Application of Molecularly Imprinted Polymers (MIP)

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Article Application of Molecularly Imprinted Polymers (MIP) and Magnetic Molecularly Imprinted Polymers (mag-MIP) to Selective Analysis of Quercetin in Flowing Atmospheric-Pressure Afterglow Mass Spectrometry (FAPA-MS) and in Electrospray Ionization Mass Spectrometry (ESI-MS)

Maria Guć* and Grzegorz Schroeder Faculty of Chemistry, Adam Mickiewicz University in Poznań,Uniwersytetu Poznańskiego 8, 61-614 Poznań,Poland * Correspondence: [email protected]; Tel.: +48-61-829-1797; Fax: +48-61-829-1555

Received: 31 May 2019; Accepted: 25 June 2019; Published: 26 June 2019

Abstract: Molecularly imprinted polymer (MIP) and magnetic molecularly imprinted polymer (mag-MIP) for solid extraction and pre-concentration of quercetin have been successfully prepared by thermal polymerization method using quercetin (Q) as a template, acrylamide (AA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a cross-linking agent. The MIP and mag-MIP were successfully applied in analysis of quercetin by mass spectrometry (MS) methods. To perform ambient plasma ionization experiments, a setup consisting of the heated crucible, a ﬂowing atmospheric-pressure afterglow (FAPA) plasma ion source, and amaZon SL ion trap (Bruker, Bremen, Germany) was used. The heated crucible with programmable temperature allowed desorption of the analytes from MIPs structure which resulted in their direct introduction into the ion stream. The results of Q-MIP/Q-mag-MIP and FAPA-MS measurements were compared with those of the analysis of quercetin by the ESI-MS method without extractions and pre-concentration of analytes on polymers. Limits of detection (LOD) for quercetin solutions in both positive and negative ESI-MS 8 7 2 were established at 10− M and 10− M, respectively. The linearity (R = 0.9999) of the proposed analytical procedure for quercetin determination in positive ions was provided in the range between 4 7 10− M and 10− M. Moreover, the same parameters were established for FAPA-MS in positive ions, reaching LOD at 0.005 mg/gMIP and the linearity of the method in the range of 0.015–0.075 mg/gMIP with the correlation coeﬃcient value R2 = 0.9850.

Keywords: magnetic molecularly imprinted polymer (mag-MIP); molecularly imprinted polymer (MIP); quercetin; mass spectrometry (MS); ESI-MS; FAPA-MS

1. Introduction Reduction of the stages of sample preparation for quantitative and qualitative determination of analytes is one of the key tasks of contemporary analytical chemistry. One of the approaches proposed is based on the use of selective methods of extraction and signiﬁcant concentration of the analyte on solid supports in combination with mass spectrometry as the method of analyte detection [1]. Recently, much progress has been made thanks to the method of molecular imprint technology (MIT). The technology of production of molecularly imprinted polymer (MIP) [2–10] and magnetic molecular imprinted polymer (mag-MIP) [11] is based on the formation of complexes between the analyte/template/guest molecule and the functional monomer/host molecule. The analyte-monomer complex is then subjected

Molecules 2019, 24, 2364; doi:10.3390/molecules24132364 www.mdpi.com/journal/molecules Molecules 2019, 24, 2364 2 of 15 to polymerization initiated by temperature or irradiation (thermal or photoinitiation) during which in the presence of a cross-linking agent, a three-dimensional polymer structure is formed. At the subsequent stage, the template is removed from the polymer structure obtained, leaving empty cavities capable of binding the template molecules as they are complementary to these template molecules in size, shape and functional group positions [12–14]. The final properties of polymer scavengers depend on a number of parameters, including the types of monomers chosen, type of their interactions with the template, quantitative ratio of the monomer to the template, conditions of polymerization and the way of removing the template from the polymer (Soxhlet extraction, dialysis, thermal desorption). The polymers obtained are chemically stable, water insoluble, which permits their multiple uses in chemical analysis, and their synthesis process is relatively simple, reproducible and cheap. The only limitation of this method in chemical analysis for obtaining selected polymers dedicated to a given analyte, is the necessity of using, in the synthesis of MIP and mag-MIP, as a template the pure analyte in the amount permitting production of a required amount of the polymer [15,16]. Quercetin (Q), a flavonoid, multiring aromatic compound, is mainly used in food industry, medicine, in textile industry for dying of cotton and in chemical analysis. Quercetin is a flavonoid most commonly met in plants. It occurs in high concentrations in black elderberries and blueberries, close to 42 mg/100 g, in slightly lower concentrations it is found in cranberries, blackcurrants, bilberries, apricots, red grapes, cherries, blackberries, apples, strawberries, and raspberries. It should be noted that quercetin is nonuniformly distributed in particular parts of plants, e.g., the content of quercetin in unpeeled apple is close to 4.4 mg/100 g, while after peeling is drops to about 0.34 mg/100 g. Moreover, the content of quercetin increases with growing maturity of the fruit. In a bit higher concentrations is quercetin found in vegetables - its content is the highest in capers (~180.77 mg/100 g) and onion. The level of quercetin concentration depends on the species, e.g., in red onion it higher (~19.93 mg/100 g) than in yellow one (~13.27 mg/100 g), moreover its concentration is higher in the outermost scales [17–19]. Flowing atmospheric pressure afterglow mass spectrometry (FAPA-MS) is an analytical chemistry technique for sensitive detection of low molecular weight small organic compound. The method of analysis by mass spectrometry with the flowing atmospheric pressure afterglow ion source in the ambient atmosphere and has been proven to be a promising tool for direct and rapid determination of many compounds. The construction of the argon plasma torch and its operating parameters have a direct impact on the quality of the spectra obtained in the mass spectrometer. Argon plasma is obtained between the 2 electrodes inside the quartz tube. The first electrode is inside quartz tube and the second electrode is outside at the end of the quartz tube. The analytes were thermally desorbed and ionized by FAPA and identified in classic way by the mass spectrometer analyzer. Thermal desorption of analytes is a specific method of introducing a sample into a plasma stream. The order of release of the compounds depends on their volatility. The method offers fast and reliable structural information, with no need of pre-separation, and can be an alternative to the: EI-MS, GC-MS, ESI-MS; for fast analysis of organic compounds. This paper presents new analytical procedures for determination of organic compounds-quercetin in real samples with the use of mag-MIP for pre-concentration and FAPA-MS with direct thermal desorption of analytes. The combination of analyte pre-concentration technique with MIP/mag-MIP and FAPA-MS analysis created new method with decreased LOD and LOQ for several trace analytes. The method proposed has been compared to that of direct determination of quercetin in water solutions based on the use of ESI-MS.

