jou rn al hom epage: www.elsev ier .com/ locate /chroma
evelopment of novel amphiphilic magnetic molecularly imprintedolymers compatible with biological fluids for solid phase extractionnd physicochemical behavior study
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, ChinaKey Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, 24 Tongjia Lane, Nanjing10009, Jiangsu Province, ChinaDepartment of Pharmacy, Stanford University Medical Center, Palo Alto, CA, USAFaculty of Pharmacy, University of Paris V, 4 Avenue de l’Observatoire, 75006 Paris, France
r t i c l e i n f o
rticle history:eceived 2 May 2013eceived in revised form 3 July 2013ccepted 19 July 2013vailable online xxx
In the present work, a novel amphiphilic magnetic molecularly imprinted polymer (M-MIP) has beensynthesized by a simple non covalent method for the loading of gatifloxacin (GTFX) in polar solvent.This nanomaterial used as sorbent has been applied to the solid phase extraction of GTFX in differentspiked biological fluids. For the first time, studies of dispersibility and solubility behaviors with differentsolvents and water were performed to demonstrate amphiphilicity and also to find the better nanoma-terial obtained. Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), transmissionelectron microscopy (TEM) and X-ray (XRD) were used to characterize the nanomaterials, and Scatchardplot analysis to demonstrate the binding kinetic. Results suggest that the dispersibility, solubility and theadsorption in water have relationships with the structure of nanomaterials prepared. The oleic acid coatedon the M-MIP combined with the washing process has enhanced the amphiphilicity of the nanomateri-
hysicochemical behavior study als. The M-MIP2 showed better selectivity and adsorption behavior with imprinted efficiency higher than(2) in water, as well as in biological fluids. Moreover, no interference with constituents of blank urineand blank serum samples for solid phase extraction (SPE) was observed. Moreover, loading recovery wasfound higher than 95% with low RSD. The novel amphiphilic magnetic nanomaterial prepared here assorbent is suitable for SPE of GTFX in biological fluids for therapeutic monitoring control. It could be alsoused as carrier in drug delivery system for experimental and clinical studies.
. Introduction
Recently, a great deal of researches on nanomaterials as adsor-ents for various compounds (drugs, biomolecules) has been
nvestigated in order to use them either as carrier in drug deliveryystem or as sorbent in drug solid phase extraction [1–3]. Theseanomaterials including molecularly imprinted polymers (MIPs)4,5], M-MIPs [6,7], carbon nanotube (CNTs) [8], magnetic-CNTs (M-
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NTs) [3] and magnetic-MIP-CNTs [9] can adsorb drugs and otheriomolecules by forming stable covalent or non-covalent bondsetween nanomaterials and analyte. Among these nanomaterials,
∗ Corresponding author at: Department of Analytical Chemistry, China Pharma-eutical University, 8 Nanjing 210009, China. Tel.: +86 025 83271505;ax: +86 025 83271505.
E-mail address: [email protected] (H. He).1 These authors contributed equally to this work.
M-MIPs seems to be the best candidate not only for drug deliverysystem in therapeutics but also for SPE in drug analysis.
The development of a nanomaterial for drug adsorption is thefirst capital step of any nanotechnology application in medicine.Therefore, the choice of nanomaterial and technique is determinantfor the success of a preparation obtained. Previously knowledge ofthe physic-chemical properties of the adsorbent prepared as wellas its behavior may be also highlighted.
Compared to the conventional extraction methods such asliquid–liquid extraction (LLE) [4] and SPE [5], magnetic solid phasemicro extraction (MSPME) is a promising technique that pro-vides cleaner extracts with less solvent consumption, and notablyimproves analytical parameters. Moreover, it combines the extrac-tion, enrichment, and sample introduction in one single step [6,10].
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However, most of MIPs used for the recognition of templatesare performed in organic media. The application in aqueous envi-ronment as well as in biological fluids of MIPs with non-covalentbonds is a difficult and challenging task [11–14]. Components of
a First washed with water, then methanol:acetic acid (8:2) until complete removb Washing process using only water several time to remove reagent and solvents
iological samples, such as proteins, could be strongly adsorbedo the polymeric surface by hydrophobic interaction with the MIP,hich adversely affects their recognition efficiency [15]. Hydrogen
onds between functional monomers and template could be bro-en [16,17]. Thus, the development of an MIP with application inoth aqueous and organic media has attracted much. Such adsor-ents must be simpler, cheaper and capable of improving extractionelectivity and efficiency [15,18–21]. However, low adsorption,oor solubility and interference with biological constituents stillemain a major challenge for the use of nanomaterials in SPE tech-ique [22].
