Green Development of Polymeric Dummy Artificial Receptors

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Green Development of Polymeric Dummy Artificial Receptors with Affinity for Amide-based Pharmaceutical Impurities Raquel Viveiros, Vasco D. B. Bonifácio, William Heggie, and Teresa Casimiro ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b02948 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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ACS Sustainable Chemistry & Engineering

Green Development of Polymeric Dummy Artificial Receptors with Affinity for Amide-based Pharmaceutical Impurities Raquel Viveirosa,b, Vasco D.B. Bonifácioc, William Heggieb, Teresa Casimiroa* aLAQV-REQUIMTE,

Departamento de Química, Faculdade de Ciências e Tecnologia,

Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal bHovione cCQFM-IN

FarmaCiencia SA, R&D, Sete Casas, 2674-506 Loures, Portugal

and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico,

Universidade de Lisboa, Av. Rovisco Pais,1, 1049-001 Lisboa, Portugal *Corresponding author: Teresa Casimiro, E-mail: [email protected]

ABSTRACT A simple, clean and sustainable technology has been applied to the development of amide-based artificial receptors for a pharmaceutical impurity - acetamide. Artificial receptors in the form of Analogue Template Molecularly Imprinted Polymers (AT-MIPs) were produced in supercritical carbon dioxide (scCO2) medium using methacrylamide and ethylene glycol dimethacrylate as functional monomer and crosslinker, respectively. AT-MIPs were obtained in high yields, easyto-handle and ready-to-use solids. The materials were characterized by Fourier Transform Infrared spectroscopy, Nitrogen Adsorption Porosimetry, Scanning Electron Microscopy and Morphologi

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G3 particle size analysis. The affinity properties of AT-MIPs for three amides (acetamide, benzamide and pivalamide) were assessed by packing the affinity materials into blank columns and evaluating their profile under static and dynamic modes. The size and shape of the cavities created on the artificial receptors had a strong impact on the material´s performance. The results suggest a potential use of 3D-shaped artificial receptors as an efficient device for the removal of amide-based compounds, whose dimensions are equal or lower than the template molecule, from pharmaceutical crude mixtures.

KEYWORDS: Supercritical carbon dioxide; Green chemistry, Genotoxic impurity; Dummy template; Molecularly imprinted polymer; Solid-phase extraction; Affinity purification; Amide-based compounds

INTRODUCTION Acetamide (ACET) is a low molecular compound, well-known as impurity at the final stages of drug manufacture.1,2 Selective removal of ACET is of special interest because of its potential genotoxic behavior.3 Since 2006, the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA)4,5,6 have imposed strict limits on content of genotoxic compounds in final drug products, which has led pharmaceutical companies to seek even more efficient procedures that could address the problem under still cost-effective processes. Therefore, there is a particular interest in processes that can efficiently remove genotoxic compounds down to levels below those defined by regulatory entities.


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Typical purification processes such as chromatography, nanofiltration, recrystallization, resin purification, extraction, activated carbon powder treatments, distillation and hybrid processes are typically adopted by the pharmaceutical industry,7,8 often with low specificity and, in some cases, at high cost. Affinity structures are appealing alternatives to address this problem due to their high specificity for the removal of contaminants from crude mixtures.9–11 Molecularly imprinted polymers (MIPs) are artificial receptors within molecularly imprinted sites for specific target compounds. This enhances the merit of adsorption materials in these purification processes. Moreover, these materials are very stable under harsh conditions; they are robust, reusable and are prepared in a cost-effective manner thus making them a suitable for application in many fields where selective affinity is involved. The most straightforward way to prepare MIPs is by using a non-covalent strategy in which, template, monomer and crosslinker are employed in the construction of a crosslinked matrix.12 In this process, the template molecule can be a real template, i.e. the molecule for which the affinity is wanted, or an analogue template molecule, a similar molecule in terms of functionality, size and conformation and is normally of the same molecular family as the real template. This last approach is also referred to as dummy templating. Dummy templating is used for a variety of reasons, which include cases where use of the real template could make the preparation process hazardous (i.e. explosive or highly toxic) and when the cleavage of the real template molecule from the matrix is problematic and thus effects the accuracy of the analysis and quantification of the final polymer.13 An additional advantage of the analogue templating approach is that it can be used to fine tune the cavity size of the artificial receptors and produce high affinity materials for a family of compounds.