2. Results and Discussion The procedure for determination of an analyte with the use of MIP/mag-MIP by FAPA-MS comprises the following stages. 1. Synthesis of Q-MIP/Q-mag-MIP. 2. Determination of the temperature of thermal desorption of analyte from Q-MIP/Q-mag-MIP structures. Molecules 2019, 24, 2364 3 of 15

3. Obtaining of the empty MIP/mag-MIP with cavities permitting selective adsorption in the system structure. 4. Selective sorption of the analyte from water solution of a studied sample on the empty MIP/mag-MIP. 5. Separation of Q-MIP/Q-mag-MIP system. 6. Determination of the analyte by FAPA-MS with thermal desorption of the analyte. To evaluate the proposed method for quercetin determination using MIP/mag-MIP by FAPA-MS, the performance of this method was compared with quercetin determination by ESI-MS.

2.1. Syntheses of Q-MIP/Q-mag-MIP At this stage of we must decide about the use of the polymers obtained, as the ability to selectively bind the analyte from the sample studied, in the conditions of competition between the analyte binding by the solvent (e.g., water) and the cavity substituents, depends on the choice of particles binding the analyte in the polymer and the character of polymerization [13]. There are two factors that have essential influence on MIP recognizability: one is steric memory-size and shape of binding cavities and the other is chemical memory-spatial arrangement of complementary functionality, both of which are correlated to the template-monomer interaction and porogen polarity. Yu and Mosbach [2] have reported that MIP for quercetin was prepared by using quercetin as template molecule and MAA or AA as functional monomer. Karaman Ersoy et al. [4] and Krnanova et al. [10] have studied the effectiveness of MIP prepared by using different functional monomers including MAA, AA, 4-vinylpyridine (4-VP), and found that the behavior of MIP towards quercetin was different in different functional monomers. Yu et al. [2], Xie et al. [5], and Song et al. [3] have prepared effective MIP for quercetin by using AA as a functional monomer. The polarity of the solvent, wherein MIP has been used for sorption of the analyte and the ability to form strong hydrogen bond between monomers-solvent is crucial for the choice of the synthesis method. The analyte and solvent molecules compete for the formation of bonds with terminal groups of monomers in the polymeric cavity. The quercetin molecule contains a carbonyl group and five phenolic hydroxyl groups, capable of forming hydrogen bonding interaction with amino-group of AA, carboxy-group of MAA and pyridyl-groups of 4-VP. On the other hand, in water the weakest hydrogen interaction with solvent molecules is observed for amide-groups of AA. From various MIP/mag-MIP systems, the MIP/mag-MIP obtained with the AA monomer is optimal for use in the analysis of quercetin in aqueous solutions. The basic parameter of the monomer selection was the type of interaction of its functional groups with the functional groups of the template. Acid-base properties of quercetin in aqueous solutions have been studied by Chabotarev [19] who also reported the dissociation constants (pK). The pK values assigned to the appropriate functional groups of quercetin are pK3-OH = 6.4, pK5-OH = 8.1, pK7-OH = 9.0, pK30-OH = 9.6, pK40-OH = 11.3, respectively. Analysis of the structure-reactivity correlation allows a conclusion that the 3-OH group in the ortho position to the electron acceptor carbonyl group is the first to undergo dissociation leading to the appearance of the first anionic form in the solution. The four -OH groups participate in the formation of bonds in the polymer cavities. Isotherms of quercetin adsorption for such a system have been presented by Yu et al. [14]. As reported, the adsorption capacity increased from 267 to 1080 ng/g when the initial concentration of quercetin was increased from 2 to 14 mg/L. The value of equilibrium dissociation constants (Kd) was determined from the Scatchard plots. Kd calculated for studied materials was 2.086 mg/g and 11.76 mg/L respectively. We obtained such a system (Figure S1). The system Q-MIPs, obtained by adsorption of quercetin from water solution by MIPs, can be separated from the water solution by filtration or centrifugation. These processes can be difficult for polymers of low molecular mass and in electrolyte solutions. A very promising solution is based on endowment of MIPs with magnetic properties and then separation of Q-mag-MIPs by a strong magnetic field. Hybrid materials with a defined structure and function find wider application in chemical analysis. Both, MIP and mag-MIP show similar sorption properties and can be used with similar Molecules 2019, 24, 2364 4 of 15 success, but mag-MIP is easier to isolate from the solution e.g., using a neodymium magnet. Magnetic hybrid materials having identical structure and properties as their non-magnetic analogues, (the same manufacturing costs) enable significant simplification and shortening of time of analytical procedures. Comparison of these two types of materials allows optimization of analytical procedures. The most commonly used molecule that is capable of endowment magnetic properties is Fe3O4. The first step to obtain mag-MIP is the preparation of magnetic nanoparticles, then the second step is to modify their surface or to functionalize the magnetic components. The third step is the polymerization in the presence of the template, functionalized monomer, and a cross-linking agent, on the surface of magnetic nanoparticles [11,13]. The process of mag-MIP synthesis is illustrated by the scheme in Figure S2. In the determination the imprinting factor of the studied MIPs next experiments was performed. 1 The standard solution (10 mL) of quercetin in water at an initial concentration of 10 µg mL− was mixed and incubated with 10 mg of Q-MIP, non-imprinted polymer (NIP), Q-mag-MIP, or magnetic non-imprinted polymer (mag-NIP), respectively at room temperature for 10 min. The concentration of quercetin in solution after separation of MIP/mag-MIP/NIP/mag-NIP was determined. The adsorption capacity (Q) of the quercetin to the imprinted polymers was determined using equation: Q = (C0 1 − CE)V/W; where C0 and CE (mg mL− ) represent the initial and the equilibrium solution concentration of the adsorbed compounds, respectively, V (mL) represents the volume of the solution, and W (mg) is the weight of the imprinted polymers. The values of imprinting factor (IF) are calculated for five repetitions 1 of measurements from the following equations: IF = QMIP/QNIP; where QMIP and QNIP (mg g− ) represent the adsorption capacity of Q-MIP and NIP, respectively. The value of IF for Q-MIP/NIP is 6.25, while for Q-mag-MIP/mag-NIP is higher and is IF = 7.75. These results indicate the higher selectivity of Q-mag-MIP versus Q-MIP and higher reliability for binding quercetin in the polymer structure. The products were MIP and mag-MIP capable of selective binding of quercetin from water 1 solutions. The solubility of quercetin in water is 0.261 mg mL− [20].