Magnetic molecularly imprinted polymer is a spherical poly-er endowed with magnetic properties. They have highly selective
inding characteristics to the target template and homologues23,24]. Polymerization of the magnetic MIP beads is usuallynduced by conventional heating [25,26] or UV light [27,28] thats time consuming. To reduce time, few reports has suggested aew process using microwave heating technique to avoid time-onsuming [18,29]. However, this process require the use of apecially design microwave which may be expensive. Actually, syn-hesized magnetic polymer is widely used as sorbent for samplereparation. This technology when used for drug extraction in bio-
ogical media is rapid and simply by applying a magnet to thereparation in order to remove magnetic polymers from sampleatrices without centrifugation or filtration as classic extractionodes. The extraction consists of the loading and elution of the
emplate.The applicability of the M-MIP depends on its solubility, dis-
ersibility [30] and template recognition in the solvent used forxtraction [31–33]. Some M-MIPs are efficient in polar solventhydrophilic polymers) [5,34] while others require non polar sol-ent (hydrophobic -polymers) [20]. Amphiphilic M-MIPs could behe best nanomaterial having both hydrophobic and hydrophilicroperty destined to extraction of drugs in biological samples.ifferent amphiphilic polymers have been cited in the literature
35–40], but as we know, no reports about the use of amphiphilicagnetic molecularly imprinted polymer were found.In the present work, a novel amphiphilic M-MIP used as sor-
ent has been synthesized by a simple non covalent method for theoading of GTFX in polar solvent. This sorbent has been applied tohe SPE of GTFX in different spiked biological fluids (urine, serum).atifloxacin is a synthetic antibiotic of the fourth generation fluo-
oquinolone family [41,42] and was selected in this study becausef its high antibacterial efficacy and also of its side effects. GTFX issed for the treatment of various bacterial infections and in certainatients with antibiotic resistance. However, its oral and IV forms
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re recently banned in USA and Canada only, due to the apparitionf diabetes [43]. For avoiding its side effects as well as for controllingts efficacy in treated patients, a therapeutic control monitor-ng of GTFX in biological fluids is necessary. Therefore, a novel
the template, finally wash with water to remove residues of washing solvent.ues.
nanomaterial compatible to biological fluids have been developedherein and used as SPE sorbent in it, with physic-chemical analysis.In our previous study we have demonstrated that GTFX could be asuitable template for the preparation of the MIP [44,45]. The tech-nique described herein, has shown net advantages as comparedto previous work because of its amphiphilic characters. Moreover,the M-MIPs obtained herein could be used as drug carrier for GTFXdelivery system in therapeutics in order to avoid its toxic effects.
2. Experimental
2.1. Materials
All chemicals and solutions used were of analytical reagentgrade. Gatifloxacin (GTFX), norfloxacin (NRFX), ciprofloxacin(CPFX), amoxicillin (AMX) powder and ferric chloride hexahy-drate FeCl3.6H2O (Fe3+) were purchased from Sinopharm ChemicalReagent Co., Ltd. (Shanghai, China). Ferrous sulfate heptahydrateFeSO4.7H2O (Fe2+) was purchased from Nanjing Chemical ReagentCo., Ltd. (Nanjing, China). Methacrylic acid (MAA), ethylene glycoldimethacrylate (EGDMA), polyvinylpyrrolidone (PVP), azobisisbu-tyronitrile (AIBN), polyvinyl alcohol (PVA), polyethylene glycol6000 (PEG-6000) and oleic acid were obtained from Aladdin Indus-trial Corporation (Shanghai, China).
2.2. Preparation of the Fe3O4, PEG-Fe3O4 and magnetic polymers
The preparation of Fe3O4 was performed by a chemical co-precipitation of Fe2+ and Fe3+ ions according to this reference withminor changes [46]. Fifty milliliters of 0.02 mol Fe2+ and 0.04 molFe3+ solutions were prepared with deionized water in two beakers,and then mixed in a 250 mL three necked flask. When the solu-tion was heated to 80 ◦C, ammonia solution (50 mL) was addeddrop wise under nitrogen gas protection with vigorous mechanicalstirring until the pH was established between 10 and 11. After addi-tion of ammonia, the solution immediately turned black indicatingthe formation of iron oxide nanoparticles (Fe3O4) in the system.The solution continued to be heated at 80 ◦C for 2 h, and then theprecipitated powders were collected by magnetic separation. Theobtained magnetic nanoparticles were washed immediately withdeionized water for several times. The final product was dried at40 ◦C under vacuum oven. The modification of the Fe3O4 nanopar-ticles was performed with surface modifiers as reported in theprocedure with minor change [18]. The Fe3O4 (2 g), distilled water(30 mL) and dimetyl sulfoxyde (DMSO) (10 mL) were mixed with
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PEG (10.0 g), oleic acid (2.0 mL), and polyvinyl alcohol (8.0 g) bywhisking for 20 min, followed by sonication for 30 min until ahomogeneously dispersed solution was obtained. The magneticparticles was separated and dried in the oven for 24 h.
The M-MIPs were prepared by co-precipitation method asollows: The required amounts of template (GTFX), monomerMAA), magnetic nanoparticles (Fe3O4 or PEG-Fe3O4) cross-linkerEGDMA), porogen (PVP), initiator (AIBN), and oleic acid areetailed in the Table 1. The preassembled solution was first pre-ared by adding the template and the monomer in 10 mL of DMSOnd stirred for 30 min. The magnetite nanoparticles (Fe3O4) wereixed with 4 mL of DMSO under ultrasound for 10 min. Then
GDMA and the preassembly solution were both added into theixture of Fe3O4 in DMSO. This mixture was sonicated again for
0 min for preparation of pre-polymerization solution. The PVPsed as dispersant was dissolved into 100 mL of DMSO: H2O (9:1,/v) in a three necked round-bottomed flask. The mixture wastirred at 300 rpm and purged with nitrogen gas to displace oxygent 60 ◦C. The pre-polymerization solution was then transferred into
three-necked flask followed by adding AIBN then oleic acid after h. The reaction was maintained at 60 ◦C for 10 h. After the poly-erization, the polymers obtained were separated, and washed as
hown in the Table 1, and finally washed with water several timesntil the template molecule in the washing liquid could not beetected by UV-vis spectrometer in the range from 250 to 400 nm.he polymers collected were dried at 60 ◦C. The M-MIPs obtainedan be directly used for extraction. In parallel, the magnetic non-mprinted polymers (M-NIPs) were prepared as above, but withoutTFX added and used as control.