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The analogue templating approach has been used in the production of affinity materials to remove contaminants, such as fluoroquinolones and sulfonamides,14 theanine,15 and bisphenol A from water,16 resveratrol from wine,17 purification of prednisolone18 and, more recently, have been applied on food, environmental and biological mediums.13 However, the major limitation of using analogue template molecularly imprinted polymers (AT-MIP) is the potential lower molecular recognition in comparison to the use of the real template. To achieve a worthy AT-MIP strategy, the choice of the analogue template and functional monomer are critical19 in assuring a high binding capacity and selectivity. In general, analogue templates are selected based on molecular structures of analogous molecules whereas the choice of monomer is empirical. Consequently, the efficiency of the final AT-MIP cannot be predicted20 and must be tested and evaluated experimentally. Recently, green solvents, such as ionic liquids, have been used in the production of AT-MIPs.21 However, to the best of our knowledge, no report of the use and development of scCO2 in the preparation of AT-MIPs has been made. scCO2 has been successfully applied in MIP synthesis22 where the final properties of the resulting materials show significant advantages such as, the high yield preparation of ready-to-use powders which are; free-flowing, sterile and easy-to-handle. Amongst the different fields where scCO2 has already shown and added value compared to traditional approaches are: drugs for oral drug delivery,23,24 removal of pollutants from fuels and environment,22–25 switchable sensors,26 separation of valuable targets from environment27 and selective removal of pharmaceutical impurity.9,28,29 In this study, AT-MIPs were developed to target ACET by using benzamide (BENZ) and pivalamide (PIV) as planar- and 3D-shaped analogue template molecules using methacrylamide (MAM) and ethylene glycol dimethacrylate (EGDMA), as the functional monomer and crosslinker


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in scCO2, respectively (Figure 1). It is expected that the cavities created by these template molecules reflect the shape of the templates themselves. The AT-MIPs are assumed to have quasiplanar and 3D-shaped affinity cavities and will be referred using this nomenclature hereinafter. All polymers were characterized by FTIR, SEM, ASAP, average particle size and particle size distribution and their performance to selectively remove ACET and analogue molecules evaluated through dynamic binding experiments.

Figure 1. Molecular structures of acetamide and dummy templates, functional monomer and crosslinker.

EXPERIMENTAL SECTION Materials. Benzamide (BENZ, 99% purity), pivalamide, (PIV, 98% purity), acetamide (ACET, 99% purity), methacrylamide (MAM, 99% purity) ethylene glycol dimethacrylate (EGDMA, 98%


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purity) were purchased from Sigma-Aldrich. Azobis(isobutyronitrile) (AIBN, 98% purity) was purchased from Fluka. HPLC grade acetonitrile (ACN) from Carlo Erba was used for chromatographic separations. SPE syringes Supelclean TM LC-Ph SPE Tubes 3 mL was purchased from Supelco. Trifluoracetic acid (TFA) was obtained from Applied Biosystems. Carbon dioxide was obtained from Air Liquide with purity better than 99.998%. All chemicals were used without further purification. Development of planar and 3D-shaped affinity cavities on AT-MIPs using scCO2. AT-MIPs were prepared in scCO2 following the methodology described previously reported.30 Typicallly, to synthesize MIPs, 1 mmol of analogue template (BENZ or PIV), 4 mmoles of MAM, 20 mmol of the EGDMA and 1 wt% of AIBN were placed inside of a stainless steel high-pressure reactor with 33 mL of capacity where two aligned sapphire windows were used and a TeflonTM magnetic stir bar. The reactor was submerged in a water bath at 65 ˚C, which is the optimum conditions for AIBN initiation and was achieved by using an open bath circulator Julabo (± 0.1 ˚C). Carbon dioxide raised up to 21 MPa and the polymerization reaction proceeded during 24 hours under stirring. Under initial conditions of temperature and pressure, all the reactants were completely dissolved, assuring a single homogeneous phase in supercritical environment. After the polymerization to be complete, the copolymer was washed with fresh stream of CO2, slowly during 1 hour to eliminate the template molecule and rinse out the reactants that have no reacted. The control polymers, non-imprinted polymers (NIPs) were produced using the same methodology without addition of template. ScCO2-assisted desorption of analogue template molecules from AT-MIP matrices. The desorption of analogue template molecules were assessed by using an extraction process apparatus in continuous mode, and carried out using methodology described in a previous publication.31