2.2. Characterization of Q-MIP/Q-mag-MIP

2.2.1. FTIR Spectra Quercetin, AA monomer and NIP, and Q-MIP were subjected to FTIR analysis. A strong and 1 broad absorption peak assigned to the stretching vibration of hydroxyl groups was found at 3300 cm− . Hydroxyl groups in the quercetin molecule formed intra- and/or inter-molecular hydrogen bonds. 1 The peak corresponding to vibrations of carbonyl group was found at 1656 cm− . In addition, the peaks assigned to stretching vibration of non-aromatic skeleton (ring C) in the quercetin molecule 1 1 1 were found at 1615 cm− , 1512 cm− , and 1432 cm− . For AA monomer, the main absorption bands 1 1 1 at around 3356 cm− , 1673 cm− , and 1613 cm− were assigned to the following vibrations: N–H stretching, being characteristic absorption peaks of primary acrylamide, C=O stretching, and C=C stretching, respectively [3,6]. The FTIR spectra of NIP/mag-NIP and Q-MIP/Q-mag-MIP were a useful tool for drawing conclusions on the structure of materials obtained (Figures S3 and S4). -1 1 For NIP and Q-MIP, the peaks at 1738 cm and 1733 cm− (strong) were attributed to the CO 1 stretching vibration of AA in materials, while the peak at 1678 cm− (strong) observed only for Q-MIP was attributed to the CO stretching vibration absorption from quercetin in materials. The broad peak 1 2830–3696 cm− was assigned to the asymmetric stretching vibration of N-H and hydrogen-bonded interaction between quercetin molecules and amid groups. For mag-NIP and Q-mag-MIP, similar signals were observed in the FTIR spectra [21].

2.2.2. Thermal Analysis Thermogravimetric curves recorded for the studied materials are shown in Figures1 and2. Molecules 2019, 24, x 5 of 15

Molecules 2019, 24, x 5 of 15 Molecules 2019, 24, 2364 5 of 15

Figure 1. Thermogravimetric curves for Q-MIP and non-imprinted polymer (NIP).

Figure 1. Thermogravimetric curves for Q-MIP and non-imprinted polymer (NIP). Figure 1. Thermogravimetric curves for Q-MIP and non-imprinted polymer (NIP).

Figure 2. Thermogravimetric curves for Q-mag-MIP and magnetic non-molecular imprinted polymer (mag-NIP).Figure 2. Thermogravimetric curves for Q-mag-MIP and magnetic non-molecular imprinted polymer (mag-NIP). The samples Q-MIP, Q-mag-MIP, and the corresponding NIP and mag-NIP have similar thermal Figure 2. Thermogravimetric curves for Q-mag-MIP and magnetic non-molecular imprinted polymer properties.The samples The polymers Q-MIP, are Q-mag-MIP, stable to about and the 250 corresp◦C, in thisonding range NIP a smalland mag-NIP mass loss have is observed similar thermal related (mag-NIP). toproperties. the loss ofThe water polymers from are the stable polymer to about structure. 250 °C, The in this greatest range massa small loss mass is observedloss is observed in the related range 300–430 C, it is more pronounced for Q-MIP and Q-mag-MIP than for NIP and mag-NIP, as it is to theThe loss samples◦ of water Q-MIP, from Q-mag-MIP, the polymer and structure. the corresp The greatestonding NIP mass and loss mag-NIP is observed have in similar the range thermal 300 – related to the release of quercetin from the materials structures. The melting point of quercetin is properties.430 °C, it is The more polymers pronounced are stable for Q-MIPto about and 250 Q-mag- °C, in thisMIP range than afor small NIP mass and lossmag-NIP, is observed as it is related related 316 C. In the range 330–350 C, the most intensive desorption of quercetin from the samples studied is toto the the◦ loss release of water of quercetin from the from ◦polymer the materials structure. struct The ures.greatest The mass melting loss pointis observed of quercetin in the isrange 316 °C.300 –In observed and in this range thermal desorption of the analyte from Q-MIP/Q-mag-MIP in FAPA-MS 430the °C, range it is more330–350 pronounced °C, the most for Q-MIP intensive and Q-mag-desorptionMIP ofthan quercetin for NIP fromand mag-NIP, the samples as it isstudied related is analysis was performed. toobserved the release and of in quercetin this range from thermal the materials desorption struct of theures. analyte The melting from Q-MIP/Q-mag-MIP point of quercetin isin 316 FAPA-MS °C. In analysis was performed. the2.2.3. range SEM 330 Images–350 °C, the most intensive desorption of quercetin from the samples studied is observed and in this range thermal desorption of the analyte from Q-MIP/Q-mag-MIP in FAPA-MS analysis2.2.3.The SEM was morphology Images performed. of the synthesized samples: Q-MIP/MIP/NIP and Q-mag-MIP/mag-MIP/mag-NIP was characterized using SEM. The SEM images (low— 500 and high— 30,000 magniﬁcation) are The morphology of the synthesized samples: Q-MIP/MIP/NIP× and Q-× mag-MIP/mag-MIP/mag- shown in Figures S5 and S6. It is seen that the surface of MIP after the template removal is cleaner than 2.2.3.NIP SEMwas characterizedImages using SEM. The SEM images (low—×500 and high—×30,000 magnification) that of polymer before the template removal of Q-MIP. are shown in Figures S5 and S6. It is seen that the surface of MIP after the template removal is cleaner TheThese morphology images reveal of the that synthesized the obtained samples: mag-MIP Q-MIP/MIP/NIP have spherical and morphology Q-mag-MIP/mag-MIP/mag- and a diameter less than that of polymer before the template removal of Q-MIP. NIPthan was 200 nm.characterized Due to their using small SEM. size, The these SEM nanoparticles images (low agglomerate—×500 and easily.high—×30,000 magnification) are shownThese in images Figures reveal S5 and that S6. the It is obtained seen that mag-MIP the surface have of MIPspherical after themorphology template removaland a diameter is cleaner less than2.2.4.than that 200 MS of Analyzesnm. polymer Due to before their small the template size, these removal nanoparticles of Q-MIP. agglomerate easily. These images reveal that the obtained mag-MIP have spherical morphology and a diameter less a) ESI-MS than2.2.4. 200 MS nm. Analyzes Due to their small size, these nanoparticles agglomerate easily. In order to establish the range of ESI-MS measurements that would permit qualitative and a) ESI-MS 2.2.4.quantitative MS Analyzes determination of quercetin in water solutions, in the range of negative or positive ions, a seriesIn of 10order standard to establish water solutions the rang ofe quercetinof ESI-MS were measurements prepared in whichthat would the concentration permit qualitative of quercetin and 4 9 a)variedquantitative ESI-MS from 10 determination− to 10− M. Theof quercetin solutions in were water introduced solutions, to in the the spectrometer range of negative using aor syringe positive pump. ions, 4 Measurements were made in the ranges of positive and negative ions. Exemplary spectra for 5 10− In order to establish the range of ESI-MS measurements that would permit qualitative× and quantitativeM concentration determination of quercetin of inquercetin solutions in are water presented solutions, in Figures in the range S7 and of S8. negative or positive ions,