.3. Characterization
The size and morphology of the M-MIPs beads obtained werexamined by S-3000 scanning electron microscopy (SEM, Hitachiorporation, Japan) and a FEI Tecnai G2 F20 transmission elec-ron microscope (TEM). Their gated structures were confirmed bynalyzing their infrared absorption spectra given by a Shimadzu IR-respige-21 FT-IR purchased from Shimadzu (Kyoto, Japan). Phase
dentification was done by the X-ray powder diffraction patternXRD), using X‘TRA X-ray diffractometer with Cu K� irradiation at
= 0.1541 nm. Their magnetic properties were tested by putting atrong magnet close to the flask containing magnetic nanoparticlesn aqueous solution.
.4. UV–visible spectrophotometric analysis
It was performed to determine different amounts of a drugfter every loading or elution process. The UV–Visible spectropho-ometer (UV-1800) was purchased from Shimadzu (Kyoto, Japan).tock standard solutions at 1000 �g mL−1 in water of GTFX wererepared and stored at 3◦C. It was diluted at appropriate concen-rations for the preparation of GTFX standard calibration curves. Allnalytical parameters (linearity, detection limit, correlation coeffi-ient, precision, relative standard deviation of intra and inter-day)ere performed for the validation of the techniques. The ratio sig-al/noise was calculated in order to determine detection limit anduantification limit using the following equations:
D = 3s
k(1)
Q = 10s
k(2)
here (s) is the standard deviation of replicate determination val-es under the same conditions as for the sample analysis in the
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bsence of the analyte and (k) is the sensitivity; namely the slope ofhe calibration graph. The UV–vis determination was set at 286 nmor GTFX. All solutions were scanned from 250 to 400 nm for theemplate using distilled water as blank zero.
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2.5. Dispersion observation experiment in water, DMSO andn-hexane
This experiment was done to demonstrate the amphiphilicproperty of different magnetic polymers, and show the relation-ship between the loading, structure and dispersibility property.The experiment was carried out using three microcentrifuge tubeswith locking for the same M-MIP. Each tube (1, 2 and 3) contained20 mg of M-MIP accurately weighed, with 1 mL of desionized water,DMSO and n-hexane respectively. Water is a prototype polar pro-tic solvent, the DMSO is a prototype of polar aprotic solvent andn-hexane a non polar solvent. Observation began after addition ofeach solvent to each tube which was shaken to obtain a good disper-sion; this moment corresponds to the beginning time. Photographswere taken at intervals of time corresponding to 0, 30, 60, 150 and180 min to observe the dispersibility behavior.
2.6. Selection of the better M-MIP by comparing the adsorptionbehavior of GTFX on the polymers in aqueous solution
To select the best M-MIPs from the group of polymers preparedin this research, the drug loading behavior of polymers with weightof 20, 30, and 40 mg were used. Three ranges of GTFX concentra-tion were also chosen (100, 500, and 1000 �g mL−1) to determinethe concentration corresponding to the highest loading behavior.The loading process was performed by suspending the correspond-ing polymers (20, 30, and 40 mg) in 1.5 mL of GTFX (100, 500, and1000 �g mL−1) aqueous solution. After ultrasonic for 5 min andshaking at room temperature for 3 h, the mixture was separatedby external magnet and the amount of GTFX was calculated by Eq.(3).
Q = (C0 − C1) × V
m(3)
where C0, C1, V and m represent the initial solution concentra-tion of the analyte, its final solution concentration (�g mL−1), thevolume of the solution (mL) and the weight of the polymer (mg),respectively.
According to the variance of GTFX concentrations before andafter adsorption, the equilibrium adsorption capacity (Q, �g mg−1)of GTFX bound to the imprinted polymers are calculated. Theadsorption recovery and imprinting efficiency were also calculated.
2.7. Selctivity behavior of the magnetic polymers 2
GTFX, NRFX, CPFX, AMX were chosen as structural analogs toinvestigated the selectivity of the M-MIP2 and M-NIP2. The experi-ment was carried out by dispersing 40 mg of the magnetic polymersin the tube containing 1.5 mL aqueous solution of each analog at500 �g mL−1, then incubated and shaking for 3 h at room temper-ature followed by the separation of the supernatant for analysis inUV–vis in the range from 190 to 400 nm. The binding amount of thefour drugs on the M-MIP2 and M-NIP2 were compared.
2.8. Solubility behavior of the magnetic polymers 2 in water,DMSO and n-hexane
Solvents (water, DMSO, n-Hexane) were selected based onpolarity. Experiments were carried out by first transferring 10 mgof the polymer to three tubes, followed by the addition of 1 ml ofwater, n-Hexane and DMSO to the tube (a, b and c) respectively. Thesecond step involved the addition of a second solvent having the
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same or different polarity, to each tube in step 1. The third step wasdone by shaking the mixtures and observed the solubility behaviorof the magnetic polymer in them. In each step, photo was taken toshow the solubility behavior.
.9. Adsorption isotherm behavior of the magnetic polymers
The adsorption isotherm was performed by suspending 40 mgf M-MIP2 or M-NIP2 in 1.5 mL of GTFX aqueous solution withifferent concentrations ranging from 100 to 1000 �g mL−1. Afterhaking at 25 ◦C for 3 h, the mixture was separated by externalagnet and the amount of GTFX was calculated. According to the
ariance of GTFX concentrations before and after adsorption, thequilibrium adsorption capacity (Q, �g mg−1) of GTFX bound to themprinted polymers are calculated by Eq. (3).
The amounts of GTFX bound to M-MIPs or M-NIPs were calcu-ated by subtracting the GTFX concentrations in the supernatantrom its concentration in the initial solution before binding pro-ess as described in previous studies [7,47]. The average data ofriplicate independent results were used for the discussion.