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Basically, a cylindrical vessel was packed with AT-MIPs (MAM-MIP-BENZ or MAM-MIP-PIV), connected with high-pressure vessel (33 mL) containing 3 mL of ACN. The carbon dioxide was loaded up to 21 MPa and the process run in continuous mode for 3 hours allowing to extract all analogue template molecules used in the polymerization process. All apparatus were submerged at 40 ˚C using a thermostatted water bath and a continuous flux of scCO2 was used. Residual amount of analogue template within the MIP was assessed by dispersing 20 mg of crushed ATMIPs in ACN (3mL) for 3 days under stirring, followed by filtration and quantification by HPLCUV as described in the Chromatographic analysis section. Characterization techniques. Scanning Electron Microscopy analysis was assessed on a Hitachi S-2400 equipment, using an accelerating voltage set to 15 kV. Copolymers were placed on aluminum stubs using carbon tape and were coated with gold. Theoretical drawings were performed in ChemDraw version 15 and were further fed in Molinspiration version 2016.02 (www.molinspiration.com), a cheminformatics online software, for the calculation of the molecular properties (total surface area and size) of the analogue templates (BENZ or PIV) and ACET. Particle size distribution and particle size diameters of the copolymers were assessed using a Morphologi G3 particle characterization equipment from Malvern Instruments Limited. Copolymers were dispersed as dry powders (triplicated samples were used) using an automated dispersion system onto a glass plate (conditions: sample volume 13 mm3, injection pressure: 4 bar, injection time: 40 ms, setting time: 120 s) and more than 30 000 particles analyzed. The particle images were acquired using a 50x magnification lens with resolution range between 0.5–100 µm. This automatic system was used to determine the size and shape of copolymers synthesized. An accelerated surface area and porosimetry system (ASAP 2010 Micromeritics) was used to determine the specific surface area and pore size diameter of the copolymers. Before 12h to the


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measurements, the samples were degassed under nitrogen flow at 70 °C. FT-IR analyses were performed on a Bruker Tensor 27, acquiring 16 scans and 1 cm-1 resolution, in the 4000-400 cm-1 region using KBr pellets (1:5 polymer/KBr mass ratio).

Analogue template MIP binding tests Static binding tests. Static binding affinity Q (mmol.g-1) was determined using equation 1:

𝑄=

(𝐶0 ― 𝐶).𝑉 𝑊

(1)

where the amide-based concentration solutions (mg.mL-1) (C0 and C) were determined in the beginning and in the end of sorption test; the volume of the solution is V and the sample weight W (g). The ability of the polymer to bind the molecules were evaluated at 0.25 mg.mL-1. To perform the binding test, 20 mg of AT-MIPs and control were weighted and placed into SnakeSkinTM dialysis membranes and were put in contact with 50 mL of template solution, for 24 h at 100 rpm stirring. In the end, the samples were filtered and evaluated on a HPLC-UV equipment. Dynamic binding tests. Dynamic binding tests were assessed on a solid phase extraction apparatus and the binding affinity Q (mmol.g−1) was determined by using equation 1. The 3 mL volume of solid-phase extraction cartridges, were introduced individually with 20 mg of each affinity polymer, planar- and 3D-shaped AT-MIP (MAM-MIP-BENZ and MAM-MIP-PIV) and the control, the MAM-NIP. The total of 6 columns were prepared (2 columns for each polymer). Columns were previously equilibrated/conditioned with 3 mL of ACN. Thereafter, 3 mL of 0.25 mg.mL-1 of ACET, BENZ, PIV solutions were passed through the column, in this specific


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sequence. Between each load solution, columns were rinsed with 10 mL of ACN. Rinsing efficiency was controlled by HPLC. A HPLC-UV method was used to quantify the samples as described in Chromatographic analysis. Chromatographic analysis Method I. Chromatographic studies of all amides (ACET, BENZ and PIV), separately solutions used in Static binding and SPE 1 experiments were assessed using a Dionex ICS-3000 with a Photo diode Array ICS series Dionex detector and Chromelleon Dionex v6.8. acer Veriton 6800 software. A VertexPlus Column 250 x 4 mm Eurospher II 100-5 C18, fitted with a pre-column, was used to analyze the samples at 220 nm. The mobile phase was run in an isocratic mode, at a constant flow rate of 0.6 mL.min-1, using 30:70 (v/v) of an aqueous buffered solution (milli-Q water buffered with Trifluoroacetic acid at pH 2.5) and ACN. Method II. Chromatographic analyses of all amides in the same solution were carried out using an Agilent 1100 with a Photo diode Array ICS series Dionex detector and Chromeleon Dionex v6.8. acer Veriton 6800 software. Experiments were carried out at 25 ºC using a VertexPlus Column 250 x 4 mm Eurospher II 100-5 C18, with a pre-column, as stationary phase. UV detection was made at 220 nm. The isocratic mobile phase was used with a combination of aqueous buffer (Milli-Q water containing TFA at pH 2.5) and acetonitrile in the ratio 5:95 (v/v). A constant flow rate of 0.6 mL.min-1 was used. The solution containing ACET, BENZ and PIV, were injected for analysis using loop volume of 25 μL. Solvents used were filtered (0.20 μm filter) and degassed.