Molecules 2019, 24, x 6 of 15 a series of 10 standard water solutions of quercetin were prepared in which the concentration of quercetin varied from 10−4 to 10−9 M. The solutions were introduced to the spectrometer using a syringe pump. Measurements were made in the ranges of positive and negative ions. Exemplary spectraMolecules for2019 5, 24× ,10 2364−4 M concentration of quercetin in solutions are presented in Figures S7 and S8.6 of 15 In the range of positive ions, the main signal is at m/z 303 [M + H]+, while in the range of negative ions-the one at m/z 301 [M − H]−. Analysis of the MS/MSn spectra permitted identification of + fragmentationIn the range pathways of positive of quercetin, ions, the in the main ranges signal of ispositive at m/z and303 negative [M + H] ions, while (Figures in theS9 and range S10). of negative ions-the one at m/z 301 [M H] . Analysis of the MS/MSn spectra permitted identification of The fragmentation pathways are consistent− − with literature data [22] and are presented in Figures S11 andfragmentation S12 for the pathways range of of positive quercetin, and in negative the ranges ions of, positive respectively. and negative To identify ions (Figuresof quercetin S9 and in S10).the samples,The fragmentation signals related pathways to the are molecular consistent peak with as literaturewell as to data the [signals22] and the are main presented fragmentation in Figures ions S11 wereand S12 used for the[23,24]. range It of was positive found and that negative the intensit ions, respectively.y of the signal To identify is higher of quercetin for negative in the samples,ions in concentrationsignals related of toquercetin the molecular to 5 × 10 peak−4 M, ashowever well as in to low the concentrations signals the main the signal fragmentation in the positive ions wereions beginsused [23 to,24 dominate]. It was found and thatthe thechanges intensity in its of theintensity signal isare higher more for linear negative in relation ions in concentrationto those in of quercetin to 5 10 4 M, however in low concentrations the signal in the positive ions begins to concentration. × − dominateIn order and to the establish changes th ine itsrange intensity of applicability are more linear of ESI in relationmethod tofor those determination in concentration. of quercetin directlyIn orderfrom water to establish solutions, the a range series ofof applicabilitymeasurements of were ESI methodperformed. for The determination limit of detection of quercetin (MS2) 2 indirectly the range from of water positive solutions, ions was a series10−8 M of and measurements in the range wereof negative performed. ions it The was limit 10−7 M. of detectionIn ESI analysis (MS ) 8 7 ofin the the lower range content of positive of quercetin ions was 10in− waterM and solutions, in the range the probability of negative of ions making it was error 10− inM. measurements In ESI analysis orof in the interpretation lower content of of results quercetin is lower in water for solutions,the measurements the probability in the of range making of positive error in measurementsthan negative ions.or in interpretationMoreover, on ofthe results basis isof lower analysis for theof the measurements peak at m/z in 303 the (range range ofof positive positive than ions), negative the signal ions. intensityMoreover, was on the linearly basis of analysisdependent of theon peak theat quercetinm/z 303 (range concentration of positive ions),in the the quercetin signal intensity solution was concentrationlinearly dependent range onfrom the 10 quercetin−4 to 10−7 M, concentration which permits in the quantitative quercetin solutiondetermination concentration of quercetin range in from this 4 7 range.10− to The 10− calculatedM, which permitscorrelation quantitative coefficient determination (R2) at 0.9999 of quercetin proves inthe this linearity range. Theof mentioned calculated 2 dependencecorrelation coe (Figurefficient S13). (R ) at 0.9999 proves the linearity of mentioned dependence (Figure S13). UsingUsing the ESI-MS methodmethod thethe content content of of quercetin querceti wasn was determined determined in waterin wate extractsr extracts of capers of capers and andonion onion to be to 0.00045be 0.00045 and and 0.000045 0.000045 M, M, which, which, expressed expressed in in the the dry dry mass mass of of capers capers and and onion onion gives gives a aquercetin quercetin content content of of 150 150 and and 15 15 mg mg/100/100 g, g, respectively. respectively. b) FAPA-MS b) FAPA-MS The ESI-MS method was applied for quercetin determination in water solutions to get reference resultsThe that ESI-MS could method be compared was applied with the for results quercetin obtained dete usingrmination MIP/ mag-MIPin water solutions as the selective to get extractionreference resultsagent, analytethat could concentration be compared and FAPA-MSwith the forresults quantitative obtained and using qualitative MIP/mag-MIP analysis ofas thethe analyte. selective In extractionthe methods agent, of flowing analyte atmospheric concentration pressure and FAPA-MS afterglow for mass quantitative spectrometry, and qualitative the molecules analysis are ionized of the analyte.by the production In the methods and of flow flowing of thermalized atmospheric ions pressure in plasma afterglow afterglow mass of sp helium.ectrometry, When the the molecules sample are ionized by the production and flow of thermalized ions in plasma afterglow of helium. When the containing the analyte is heated to 350 ◦C, it liberates the analyte which in the ionized form in a stream sampleof plasma containing is fed to thethe massanalyte spectrometer is heated to analyzer. 350 °C, Theit liberates structure the of analyte the measuring which in system the ionized is presented form inin a an stream earlier of paper plasma [25 is– 27fed]. to The the MS mass spectrum spectromet of quercetiner analyzer. recorded The structure in the FAPA of the technique measuring shows system one issignal presented at m/z 303in an in theearlier range paper of positive [25–27]. ions The (Figure MS spectrum3). of quercetin recorded in the FAPA technique shows one signal at m/z 303 in the range of positive ions (Figure 3).

Figure 3. Flowing atmospheric-pressure afterglow (FAPA) spectrum of quercetin in the ranges of + positive ions (m/z 303 [M + H] ). In the range of negative ions, the intensity of signals from quercetin m/z 301 is approximately 10 times smaller than that of the signal m/z 303 in positive ions (Figure4). Molecules 2019, 24, x 7 of 15 Molecules 2019, 24, x 7 of 15 Figure 3. Flowing atmospheric-pressure afterglow (FAPA) spectrum of quercetin in the ranges of positiveFigure 3. ions Flowing (m/z 303 atmospheric-pressure [M + H]+). afterglow (FAPA) spectrum of quercetin in the ranges of positive ions (m/z 303 [M + H]+).

MoleculesIn the2019 range, 24, 2364 of negative ions, the intensity of signals from quercetin m/z 301 is approximately7 of 10 15 timesIn smaller the range than of that negative of the ions, signal the m/z intensity 303 in positiveof signals ions from (Figure quercetin 4). m/z 301 is approximately 10 times smaller than that of the signal m/z 303 in positive ions (Figure 4).

