solutions of all compounds were prepared individually with the concentration of 0.5 μg mL 1. Extraction solvent was 3 mL methanol. The extraction yields of the selected compounds with the MIP and NIP are shown in Fig. 7. Obviously, this demonstrated that warfarin based-MIP possessed better affinity to the structural analogs of the template molecule.
Figure 7. Extractions of warfarin, dicoumarol, acenocumarol and 7hydroxy-methylcoumarin with MIP and NIP.
Analytical performance of the MIP-SPE-HPLC For determination of warfarin under optimum condition, figures of merit of the proposed method, consisting of linear range, determination coefficient (r2), limit of detection, limit of quantification, extraction recovery and preconcentration factor were studied in blank plasma and urine samples. Limits of detection and quantification of the method for each matrix were determined by spiking samples with standard warfarin at low concentrations, extracted by the described MIP-SPE method and calculated as the concentration giving peaks for which the signal-to-noise ratio was 3 and 10, respectively. The preconcentration factor was defined as the ratio of the concentrations of analyte in the final extraction phase (concentration after preconcentration) and in the initial aqueous sample solution (concentration before dilution). As shown in Table 1, the intra- and inter-day precisions of the method in different spiked real samples were determined as relative standard deviation (RSD, %). Intra-day precision was assessed by five determinations per concentration in 1 day, while inter-day precision was evaluated by five determinations at each of the three levels on three different days in a week. For evaluating the performance of the MIP-SPE-HPLC method for the extraction of warfarin from biological samples, calibration curves were drawn by spiking the standards directly into urine and plasma and water samples (Table 2). Figure 8 shows the chromatograms obtained from plasma and urine samples by MIP-SPE-HPLC-UV. Table 1. Inter- and intra-day precision and recovery of distilled water, plasma and urine spiked with warfarin after MIP-SPE-HPLC (n = 5) Sample
Concentration added (μg mL 1)
Plasma
Urine
0.05 0.15 1.00 0.05 0.15 1.00
Inter-day
Intra-day
ER (%) ± preci- ER (%) ± precision sion (RSD, %) (RSD, %) 95.0 ± 7.3 96.1 ± 7.0 96.5 ± 6.8 96.8 ± 6.8 97.0 ± 6.5 97.5 ± 6.3
96.5 ± 4.5 97.0 ± 4.2 97.9 ± 4.0 97.5 ± 4.2 98.5 ± 3.9 98.8 ± 3.7
1626
ER, Extraction recovery. Figure 6. Chemical structure of investigated organic compounds and drugs.
wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1623–1628
Molecularly imprinted polypyrrole for warfarin extraction Table 2. Analytical performance of MIP-SPE-HPLC for determination of warfarin in biological samples Sample
Sample volume (mL)
Plasma Urine
LOD (μg mL 1)
LOQ (μg mL 1)
LR (μg mL 1)
0.0050 0.0035
0.0165 0.0115
0.0165–10.0000 0.0115–10.0000
2 10
R2 0.9985 0.9975
ER ± RSD (%) 98.8 ± 4.5 99.2 ± 3.7
Calibration equation y = 3662.8x + 166.95 y = 3609.1x + 213.36
PF 10 51
MIP, Molecularly imprinted polymer; SPE, solid-phase extraction; LOD, limit of detection; LOQ, limit of quantitation; LR, linear range; PF, preconcentration factor.
Figure 8. Representative chromatograms of (a) blank plasma sample, (b) spiked plasma sample, (c) blank urine sample and (d) spiked urine sample.
Table 3. Comparison of the present method with other reported methods for the determination of warfarin Method
LOD (μg mL 1)
Linear range (μg mL 1)
HF-LPME-HPLC-UVa
0.0050
0.0150–0.5500
LD-DLLME HPLC-UVb
0.0050
0.0050–3.0000
SPE-MEKC-ESI-MSc LLE-HPLC-ESI-MSd LLE-HPLC-UVe Stripping voltammetry
0.100 — — 0.0003
MIP-HPLC-UVf
0.0050
RSD (%) (intra-day)
RSD (%) (inter-day)
Real sample
Reference
4.2
11.1
Plasma
2.8
6.5
Plasma
0.2500–5.0000 0.0005–0.10000 0.1200–3.0000 0.0150–0.1230
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