RESULTS AND DISCUSSION Selection of planar- and 3D-shaped analogue template molecules and monomer. BENZ and PIV were selected as analogue template molecules since, like ACET, are both primary amides.


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Additionally, the aromatic phenyl group of BENZ imparts a planar structure to the molecule, whereas the bulky tertiary butyl group of PIV results in a three-dimensional molecule (see Figure 2). Whilst BENZ is planar it has a higher molecular weight and hence is slightly more voluminous (114.32 Å³) than three-dimensional PIV (109.09 Å³). The functional monomer (MAM) contains an amide moiety, which would be expected to form a strong complex with ACET and both of the analogue templates (BENZ and PIV). This is crucial as it is known that the template-monomer complex is key to the success of molecular imprinting32 and may interfere on the ability of the final polymer. The use of CO2 is also advantageous as it is an aprotic solvent33 meaning that it has negligible interference on the formation and stability of the complexes.

Figure 2. Spatial conformation and occupied volume of ACET (a), BENZ (b) and PIV(c). Data obtained using the Molinspiration software.

Preparation and characterization of planar- and 3D-shaped AT-MIPs. Planar- and 3D-shaped AT-MIPs were produced using scCO2 and were obtained as free-flowing and dry,


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homogeneous powders, with yields above 90%. These features are in accordance with polymer synthesis in scCO2 reported in literature.24,28 MIPs produced using this green technology are obtained prompt-to-use and are easily manageable. Other advantages in comparison to those polymers obtained by conventional approaches are: no residual solvents and no necessity to be milled and sieved34 (which may compromise the affinity recognition sites created within the polymer). The morphological appearance of planar- and 3D-shaped AT-MIPs are presented in Figure 3.

Figure 3. The morphological characteristics by Scanning Electron Microscopy of planar- and 3Dshaped AT-MIPs: MAM-MIP-BENZ (a) and MAM-MIP-PIV (b).

FTIR analysis (raw data not shown) was performed for planar- and 3D-shaped AT-MIPs where the characteristic absorption bands28,35 could be found as expected, such as the bands at 2955 cm−1 (C–H stretching), 1732 cm−1 (ester C=O stretching) and 1646 cm−1 (amide C=O stretching). The specific surface areas, pore volumes, and pore sizes of planar- and 3D-shaped AT-MIPs, were determined by nitrogen porosimetry. The Table 1 list the values obtained. In general, planar-


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and 3D-shaped AT-MIPs presented higher BET surface areas than NIP, however showed similar values for total average pore diameter and pore volume. Table 1. Physical features of planar- and 3D-shaped AT-MIPs. BET surface area

Pore volume

Average pore

Average particle

(m2.g-1)

(cm3.g-1)

diameter (nm)

diameter (µm)

MAM-NIP

26.1

0.03

4.2

4.4±0.4

MAM-MIP-BENZ

69.2

0.07

4.4

3.3±0.7

MAM-MIP-PIV

39.4

0.04

3.9

5.0±0.1

Control

Copolymers

AT-MIPs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Particle size diameter, as well as particle size distribution, were analyzed through the Morphologi G3 particle characterization system. Average particle size diameters for planar- and 3D-shaped AT-MIPs are listed in Table 1 and in Figure 4, it is shown the particle size distribution. Comparing the results herein reported with the MAM-NIP (control polymer), with an average particle size diameter of 4.4±0.4, it can be seen that MAM-MIP-BENZ shows a slight decrease in the average particle size diameter, 3.3±0.7. On the other hand, MAM-MIP-PIV has a larger average particle size diameter, 5.0±0.1. Different particle sizes are influenced by different solubility of the products in scCO2 and the polymer nucleation and growth.


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Figure 4. Particle size distribution of planar- and 3D-shaped AT-MIPs: MAM-MIP-BENZ (a) and MAM-MIP-PIV (b).