− FigureFigure 4. 4. FAPAFAPA spectrum of quercetin in the ranges of negative ions (mm/z/z 301301 [M[M − H] ).). Figure 4. FAPA spectrum of quercetin in the ranges of negative ions (m/z 301 [M− − H]−). TheThe FAPA-MS FAPA-MS analysis of of the the ions generated generated as as a a result result of thermal desorption desorption of of the the analyte analyte The FAPA-MS analysis of the ions generated as a result of thermal desorption of the analyte moleculesmolecules from MIPMIP/mag-MIP/mag-MIP (Figures(Figures5 and5 and6) or6) NIPor NIP/mag-NIP/mag-NIP (Figures (Figures7 and 7 and8), shows 8), shows that nothat ions no molecules from MIP/mag-MIP (Figures 5 and 6) or NIP/mag-NIP (Figures 7 and 8), shows that no ionsat m /atz 303m/z positive 303 positive ions andionsm and/z 301 m/z negative 301 negative ions are ions generated, are generated, while the while analysis the analysis of the systems of the ions at m/z 303 positive ions and m/z 301 negative ions are generated, while the analysis of the systemscontaining containing the analyte the Q-MIP analyte/Q-mag-MIP Q-MIP/Q-mag-MIP shows that shows the ions that at them/z ions303 inat them/z range 303 in of the positive range ions of systems containing the analyte Q-MIP/Q-mag-MIP shows that the ions at m/z 303 in the range of positive(Figure9 ions), at (Figurem/z 301 9), in theat m/z range 301 of in negative the range ions of negative (Figure 10 ions) characteristic (Figure 10) characteristic of quercetin, areof quercetin, generated. arepositive generated. ions (Figure 9), at m/z 301 in the range of negative ions (Figure 10) characteristic of quercetin, are generated.

Molecules 2019, 24, x 8 of 15 FigureFigure 5. 5. FAPA-MSFAPA-MS spectrum spectrum of of MIP MIP in in the the range range of of positive positive ions. ions. Figure 5. FAPA-MS spectrum of MIP in the range of positive ions.

Figure 6. FAPA-MS spectrum of MIP in the range of negative ions. Figure 6. FAPA-MS spectrum of MIP in the range of negative ions.

Figure 7. FAPA-MS spectrum of NIP in the range of positive ions.

Figure 8. FAPA-MS spectrum of NIP in the range of negative ions.

Molecules 2019, 24, x 8 of 15 Molecules 2019, 24, x 8 of 15

Molecules 2019, 24, 2364 Figure 6. FAPA-MS spectrum of MIP in the range of negative ions. 8 of 15 Figure 6. FAPA-MS spectrum of MIP in the range of negative ions.

FigureFigure 7. 7. FAPA-MSFAPA-MS spectrum spectrum of NIP in the range of positive ions. Figure 7. FAPA-MS spectrum of NIP in the range of positive ions.

Molecules 2019, 24, x 9 of 15 FigureFigure 8. 8. FAPA-MSFAPA-MS spectrum spectrum of of NIP in the range of negative ions. Figure 8. FAPA-MS spectrum of NIP in the range of negative ions.

FigureFigure 9. 9. FlowingFlowing atmospheric-pressure atmospheric-pressure afterglow afterglow mass mass spectrometry spectrometry (FAPA-MS) (FAPA-MS) spectrum spectrum of of Q-MIP Q-MIP + inin the the ranges ranges of of positive positive ions ions ( (m/zm/z 303 [M + H]H]+).).

Figure 10. FAPA-MS spectrum of Q-MIP in the ranges of negative ions (m/z 301 [M − H]−).

The analytical results of helium plasma ionization source FAPA-MS experiments were obtained with the use of a solution of pure quercetin. Experiments were repeated three times. The relative standard deviation (RSD) obtained for all data did not exceed 15%. In order to determine the background signals, the spectra of particular matrices (NIPs) without the analytes were recorded. The limit of detection (LOD) is the concentration of a substance below which its identity cannot be distinguished from analytical artefacts. The limit of detection (LOD) was calculated according to the definition: LOD = mean blank value + 3 × standard deviation. The m/z signal of the analyte was three times higher than that of the noise level. In the experiments in which the LOD of pure compounds was determined, 20 μL of the solution of the analytes at different concentrations in methanol were introduced into the crucible and heated to about 300 °C. In the context of an analysis system, the linearity means that we assume a linear relationship between the concentration of analyte (x) and intensity of m/z signals (y), which can be written mathematically as y = f(x). It is a common practice to check the linearity of a calibration curve by inspection of the correlation coefficient (R2). A correlation coefficient close to unity (R2 = 1) is considered sufficient evidence to conclude that a perfect linear calibration has been achieved. Using different concentrations of pure analytes, different weights of the solid matrices with the analyzed compounds, it was found that the linearity of the analyte determinations was satisfied when the correlation coefficient of the function y = f(x) was greater than R2 = 0.9850. On the basis of measurements made for Q-MIP and Q-mag-MIP systems containing different amounts of quercetin, it was established that the signal intensity dependence on

Molecules 2019, 24, x 9 of 15

MoleculesFigure2019, 249. ,Flowing 2364 atmospheric-pressure afterglow mass spectrometry (FAPA-MS) spectrum of Q-MIP 9 of 15 in the ranges of positive ions (m/z 303 [M + H]+).

Figure 10. FAPA-MS spectrum of Q-MIP in the ranges of negative ions (m/z 301 [M H]− ). Figure 10. FAPA-MS spectrum of Q-MIP in the ranges of negative ions (m/z 301 [M − H]− ). −