The specific recognition property of M-MIPs can be evaluatedy imprinting factor (˛), which is defined as:
= QM−MIPs
QM−NIPs(4)
here QM-MIPs and QM-NIPs are the equilibrium adsorption capac-ty of the template on M-MIPs and M-NIPs respectively. Imprintingactor (˛) represents M-MIPs special recognition performance forTFX, and its larger value means that M-MIPs have better recogni-
ion characteristic and stronger imprinting effect.
.10. Magnetic solid phase extraction of GTFX spiked in biologicaluids toward the polymers
The human urine and serum sample were chosen in this study,ecause GTFX is mostly eliminated in urine and mainly present
n human serum [40]. Another reason for the selection of theseamples is to understand how the selectivity and amphiphilicityroperties of M-MIPs for the drug in the two complexes sampleith presence of many biomolecules (protein, lipids, urea, water
tc.). The urine and serum from non-treated humans were spikedith stock standard GTFX and diluted at final concentration of
00 �g mL−1 with the corresponding medium. 1.5 ml of those sam-les was mixed with 40 mg of M-MIPs or M-NIP in a flask byltrasound for 5 min. This was then kept for 3 h at room tempera-ure with shaking. After this contact time, the sorbent with capturedTFX was separated from the suspension with a magnet and theupernatant was then measured by UV–vis spectrometry at 286 nmith spectral analysis (240–400 nm) to determine the amount ofTFX non-adsorbed. Further dilution of the supernatant with wateras eventually made to ensure that the concentrations of the drug
n the sample solutions fell within the linear range of the standardalibration curve. In parallel, the GTFX analyte adsorbed onto the-MIPs was consequently eluted from the sorbent by ultrasonic
esorption with 1.5 mL of methanol: acetic acid (9:1, v/v). Afterotovaporation of the solvent, 1.5 ml of distilled water was addedor UV–visible determination. The elution was repeated three timesor cumulative release. Finally, after the extraction process, with theim to reuse in the future the same sorbent for new extraction ofhe GTFX, the sorbent was washed with only water several times toemove any residues of solvents, biological fluids and trace of therug. The polymers separated from the solvent, were dried at 60 ◦Cnd kept to be used for one new other extraction. The control ofuman urine and serum without spiked with GTFX was also per-
ormed. All samples were stored at 3 ◦C in a refrigerator. The aimf this section is to compare and evaluate the adsorption efficiencyf the amphiphilic M-MIP2 in the biological fluids and to observe
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he possible interferences from different natural compounds orngredients present in healthy human urine or serum, on the GTFXdsorption on the polymers obtained. This section also consideredhe recognition characteristic of the sorbent.
Fig. 1. Schematization of the preparation steps of the four kinds of M-MIPs, M-NIPsand Fe3O4 nanoparticles.
3. Results and discussion
3.1. Preparation of the Fe3O4 nanoparticles and the magneticpolymers
The preparation of several M-MIPs and M-NIPs described hereinis novel because it uses a high amount of oleic acid and a specialwashing process after preparation. These nanomaterials have beensuccessfully synthesized by a co-precipitation [10] method andthe difference between, imprinted and non-imprinted have beenobserved through affinity for a template. The recognition proper-ties of M-MIPs were higher than M-NIPs because of the presenceof binding sites “memory” or selective sites for GTFX on the M-MIPs. The hydroxide groups in the cavities were responsible tothe non-covalent bond (hydrogen-bonding) formed between thetemplate and sorbent. The preparation involved two kind of Fe3O4(unmodified and modified) previously prepared. The unmodifiedFe3O4 magnetite which was used to separately prepare M-MIP1and M-MIP2 was successfully coated with the MIPs matrix com-ponents (Table 1, Fig. 1 M-MIP1 and M-MIP2) while the Fe3O4modified with oleic acid and polyethylene glycol was also sepa-rately coated by MIP3 and MIP4 shells (Table 1, Fig. 1 M-MIP3 andM-MIP4). The change of Fe3O4 magnetite (in powder and suspen-sion form) color from black to brown during M-MIPs and M-NIPspreparation was one of a good visual indicator of the success ofthe coating process. It was found that the washing process whichis usually done to remove unreacted reagents, template and sol-vent residues, associate to the coating of oleic acid on the M-MIP,
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could be helpful in enhancing dispersibility solubility and recog-nition properties. During the last washing step, water was usedseveral times until the precipitation of the material in water wasvery slow. The polymers obtained by this technique were stable and
P. Dramou et al. / J. Chromatogr. A xxx (2013) xxx– xxx 5
F MIP4.F (a) puM
eafs(b
dttgnMawbto
ig. 2. (A) SEM of magnetic polymers: (a) M-MIP1, (b) M-MIP2, (c) M-MIP3, (d) M-e3O4, (c) M-MIP1, (d) M-MIP2, (e) M-MIP3 and (f) M-MIP4. (D) XRD patterns of
-MIP4.
ndowed with high magnetic property in water, DMSO, n-hexanes well as in other mixtures. The important limitations to consideror the applications of M-MIPs were in part solved herein by synthe-izing the amphiphilic magnetic polymer in polar aprotic solventDMSO) and successfully use it, in aqueous environment as well asiological fluids.