Affinity evaluation of planar- and 3D-shaped AT-MIPs Static affinity tests. In order to evaluate the affinity capacity, static binding tests were performed on planar- and 3D-shaped AT-MIPs, in static mode. Data obtained for all amide-based compounds (ACET, BENZ and PIV) are shown in Figure 5.

Figure 5. Static binding tests for planar- and 3D-shaped AT-MIPs using solutions of 0.25 mg.mL-1 amide-based compounds.


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High adsorption capacity of all amide-based compounds were reached by 3D-shaped MIP than planar-shaped MIP and control material. This result is expected because although 3D-shaped MIP have demonstrated to have lower spatial conformation than planar-shaped MIP, has a 3D cavity meaning more accessible than 1D cavity. Then, 3D-shaped MIP showed ability to be potentially used as a device to remove amide-based compounds, successfully. Previous work on MAM-based copolymers designed to be specific for ACET,28 and prepared under the same scCO2-assisted conditions, gave an adsorption value of 2.0 mmol.g-1 for ACET (static binding). On the other hand, in

this

work,

planar-

and

3D-shaped

AT-MIPs

rendered,

respectively,

1.26

and

1.33 mmol.g-1adsorption values for ACET. Dynamic binding tests – SPE. To assessed the affinity behavior of the polymers solid phase extraction measurements were performed in dynamic mode (duplicated samples were used). 20 mg of each polymer, planar- and 3D-shaped AT-MIPs (MAM-MIP-BENZ, MAM-MIP-PIV) and the control polymer (MAM-NIP), were packed in empty columns and the behavior assessed by two different approaches: SPE-1: a single amide solution at 0.25 mg.mL-1 (ACET, BENZ, PIV) was delivered separately passing along the column. Firstly, each polymer was loaded into separate columns and equilibrated/aconditioned with ACN. Then, each amide solution was dropped on the top and collected on the column bottom. Between loadings the columns were exhaustively cleaned with ACN. SPE-2: A mixture amide solution at 0.25 mg.mL-1 containing the three amides (ACET, BENZ, PIV). Each polymer was loaded into separate columns and equilibrated/aconditioned with ACN. Then, the solution was dropped on the top and collected on the column bottom, and next columns were exhaustively scavanged with ACN.


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Figure 6. Solid-phase extraction (SPE-1) performance evaluation of planar- and 3D-shaped AT-MIPs using solutions of 0.25 mg.mL-1 amide-based compounds.

From Figure 6 (SPE-1), it can be seen that both planar- and 3D-shaped AT-MIPs (BENZ and PIV) showed different selectivity toward the different amides, ACET, BENZ and PIV. The higher specificity of the planar-shaped MIP over control polymer suggest that molecular imprinting has been successful and presumably the cavities created in the MIP generated in scCO2 reflect the different size and shape of the templates, whilst maintaining the same bonding affinities common to the family of amides. Hence the assumption that both planar and 3D cavities have been produced in the AT-MIPs. As stated before, AT-MIP strategy typically leads to loss of specificity resulting in lower molecular recognition capacity in comparison to the strategy in which the real template is used. However, BENZ and PIV are larger molecules than ACET, so it was expected that planar- and 3D-shaped AT-MIP might be capable to retain high amounts of ACET. Contrarily to the observed in static binding experiments, in general planar-shaped AT-MIP could adsorb higher amounts of all amides than 3D-shaped AT-MIP, from 0.13 mmol.g-1 (ACET) to 0.16 mmol.g-1 (PIV), that can


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be due to the high surface area of planar-shaped AT-MIP (2 times higher than 3D-shaped AT-MIP) and also, obviously, spatial requirements are more important than the absolute size of the cavity. Mobility and diffusion of the molecules through the polymer must also play an important role in these dynamic experiments. Nevertheless, none of these AT-MIPs were able to achieve the high binding value of MAM-MIP-ACET reported previously (2.0 mmol.g-1).28 In addition, these columns were assessed at 0.25 mg.mL-1 solution containing the mixture of the three amides (ACET, BENZ and PIV) in the same solution, in order to evaluate the competitiveness of the cavities in the presence of all amides. For this purpose, a HPLC-UV method was optimized (Method II).

Figure 7. Solid-phase extraction (SPE-2) performance evaluation of planar- and 3D-shaped AT-MIPs using a 0.25 mg.mL-1 solution containing a mixture of three amide-based compounds.