TheThe analytical analytical results results ofof heliumhelium plasma ionization ionization source source FAPA-MS FAPA-MS experiments experiments were were obtained obtained withwith the the use use of of a a solution solution of of pure pure quercetin.quercetin. Ex Experimentsperiments were were repeated repeated three three times. times. The The relative relative standardstandard deviation deviation (RSD)(RSD) obtainedobtained for all data did not not exceed exceed 15%. 15%. In Inorder order to todetermine determine the the backgroundbackground signals, signals, thethe spectraspectra of particular matr matricesices (NIPs) (NIPs) without without the the analytes analytes were were recorded. recorded. TheThe limit limit of of detection detection (LOD) (LOD) isis thethe concentrationconcentration of of a a substance substance below below which which its its identity identity cannot cannot be be distinguisheddistinguished from from analytical analytical artefacts.artefacts. The limit limit of of detection detection (LOD) (LOD) was was calculated calculated according according to the to the definition:definition: LOD LOD= =mean mean blankblank valuevalue + 33 × standardstandard deviation. deviation. The The m/zm /signalz signal of ofthe the analyte analyte was was three three × timestimes higher higher than than that that of ofthe the noisenoise level.level. In In th thee experiments experiments in in which which the the LOD LOD of ofpure pure compounds compounds waswas determined, determined, 20 20µ μLL ofof thethe solutionsolution of the analytes at at different different concentrations concentrations in inmethanol methanol were were introduced into the crucible and heated to about 300 °C. In the context of an analysis system, the introduced into the crucible and heated to about 300 ◦C. In the context of an analysis system, the linearitylinearity means means that that we we assumeassume aa linearlinear relationship between between the the concentration concentration of ofanalyte analyte (x) (x)and and intensityintensity of ofm m/z/z signals signals (y), (y), which which can can be be written written mathematically mathematicallyas as yy= = f(x).f(x). It is a common practice practice to to check the linearity of a calibration curve by inspection of the correlation coefficient (R2). A check the linearity of a calibration curve by inspection of the correlation coefficient (R2). A correlation correlation coefficient close to unity (R2 = 1) is considered sufficient evidence to conclude that a perfect coefficient close to unity (R2 = 1) is considered sufficient evidence to conclude that a perfect linear linear calibration has been achieved. Using different concentrations of pure analytes, different calibration has been achieved. Using different concentrations of pure analytes, different weights weights of the solid matrices with the analyzed compounds, it was found that the linearity of the of the solid matrices with the analyzed compounds, it was found that the linearity of the analyte analyte determinations was satisfied when the correlation coefficient of the function y = f(x) was determinations was satisfied when the correlation coefficient of the function y = f(x) was greater than R2 greater than R2 = 0.9850. On the basis of measurements made for Q-MIP and Q-mag-MIP systems = containing0.9850. On different the basis amounts of measurements of quercetin, made it was for established Q-MIP and that Q-mag-MIP the signal systems intensity containing dependence diff onerent amounts of quercetin, it was established that the signal intensity dependence on the concentration of quercetin was linear in the quercetin solution concentration range 0.015–0.075 mg Q/g MIP, while the limit of detection of the analyte in Q-MIPs was 0.005 mg/g MIP. The procedure for determination of quercetin in water solutions with the use of MIP/mag-MIP. A portion of 100 mg of empty MIP, from which quercetin was removed by extraction with a solvent, should be added to 10 mL of the extract containing quercetin. After 30 min of vigorous stirring, the suspension should be centrifuged and the obtained Q-MIP system should be dried at a temperature up to 40 ◦C. Then the system should be placed on a heating table and analyzed by FAPA-MS. The analogous procedure with mag-MIP is similar, but the difference is that the Q-mag-MIP system is separated from the solution with the use of a neodymium magnet, which facilitated the analysis and shortened its time. Following this analytical procedure with MIP/mag-MIP, the content of quercetin in solutions was 9 determined up to the concentration of 10− M. The limit of detection FAPA-MS in the range of positive 9 8 ions was 10− M and in the range of negative ions it was 10− M. This concentration of quercetin solution should be assumed as the limit of quantification of the analyte by the method MIP/mag-MIP combined with FAPA-MS. The method was applied for determination of quercetin in negative ions FAPA-MS, in water solutions of capers (Figure 11) and onion (Figure 12) to be of 0.00055 and 0.000049 M, which, expressed Molecules 2019, 24, x 10 of 15 Molecules 2019, 24, x 10 of 15 the concentration of quercetin was linear in the quercetin solution concentration range 0.015–0.075 mgthe Q/gconcentration MIP, while of the quercetin limit of wasdetection linear of in the the analyte quercetin in Q-MIPs solution was concentration 0.005 mg/g MIP.range 0.015–0.075 mg Q/gThe MIP, procedure while thefor limitdetermination of detection of quercetinof the analyte in water in Q-MIPs solutions was with 0.005 the mg/g use MIP.of MIP/mag-MIP. AThe portion procedure of 100 for mg determination of empty MIP, of quercetin from whic inh water quercetin solutions was withremoved the use by ofextraction MIP/mag-MIP. with a solvent,A portion should ofbe 100 added mg ofto empty10 mL MIP,of the from extract whic containingh quercetin quercetin. was removed After by30 extractionmin of vigorous with a stirring,solvent, theshould suspension be added should to 10 be mL centrifuged of the extract and thecontaining obtained quercetin. Q-MIP system After should30 min be of driedvigorous at a temperaturestirring, the suspensionup to 40 °C. shouldThen the be system centrifuged should and be placedthe obtained on a heating Q-MIP table system and should analyzed be bydried FAPA- at a MS.temperature The analogous up to 40 procedure °C. Then the with system mag-MIP should is be simi placedlar, but on athe heating difference table andis that analyzed the Q-mag-MIP by FAPA- systemMS. The is analogous separated procedurefrom the solution with mag-MIP with the is use simi oflar, a neodymiumbut the difference magnet, is whichthat the facilitated Q-mag-MIP the analysissystem is and separated shortened from its thetime. solution with the use of a neodymium magnet, which facilitated the analysisFollowing and shortened this analytical its time. procedure with MIP/mag-MIP, the content of quercetin in solutions was determinedFollowing this up analyticalto the concentration procedure ofwith 10− 9MIP/mag-MIP, M. The limit of the detection content FAPA-MS of quercetin in thein solutionsrange of positivewas determined ions was up10− 9to M the and concentration in the range of of negative 10−9 M. ionsThe itlimit was of 10 detection−8 M. This concentrationFAPA-MS in theof quercetin range of solutionpositive ionsshould was be 10 assumed−9 M and inas thethe rangelimit of of quantificationnegative ions it of was the 10analyte−8 M. This by theconcentration method MIP/mag-MIP of quercetin Moleculescombinedsolution2019 should, with24, 2364 FAPA-MS.be assumed as the limit of quantification of the analyte by the method MIP/mag-MIP10 of 15 combinedThe method with FAPA-MS. was applied for determination of quercetin in negative ions FAPA-MS, in water solutionsThe methodof capers was (Figure applied 11) for and determination onion (Figure of quercetin12) to be inof negative0.00055 ionsand 0.000049FAPA-MS, M, in which, water inexpressedsolutions the dry mass ofin capersthe of dry capers (Figure mass and of 11) onioncapers and givesandonion on a (Figureion quercetin gives 12) a content quercetinto be of of 0.00055 158content and andof 17 158 mg0.000049 and/100 17 g, M,mg/100 respectively. which, g, Therespectively.expressed results obtained in Thethe dryresults were mass obtained adequate of capers were in and relation adequate onion to gives thein relation classic a quercetin to ESI-MS the classiccontent method, ESI-MS of but158 method, lowerand 17 LOD mg/100but lower allow g, of determinationLODrespectively. allow of The of determination compounds results obtained of with compounds were lower adequate concentrations with lowein relationr concentrations inenvironmental to the classic in environmentalESI-MS samples. method, samples. but lower LOD allow of determination of compounds with lower concentrations in environmental samples.