The recognition properties of MIPs in aqueous environmentseserve high standing, as water is the most important solvent sys-em for applications of M-MIPs. Most of the M-MIPs are formed andested in non-polar organic solvents because, they rely on hydro-en bonding and electrostatic interactions that are strongest inon-polar organic solvents. The not commonly appreciated is that-MIPs formed in organic solvents often retain their selectivity in
queous solvent systems. For this time, the selectivity of the MIPs
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as enhanced in aqueous solvent systems. This is surprisingly newecause, it is know that, that MIPs usually show optimal recogni-ion properties when they are tested in the same solvent as thene used in their preparation. Best reproduction and repeatability
(B) TEM images of M-MIP. (C) FTIR spectra of (a) the Fe3O4 nanoparticles, (b) PEG-re Fe3O4 nanoparticles; (b) PEG-Fe3O4; (c) M-MIP1; (d) M-MIP2; (e) M-MIP3; (f)
results in the nanomaterial preparation have been obtained withrelative standard deviations (RSD) less than 10% (n = 4).
3.2. Characterization of the Fe3O4 and the magnetic polymers
3.2.1. SEM and TEM observationThe nanoparticles forms of Fe3O4 magnetite and of M-MIPs or
M-NIPs obtained were spherical and smaller, assuming to be inthe range of nanometer. The SEM images of M-MIPs illustratedin (Fig. 2A) show different amplifications of spherical bead con-glomerated. The morphology is differing from the M-MIP1, 2, 3to the M-MIP4 corresponding respectively to (Fig. 2Aa–Ad). In the(Fig. 2Aa and Ab), the polymers beads are spherical with good shapeand let show some porosity.
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This was confirmed by experimental results with the powderobtained in the two polymers; M-MIP1 was dusty and very mildcompared to the other polymers, and then follows by the M-MIP2.Magnetic MIP-4 had the highest weight due to the double coating;
ig. 3. Three states of the M-MIP2: (A) precipitation state, (B) dispersion form, (C)agnetic separation.
s shown in the SEM image (Fig. 2Ad) some of its spheres were big-er when compared to other spheres. All the M-MIPs had internavities and were porous and spherical. This is due to the use of theispersant agent (PVP) during the polymerization process. More-ver, this observation confirms the successful grafting process onhe iron nanoparticles. It is well-known that the cavities play anmportant role in adsorption. They can increase the adsorptionapacity of polymers and improve the mass transfer rate for releas-ng and rebinding analytes. The TEM images shown in (Fig. 2B),llow us to assume that the magnetic polymers obtained fromhe iron nanoparticles are spherical with estimated size inferioro 500 nm. The magnetic nanoparticles (Fe3O4) forms the core, ofhe M-MIP while, the MIP forms the shell.
.2.2. Magnetic property observationAll the polymers generally have magnetic property. However,
here is variation in magnetic property which is due to differingmount of iron oxide used during the preparation of M-MIPs, theype of Fe3O4 (unmodified or modified), and the preparation mode,s shown; see Table 1. Therefore, the magnetic properties of theIPs are slightly inferior to that of the pure iron oxide. This is due
o the MIPs coating on iron oxide of the M-MIPs that tends to shieldts magnetic property. This property in M-MIPs was tested by pla-ing a magnet close to the assay tube containing aqueous solutionf M-MIPs (Fig. 3). It was observed that the materials respondedagnetically to an external magnetic fields when the magnet is
lose to the tube. The magnetic response vanished when the mag-et was moved far the tube. After encapsulation of the M-MIPs,his magnetic response of the polymers dropped but did not showny negative influence in the magnetic function. This was expectedecause the polymeric coating had effectively shielded the mag-etite. The polymers endowed with magnetic property could beuspended under common magnetic stirring during the loadingrocess. Moreover, they could also be rapidly isolated from the sam-le solution within a short time by placing a strong magnet closeo the sample in the separation step.
.2.3. Infrared observationThe infrared spectra of Fe3O4, PEG-Fe3O4 and M-MIP1 to M-
IP4 obtained, showed different main functional groups of theredicted structures with corresponding infrared absorption peakshown in (Fig. 2Ca–f) respectively. All compounds have many peaksn common, indicating that the main structure was not changed byhe modification. The peak at 3420 cm−1 is common to all com-ounds: and corresponds to the stretching vibration of O H bondf the hydroxyl groups. It can therefore be deduced that Fe(OH)2,
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e(OH)3 and FeOOH formed resulting from hydroxylation on theurface of Fe3O4. The peaks at 1632 cm−1, 1382 cm−1, 1080 cm−1
lso show the existence of Fe3O4 and derived elements. The sharp,trong of Fe-O stretching peak (590 cm−1) observed in the six
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spectra confirm the presence of iron oxide but in variable amount.This is shown by the slightly variations in peak (a–f). The typi-cal bands of PEG-Fe3O4 and M-MIPs were located at 2920 cm−1,2851 cm−1, due to the C H stretching vibrations of polymer chainsfrom the oleic acid and the PEG mixture. Other common absorp-tion bands are 1717 cm−1 (stretching vibration of C O bonds oncarbonyl groups) in which the O H bonds indicate the existence ofcarboxylic groups in the polymer. All these bands indicate the suc-cessful preparation of the M-MIP or M-NIP shell around the ironoxide beads.