3D-shaped AT-MIP was able to recognize all amides in double than planar-shaped AT-MIP Figure 7 (SPE-2). This tendency is different than the previous SPE experiments showed in Figure 6 (SPE-1). In this case we evaluated the effect of having a mixture of the three amides in the solution in comparison to the previous single amide solution experiment.


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Additionally, assessment of the physical properties (see Table 1) for both polymers show that planar-shaped AT-MIP has a higher BET surface area, pore volume and average pore diameter than 3D-shaped AT-MIP. In SPE-1, all amides had the opportunity to interact with the cavity separately, and in SPE-2 the competition for the cavity was investigated. In the first case, the amides have more time to contact with the polymer, as compared with the second scenario where all of them were preferentially adsorbed by the 3D-shaped AT-MIP, which possess a larger cavity. Planar-shaped AT-MIP exhibited the best performance in dynamic mode (SPE-1) and is an interesting option not only for ACET selective removal but also for BENZ and PIV as well. This MIP may have industrial potential for use in devices to remove separately amide-based compounds/impurities. However, 3D-shaped AT-MIP (SPE-2) showed to be more suitable for the removal of several amides from pharma processes. Impact of template molecular arrangement on the molecular imprinting influence (IF). The chemical configurations of BENZ as well as PIV, see Figure 2, reveals that the spatial arrangement of the O=C-NH2 groups of the three amides are similar. Hence it is to be expected that the affinity, selectivity (Figure 5) and IF values (Table 2) of planar- and 3D-shaped AT-MIPs towards to BENZ and PIV should be influenced primarily by cavity size and shape.

Table 2. Imprinting factor obtained in SPE experiments for BENZ and PIV.

IFa AT-MIPs

SPE-1b

SPE-2c

Planar-shaped

2.04

1.08

3D-shaped

0.88

1.64

aCalculated

by dividing QMIP by QNIP. bSPE-1, solution containing a single amide. cSPE-2,

solution containing a mixture of all amides.


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Our data highlight the critical role of two parameters in the success of AT-MIPs: the molecular similarity of the analogue template and the molecule that affinity is required, and as high as possible the Impact Factor to be for the analogue target oneself.20 AT-MIPs herein produced, are suitable for efficient removal of amides from crude mixtures, specially 3D-shaped AT-MIP under SPE-2 conditions.

CONCLUSION Herein, the relevancy and versatility of AT-MIPs produced using scCO2 technology for the selective removal of amide-based compounds, in particular the genotoxic compound acetamide. The size and shape of the cavity (planar or 3D) are important parameters on the material’s performance, and showed to have a huge influence on the type of performed experiments (static or dynamic modes). However, from an industry point of view, 3D-shaped

AT-MIP has potential

applicability for the removal of amide-based genotoxins from pharmaceutical crude mixtures since are able to capture 32% more acetamide than planar-shaped AT-MIP, in the presence of a mixture of amides (SPE-2).

ACKNOWLEDGMENTS This article was presented at the 13th International Chemical and Biological Engineering Conference (CHEMPOR 2018). The authors acknowledge the Scientific and Organizing Committees of the Conference for the opportunity to present this work. Financial support from Fundação para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior


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(FCT/MCTES), Portugal, through projects PTDC/QEQ-PRS/2757/2012 and PTDC/EQUEQU/32473/2017 and principal investigator contract IF/00915/2014 (T.C.) is acknowleged. The Associate Laboratory Research Unit for Green Chemistry - Clean Technologies and Processes LAQV is financed by national funds from FCT/MCTES (UID/QUI/50006/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER – 007265). The doctoral grant SFRH/ BDE/51907/2012 as a partnership from FCT/MCTES and HOVIONE, is acknowledged by R.V. The high quality level of analytical services from REQUIMTE, particularly Nuno Costa, on amides quantification using an HPLC methodology.

Author Information section Raquel Viveiros (ORCID:0000-0002-0449-2343) Vasco D.B. Bonifácio (ORCID: 0000-0003-2349-8473) William Heggie Teresa Casimiro (ORCID:0000-0001-9405-6221) Tel.: +351 212949621

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(98), 54948–54952. https://doi.org/10.1039/c4ra11666f.

For Table of Contents Use Only

Planar and 3D-shaped dummy artificial receptors, with affinity for amide-based pharmaceutical impurities developed under supercritical carbon dioxide technology


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Green Development of Polymeric Dummy Artificial Receptors

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