Figure 11. FAPA spectrum of Q-MIP after concentration of quercetin from solution of capers (m/z 301 Figure 11. FAPA spectrum of Q-MIP after concentration of quercetin from solution of capers (m/z [MFigure − H] 11.−). FAPA spectrum of Q-MIP after concentration of quercetin from solution of capers (m/z 301 301 [M H] ). [M − H]− −). −

Figure 12. FAPA spectrum of Q-MIP after concentration of quercetin from solutionof yellow onions Figure 12. FAPA spectrum of Q-MIP after concentration of quercetin from solution of yellow onions (m/z 301 [M H] ). (Figurem/z 301 12. [M −FAPA − H]−− ).spectrum of Q-MIP after concentration of quercetin from solution of yellow onions (m/z 301 [M − H]−). To comparison of two methods ESI- and FAPA-MS, the t-test was used for analysis pure quercetin in water solution and quercetin adsorbed in selective cavities in MIP polymers. This statistic test used to determine if there is a significant difference between the means of two groups, which may be related in certain features as intensity of signals m/z, LOD, the intensity of background signals and intensity of signals of ion fragmentation. For comparable of 10 parameters the two-tailed p value equals 0.3296. By conventional criteria, this difference is considered to be not statistically significant. Despite the lack of statistically significant difference in the results for ESI- and FAPA-MS methods, these methods fined application in the analysis of compounds in two different matrices. ESI-MS allows analyzes analytes in solution only, whereas in the FAPA-MS technique, in addition to analyzing solutions, it is possible to analyze compounds, without pre-treatment from a solid or polymer matrix.

3. Materials and Method

3.1. Materials and Chemicals All reagents used were commercial products. FeCl 4H O, FeCl 6H O, tetraethoxysilane (TEOS) 2· 2 3· 2 [H226, H319, H332, H335], hydrochloric acid (HCl), citric acid, sodium hydroxide (NaOH), ammonia solution (NH4OH), acrylamide (AA) [H350, H340, H301, H361f, H372], ethylene glycol dimethacrylate Molecules 2019, 24, 2364 11 of 15

(EGDMA) [H315, H319, H335], 2,20-azobisisobutyronitrile solution 0.2 M in toluene (AIBN) [H242, H302, H412], Aluminum Oxide Activated, basic, Brockman I, 3-metacriloxipropiltrimetoxissilano (MPS), quercetin (Q) [H301], and all solvents (toluene [H225, H304, H336], chloroform [H302, H331, H351], N,N-Dimethylformamide (DMF) [H226, H360D, H312], ethanol, acetic acid of the purity grade p.a.), were obtained from Sigma-Aldrich (St. Louis, MO, USA).

3.2. Instruments Almost all materials studied were tested by Fourier transform infrared spectroscopy and thermogravimetric method. The infrared spectra were taken on an IFS 66 v/s Fourier Transform Infrared (FTIR) spectrophotometer from Bruker (Hamburg, Germany), equipped with an MCT detector (Bruker, 1 1 Hamburg, Germany, 125 scans, resolution 2 cm− ). The spectra were recorded in the 400–4000 cm− range for KBr pellets. In order to confirm the process of functionalization of studied materials, they were subjected to thermogravimetric analysis (TGA). The measurements were performed on a Setsys 1200 1 thermogravimetric analyzer (Setaram, Caluire-et-Cuire, France) with a heating rate of 10 ◦C min− from room temperature to 1000 ◦C under helium atmosphere. Scanning electron microscopy (SEM) images were acquired with QUANTA 250 FEG, FEI (Quanta, Livonia, MI, USA). ESI-MS and ESI-MSn spectra were recorded using an amaZon SL ion trap (Bruker, Bremen, Germany) equipped with an electrospray ion source in infusion mode. The sample solution was 1 introduced into the ionization source at a flow rate of 5 µL min− using a syringe pump. The apparatus was operated using the so-called “enhanced resolution mode” (mass range: 50–2200 m/z, scanning rate: 8100 m/z per second). The capillary voltage was set at 4.5 kV and the endplate offset at 500 V. − − The source temperature was 80 ◦C and the desolvation temperature was 250 ◦C. Helium was used as 1 1 the cone gas and desolvating gas (nitrogen) at flow rates of 50 L h− and 800 L h− , respectively. The mass spectrometer (Bruker, Germany) was operated in the ESI positive and negative ionization mode. In MSn experiments, the width of the selection window was set at 2 Da and the amplification of the excitation was set according to the experiment (from 0.2 to 1.5 V). Mass spectrometers were equipped optionally in FAPA ambient plasma source (Figure 13) NOVA011 (ERTEC, Wroclaw, Poland). The experimental details for FAPA method are presented in earlierMolecules publications 2019, 24, x [26,27]. 12 of 15

a c

FigureFigure 13. The 13. systemThe system used used to carry to carry out theout experiment:the experiment: (a) mass(a) mass spectrometer spectrometer inlet, inlet, (b) ( heatingb) heating system, (c) FAPA-ionsystem, (c source.) FAPA-ion source.

3.3. Synthesis3.3. Synthesis

3.3.1.3.3.1. MIP MIP MIPMIP was was synthesized synthesized by the the method method described described in literature in literature [15]. The [115 mmol]. The template 1 mmol molecule- template molecule-quercetinquercetin and the and4 mmol the functional 4 mmol monomer-AA functional monomer-AA were dissolved in were a mixture dissolved of 5 ml in chloroform a mixture of and 5 mL DMF. Subsequently, previously purified with Aluminum Oxide Activated, basic, Brockman I, 20 mmol cross-linking agent-EGDMA were added. The ratio of the template:monomer:crosslinking agent was 1:4:20, respectively. The pre-polymerization solution was sonicated and purged with nitrogen for 40 min. Afterwards, 1.25 mL of the initiator-AIBN was added. The tube was sonicated and purged with nitrogen for additional 20 min, sealed and placed in an oven for 24 h at 60 °C. After polymerization, Q-MIP was dried under reduced pressure, and ground using mortar and pestle. The particles of quercetin were extracted with a mixture of ethanol and acetic acid (9:1, v:v) via Soxhlet extraction for 90 h. Then, the final product MIP was dried at 60 °C under vacuum and ground.

Molecules 2019, 24, 2364 12 of 15

5 ml chloroform and 5 mL DMF. Subsequently, previously puriﬁed with Aluminum Oxide Activated, basic, Brockman I, 20 mmol cross-linking agent-EGDMA were added. The ratio of the template:monomer:crosslinking agent was 1:4:20, respectively. The pre-polymerization solution was sonicated and purged with nitrogen for 40 min. Afterwards, 1.25 mL of the initiator-AIBN was added. The tube was sonicated and purged with nitrogen for additional 20 min, sealed and placed in an oven for 24 h at 60 ◦C. After polymerization, Q-MIP was dried under reduced pressure, and ground using mortar and pestle. The particles of quercetin were extracted with a mixture of ethanol and acetic acid (9:1, v:v) via Soxhlet extraction for 90 h. Then, the ﬁnal product MIP was dried at 60 ◦C under vacuum and ground.