3.2.4. X-ray observationThe X-ray power diffraction of the Fe3O4 nanospheres, PEG-
Fe3O4 and from M-MIP1 to M-MIP4 is shown in Fig. 2D.Comparatively, it indicates that Fe3O4 is the dominant phase in allthe samples JCPDS no. 19-0629. Five characteristics peaks for Fe3O4in the 2� region of 20–70◦, marked by their indices (2 2 0), (3 1 1),(4 0 0), (4 2 2), (5 1 1), and (440), were observed for all samples and itrevealed that the resultant particles were pure Fe3O4 with a spinelstructure [26]. It was also observed that, the polymerization pro-cess did not cause any phase change of Fe3O4. The intensity of thepeaks decreased slightly [26] from the Fe3O4 (Fig. 2Da) to PEG-Fe3O4 (Fig. 2Db) which show one additional new peak due to thecoating process. The peaks were generally decreasing from ‘a’ to ‘f’but were not completely disappeared in the M-MIPs (Fig. 2Dc–f);this is also due to the coating of MIP on the magnetic core. Accordingto the Scherrer equation [48], the average crystallite size after cal-culation which is based on the XRD pattern (3 1 1), is in the order ofnanometer value for the Fe3O4 and inferior to 500 nm for M-MIPs.This observation agrees well with the TEM images shown in Fig. 2B.
3.3. Validation of the UV-visible spectrophotometer analysis
Linear relation was observed between absorbance and concen-tration in accordance with Beer’s law [43]. The following calibrationcurve of equation Y = 0.0661X + 0.0038 with correlation coeffi-cient r2 = 0.9999 for GTFX, intercepts equal to 0.0038 and theslopes 0.0661 were found. Adsorption spectra for validation ofthe method as well as the calibration curve are shown in suppor-ting information (Fig. 1S). The standard calibration curves of GTFXdetermination exhibited good linearity over the range of concen-trations tested (0.25 to 30 �g mL−1). Sensitivity parameters suchas limits of detection (LOD) and of quantification (LOQ) were cal-culated to be: 0.075 �g mL−1 (S/N > 3) and 0.25 �g mL−1 (S/N > 10)respectively. This demonstrates the high sensitivity of the methods.
Precision was evaluated by measuring relative standard devia-tions (RSD) of intra and inter-day tests. The intra-day precision wasperformed by analyzing the drug solutions four times in one day.The inter-day precision was performed over four days. The resultsobtained prove that RSD of intra and inter-day tests were in theranged of 0.23 to 3.44% and from 0.92 to 4.2%, respectively.
3.4. Dispersion behavior in water, DMSO and n-hexane
All M-MIPs were well dispersed in three different solventpolarities (water, DMSO, n-Hexane) at the initial time seeFig. 4B. Homogeneous solution was observed when (water, DMSO,n-Hexane) was added to the M-MIPs powder (Fig. 4A). The obser-vation has been materialized by taking photo during different timeintervals, at 27 ◦C. Precipitation was observed after 30 min (Fig. 4C).The speed of precipitation differed from one M-MIP to another,and from one solvent to another. The variation was a function of
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the time. Fig. 4D–F are the photos taken after 60, 150 and 180 minrespectively. In general we have observed that, the dispersibility ofall M-MIPs decreased with time but the loading process was notaffected. Since precipitation is opposed to dispersibility, as time
P. Dramou et al. / J. Chromatogr. A xxx (2013) xxx– xxx 7
F inds oa e of th
etsiswMessrato
3
ihc(t
ig. 4. Photograph of the dispersion experiment in function of time through three kssays tube with de different powder of polymer (20 mg), B, C, D, E, F represent stat
lapsed increased, precipitation was observed for all M-MIPs inhree corresponding solvents. Precipitation speed decreased witholvent used in the following order: n-Hexane > H2O > DMSO. Thiss due to their physical and chemical properties (see Table 1S inupporting information). The precipitation decrease of all M-MIPsas also observed in the following order: M-MIP2 > M-MIP1 > M-IP3 > M-MIP4. This was also due to the specific characteristics of
ach compound. These results demonstrate the existence of thetructure-activity relationship of each M-MIPs prepared in thistudy. The dispersion behavior studied herein is original and noteported elsewhere. Few studies have made a brief dispersionssessment of some other nanomaterials such as (TiO2 nanopar-icles) [48], magnetic nanoparticles [30,32,49] but not deeply asur work developed herein.
.5. Selection of the better M-MIP
This process was done by comparing the adsorption behav-or of GTFX on polymers in aqueous solution. This experiment
Please cite this article in press as: P. Dramou, et al., J. Chromatogr. A (2013
as demonstrated the relationship between structure and physi-ochemical property of different polymers obtained. As shown inFig. 5) the results present that these M-MIPs have different adsorp-ion behaviors. Their difference is due to the different preparation
f solution 1, 2 and 3 are (1 mL) of water, DMSO and n–hexane respectively; A is thee mixture in 0, 5, 30, 60, 150 and 180 min.
mode, kind of product obtained, polymer amount used for load-ing process and drug concentration added in the solution. In thegraphic of Fig. 5, the concentration variation of four M-MIPs kinds(Fig. 5a, d, g), or of four M-NIPs (Fig. 5b, e, h) was plotted againstthe adsorption recovery. The last three graphics (Fig. 5c, f, i) arethe adsorption efficiency of the polymers. In Fig. 5 with graphicsa, d and g, the amount of the polymers used was 20, 30 and 40 mgrespectively. It was found that for all polymers, the loading recoveryincreased with increasing mass weight, while the adsorption effi-ciency decreased slightly from (c) toward (i). For the same polymer,having the same mass weight but at different drug concentrations,the loading recovery decreased from 1000 �g mL−1 to 100 �g mL−1
drug concentrations. This behavior could be explained by the exist-ence of two kinds of adsorption mode on the M-MIPs and M-NIPs.One is due to the imprinted recognition site (specific interactionscharacterized by low and high affinity sites) case of the M-MIPs andthe second is due to the powder surface adsorption ability (non spe-cific interactions) observable in the M-MIPs and M-NIPs. When thematerial amounts increase in the same solution, the second type of
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adsorption have a tendency to re-increase more slightly than thefirst. This is due to low availability of imprinted binding site. Thatmeans also the M-MIP3 and the M-MIP4 doesn’t have imprintingeffect, or it is low in the case of M-MIP1. Graphic g corresponds to
8 P. Dramou et al. / J. Chromatogr. A xxx (2013) xxx– xxx
F eous ca
tMhtDpf
3
tstw
Fn
ig. 5. Study on recovery and adsorption efficiency of 4 polymers with different aqu, b and c correspond to 20 mg, d, e, f to 30 mg at end g, h, I to 40 mg of polymers.