3.3.2. Mag-MIP Mag-MIP was synthesized by the method described in literature [11]. The magnetite nanoparticles (Fe3O4) were synthesized in a two-step procedure. Firstly, magnetite (Fe3O4) was synthesized by the method described in literature [28] by using FeCl 4H O and FeCl 6H O as precursors. Secondly, 2· 2 3· 2 the surface-functionalized magnetite was produced via hydrolysis and condensation of the TEOS organosilane agents. FeCl 4H O (2 g) and FeCl 6H O (5.2 g) were dissolved into 25 mL deoxygenated 2· 2 3· 2 water, and then 0.85 ml of concentrated HCl were added. The resulting solution was added dropwise into 250 mL of 1.5 M NaOH solution upon vigorous stirring and N2 protection at 80 ◦C. The synthesized magnetic nanoparticles (MNPs) were separated from the solution by a powerful magnet and washed with 200 mL deionized water three times. Fe3O4 was used for the preparation of a stable aqueous suspension. A total of 10 mL of an aqueous solution of citric acid (0.5 g/mL) was added to the rigorously stirred suspension of washed nanoparticles. The pH value was set to 5.2 with a concentrated ammonia solution and heated to 80 ◦C. After 90 min, the pH value of the solution was elevated to 10. Lastly, the suspension was centrifuged for 5 min at 4000 rpm to remove any agglomerated nanoparticles. Next, silica coat was fabricated on the surface of Fe3O4 MNPs by the sol-gel method. 100 mL of ethanol containing 4 mL TEOS were added in the above prepared stable aqueous suspension of Fe3O4 MNPs, followed by stirring at ambient temperature for 6 h. After rinsing with ethanol and water several times, the obtained Fe3O4@SiO2 MNPs were dried at 60 ◦C. In the next step, Fe3O4@SiO2 was modified by MPS. For this purpose 250 mg of this material was dispersed in 50 mL of anhydrous toluene containing 5 mL of MPS and the mixture was allowed to react at 60 ◦C for 24 h under dry nitrogen. The mixture was filtered through a membrane, washed with toluene and dried in vacuum. The silanized material Fe3O4@SiO2-MPS was obtained. Then, mag-MIP was prepared by polymerization of 0.2 mmol template-quercetin and 0.8 mmol functional monomer-AA in 30 mL of a porogenic solvent-ethanol. The mixture was shaken in ultrasound bath at 25 ◦C for 1 h, then 200 mg of Fe3O4@SiO2-MPS were added into the system and the mixture was shaken for another 1 h. Furthermore, 4.0 mmol of the cross-linking agent-EGDMA and 1 mL of the initiator-AIBN were added into the system and the mixture was sonicated at 80 ◦C for 24 h. After polymerization, Q-mag-MIP was dried under reduced pressure, and ground using mortar and pestle. Afterwards, the template was removed after polymerization by Soxhlet extraction using a mixture of ethanol: acetic acid (9:1, v:v) as an eluent, which was replaced every 90 h. The obtained final product, mag-MIP was dried and ground. However, some water remained bound in the polymer structure. The level of the quercetin/template elimination was controlled by two techniques in the solution after extraction-using the ESI-MSMS technique and the FAPA-MSMS technique in dry MIPs and mag-MIPs. It was determined that after the extraction process, the materials contained about 1% and 5% of quercetin in the MIP and mag-MIP, respectively. Further extraction or solvent change did not remove the template in 100%.

3.3.3. NIP and mag-NIP As a control experiment, non-imprinted polymer (NIP) in the absence of quercetin during the polymerization was also prepared and treated in the identical manner. The magnetic non-molecular Molecules 2019, 24, 2364 13 of 15 imprinted polymer (mag-NIP) was also prepared in order to compare its behavior with that of mag-MIP. The procedures were the same until the polymerization with AA, which was carried out in the absence of quercetin, unlike for mag-MIP.

4. Conclusions A new method for determination of quercetin, based on the use of MIP/mag-MIP dedicated to this analyte, was proposed. Application of the FAPA-MS method, a modern tool enabling one-step, direct testing of organic substances contained in the solid phase, allows not only their detection, but also in combination with molecularly imprinted techniques it allows development of optimal analytical methods for trace analytes determination in diluted solutions. The range of this method application was established and the results obtained using this method was compared with those achieved by the classical ESI-MS method for determination of quercetin in water solutions. On the basis of a combined use of molecular imprinting technique and the innovative FAPA-MS analytical method, a new, fast and eﬀective method for determination of bioactive substances of low molecular mass. High-quality results in the present methods are obtained in particular for compounds of molecular mass up to 500 D as quercetin, because this technique uses the thermal desorption of analytes from a polymer matrix. The compounds not thermal desorbed signiﬁcantly from polymer matrices do not ionize in the plasma stream and do not generate signals in the mass spectrometer. Compounds with higher molecular weights can be analyzed using the FAPA-MS technique by introducing the sample directly into the plasma stream.

Supplementary Materials: The following are available online, Figure S1: The steps for the preparation of molecularly imprinted polymer (MIP). Figure S2: General steps for the preparation of magnetic molecularly imprinted polymer (mag-MIP). Figure S3: The FTIR spectra of NIP (a) and Q-MIP (b). Figure S4: The FTIR spectra of mag-NIP (a) and Q-mag-MIP (b). Figure S5: SEM images of the Q-MIP (a,b); MIP (c,d); NIP (e,f). Figure S6: SEM images of Q-mag-MIP (a,b); mag-MIP (c,d); mag-NIP (e,f). Figure S7: ESI spectrum of quercetin in the range of positive ions (m/z 303 [M + H]+ and 325 [M + Na]+). Figure S8: ESI spectrum of quercetin in the range of negative ions (m/z 301 [M H]− and 603 [2M H]−). Figure S9: ESI fragmentation spectrum of quercetin in the range of positive ion (m−/z 303 [M + H]+). Figure− S10: ESI fragmentation spectrum of quercetin in the range of negative ion (m/z 301 [M H]−). Figure S11: Fragmentation pathways of quercetin in the range of positive ions. Figure S12: Fragmentation− pathways of quercetin in the range of negative ions. Figure S13: Intensity of the positive signal at m/z 303 versus quercetin concentration in solution (M). Author Contributions: Conceptualization, M.G. and G.S.; methodology, M.G. and G.S.; software, M.G.; validation, M.G.; formal analysis, M.G.; investigation, M.G. and G.S.; resources, M.G. and G.S.; data curation, M.G.; writing—original draft preparation, M.G.; writing—review and editing, G.S.; visualization, M.G.; supervision, G.S.; project administration, Grzegorz Schroeder; funding acquisition, G.S. Funding: This work was supported by National Grant no. 2016/21/B/ST4/02082, provided by the National Science Centre. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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