he highest recoveries for all polymers. However the M-MIP1 and-MIP2 were the best polymers due to their own properties. The
ighest adsorption efficiency is shown in the Graphic c. Thereforehe M-MIP2 shows the best efficiency due to its preparation mode.espite to all comparative studies, M-MIP2 was chosen as the bestolymer in this study and was used in the next experimental stepor its application in SPE of GTFX.
.6. Selectivity behavior of the magnetic polymers 2
The adsorption amount of GTFX, NRFX, CPFX, AMX representing
Please cite this article in press as: P. Dramou, et al., J. Chromatogr. A (2013
he selectivity behavior of the M-MIP2 and M-NIP2 and the corre-ponded chemical structure are shown in Fig. 6. It can be observedhat, the adsorption amounts of GTFX and its analogs on the M-MIPsere higher than those on the M-NIPs. That means the M-MIPs
ig. 6. (A) Selectivity behavior graphic of the magnetic polymers 2. Experimental condanomaterials for 3 h at room temperature. (B) Chemical structure of the template and an
oncentrations of GTFX in standard conditions to optimize the amphiphilic polymer;
provided high selectivity to GTFX and its structural analogs. Com-paring the adsorption amount of the analogous only on the M-MIP,it can be observed that the affinity toward the binding site decreasesfrom GTFX to the other analogous that is due to the use of abovechemical structure as template molecule during synthesis of the M-MIP2. The magnetic polymers selectivity decreases in function ofthe structural relation or similitude of the concerning drug withthe template (GTFX). The imprinted efficiency found by the Eq.(4) shown the following order for selectivity 2.28, 2.25, 2.01, and1.02 for GTFX, NRFX, CPFX, AMX respectively. The high selectiv-ity toward GTFX and analogous is mainly due to the affinity of the
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binding site of the M-MIP2 to the chemical structure of the templateand its analogs. It is also due to the oleic acid coated on the surfaceof the M-MIP2 which, one of the principal functions is to preserveto water, the rapid destruction of the hydrogen bonding formed by
itions: 1.5 mL of 500 �g mL−1 of analyte incubated by shaking with 40 mg of thealogous drugs.
P. Dramou et al. / J. Chromatogr. A xxx (2013) xxx– xxx 9
F ixed
totb
3
spianeHd1mwbpw(MtspisdsnTTt
ig. 7. Photograph of the solubility behavior of M-MIP2 in (A) one solvent, (B) Two m
he analyte with the matrix of the polymer. The adsorption amountf AMX is low because of the difference in its structure than theemplate molecule GTFX. All these results confirm the selectivityehavior of the M-MIP2.
.7. Solubility behavior of the magnetic polymers 2
Fig. 6 shows the solubility behavior of the M-MIP2 in differentolvents (water, DMSO, n-Hexane). Graphic (A) corresponds to theolymer in a single solvent while (B) and (C) show the polymer
n a mixture of two solvents before and after shaking. The images,, b and c correspond to the same amount of M-MIP2 in water;-hexane and DMSO respectively. Because of its amphiphilic prop-rty, the MIP2 was found to be soluble in all three solvents (Fig. 6A).owever when the polymer was added to a mixture of solvents ofifferent nature (polar aprotic, polar protic or apolar,) (see TableS in supporting information), the solubility behaviors of the poly-er changed considerably. When n-hexane was added to a, andater to b, B1 was obtained. The mixture formed two liquid phases
ecause of the difference in polarity of combining solvent. The nonolar phase (n-hexane) due to its low density when compared toater remains up in the two flasks. The addition of a second solvent
to a or b) did not change M-MIP dissolution in the medium. The-MIP remains dissolved in the original solvent. However, when
he dissolution medium was swapped with both flasks shacked iname time, thus, C1 was obtained. The behavior observed here is ahenomenon called Liquid-liquid phase-transfer particles [50,51],
t has been attributed to the change of the pH [52]. However, weuggest that pH is not the only factor. This behavior could also beue to the material properties (amphiphilicity). Tang et al. reportedimilar result by demonstrating the solubilization effect of gold
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anoparticles in two different solvents (water and chloroform) [53].he same mixture was utilized also in the report of Sasaki et al. [54].his behavior could be helpful for the selection of the right solventhat results in better extraction while taking into consideration the
solvent, (C) after shaking the two mixed solvent; (a) water, (b) n-hexane, (c) DMSO.
solubility medium of the template. In A2, both solvent are polar but(a) is protic and (b) aprotic. No phase separation (B2) was thereforeobserved after the addition of a second solvent (to a and c). TheM-MIP remains in the mixture solvent even after shaking (C2). InA3, c and d have different polarity. Besides, d is a polar aprotic; thusa separation phase was observed in B3 and the M-MIP remain dis-solved in the original solvent, even after shaking. The n-hexane hasa lower density than DMSO, which means that it is lighter and willform a separate upper layer. Unexpectedly, the polymer remainsin the lower layer of the flask even after shaking. We impute thisbehavior to the affinity of the polymer toward DMSO because it wasthe solvent used in the preparation of the polymer.
3.8. Adsorption isotherm behavior of the magnetic polymers 2toward GTFX
The M-MIP2 and M-NIP2 were selected for the determinationof the adsorption isotherm behavior toward GTFX because of theirsuperior properties cited above. The binding isotherms plotted inFig. 7A indicated that the amount of GTFX bound to the M-MIP2and M-NIP2 at binding equilibrium increased with increasing con-centration of GTFX. However, the amount of GTFX bound to theM-MIP2 was higher than that bound to the M-NIP2. This result isin agreement with our previous article [44]. Scatchard analysis wasalso used for evaluation of the adsorption of M-MIP2 and M-NIP2according to the following Eq. (5):
Q
[GTFX]= Qmax − Q
Kd(5)
Where Q is the amount of GTFX bound to the polymers at equi-librium; [GTFX] is the free GTFX concentration at equilibrium; Kd is
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the dissociation constant and Qmax is the apparent maximum bind-ing amount. The values of Kd and the Qmax can be calculated fromthe slope and intercept of the linear line plotted in Q/[GTFX] versusQ.
10 P. Dramou et al. / J. Chromatogr. A xxx (2013) xxx– xxx
Table 2Result of Scatchard analysis for M-MIP2 and M-NIP2.
M-MIP M-NIP
Binding site Ia IIb ILinear equation Y = −0.0135X + 0.2232 Y = −0.0002X + 0.0744 Y = −0.001X + 0.0396Apparent maximum binding/Qmax (�g mg−1) 16.532 372 39.6Equilibrium dissociation constant/Kd (�g mL−1) 74.07 5000 1000
a Lower binding capacity and highest binding affinity site corresponding to the left part of the fig b.b Higher binding capacity site and lowest binding affinity site corresponding to the right part of the Fig c.
Table 3Result of the magnetic solid phase extraction of gatifloxacin on the M-MIP2 and M-NIP2 in the humane urine and serum samples (n = 5).
a The adsorption efficiency of the magnetic molecularly imprinted polymer 2 calculated using the following equation: ̨ = QM-MIPs/QM-NIPs.
f the
ssTSa
3fl
esUeattc
aeaptpo
Fig. 8. Binding isotherms (a) Scatchard plot analysis o
As shown in (Fig. 7B), the Scatchard plot for M-MIP2 was not aingle linear curve, but consisted of two linear parts with differentlopes. Results of parameters studied in this section appear in theable 2. The binding of GTFX to the M-NIP2 was also analyzed bycatchard method (Fig. 7C). It revealed homogeneous binding sitess also shown in Table 2.
.9. Magnetic solid phase extraction of GTFX in spiked biologicaluids by the magnetic polymers 2
The Table 3 shows different results obtained by solid phasextraction (loading, elution) of GTFX in spiked human urine anderum samples using M-MIP2 or M-NIP2 as sorbent phase andV–vis spectrometry as analytical method. Good extraction recov-ry of this SPE technique using amphiphilic magnetic polymerss sorbent was obtained (Table 3). It is observed that the adsorp-ion efficiency � in the two matrices is superior to 2; this proveshe effective recognition property and selectivity of the M-MIP2ompare to M-NIP2 or other nanomaterials prepared herein.
Loading and elution recovery obtained herein is superior to 95%nd better than that obtained in our previous report [44,45]. Thextraction behavior of the sorbent used here was enhanced by itsmphiphilicity. The elution process was also good by this SPE. No
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retreatment such as filtration, centrifugation or pH adjustment ofhe biological fluids was needed and no interference of natural com-ounds (macromolecules, proteins, lipids etc.) present in the serumr urine samples was observed during extraction. The control of
binding of GTFX onto the M-MIP2 (b) and M-NIP2 (c).
urine and serum blank samples (i.e. without GTFX) was also good.This extraction technique is also simpler and faster than classicalSPE thanks to the presence of magnetic iron oxide in the polymer[55]. The separation of eluent from sorbent is easily performed byplacing an external magnet close to the tube. The analytical con-trols of GTFX in biological fluids are necessary for the treatmentefficacy of this drug by adjusting dosage and for avoiding its sideeffects. The magnetic nanomaterials tested here may be applied tothe extraction of other drugs and also used in the drug deliverysystem for nanotherapy Fig. 8.
4. Conclusion
This paper has reported the preparation of a novel amphiphilicmagnetic molecularly imprinted polymer selective for GTFX inaqueous media performed by a co-precipitation method. This syn-thetized nanomaterial obtained has been used as sorbent for SPE ofGTFX in biological fluids with good selectivity and high recovery.Behavior studies (dispersibility, solubility) in different solvents andwater demonstrate their good amphiphilicity, an important factorfor an efficient sorbent used in SPE. Briefly, due to its sensitive mag-netic property, high adsorption capacity and good release process,
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the M-MIP synthetized in this study is suitable to be used for SPE ofGTFX in biological fluids. The extraction technique is also simplerand faster than classical SPE thanks to the presence of magneticiron in the polymer. Moreover, this technique could be extended
o the preparation of GTFX-MMIP conjugate used in drug deliveryystem for different experimental and clinical studies of GTFX later.
cknowledgements
This work was supported by China Scholarship Consul (CSC)rant (No. 2009324T15), Guizhou Provincial Natural Science Foun-ation of China (Grant No. 20122288), the Graduate Students
nnovative Projects of Jiangsu Province (Program No CXZZ11 0812)nd by the Fundamental Research Funds for the “Central Uni-ersities” Program No. JKY2011008. The authors are delighted tocknowledge discussions with colleagues in their research group.
ppendix A. Supplementary data
Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.chroma.2013.7.075.
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