Adsorptive elimination of paracetamol from physiological solutions: Interaction with MFI-type zeolite

Accepted Manuscript Adsorptive elimination of paracetamol from physiological solutions: Interaction with MFI-type zeolite Laurence Tortet, Emanuelle Ligner, William Blanluet, Pierre Noguez, Claire Marichal, Oliver Schäf, Jean-Louis Paillaud PII:

S1387-1811(17)30430-4

DOI:

10.1016/j.micromeso.2017.06.027

Reference:

MICMAT 8402

To appear in:

Microporous and Mesoporous Materials

Received Date: 10 February 2017 Revised Date:

28 April 2017

Accepted Date: 13 June 2017

Please cite this article as: L. Tortet, E. Ligner, W. Blanluet, P. Noguez, C. Marichal, O. Schäf, J.-L. Paillaud, Adsorptive elimination of paracetamol from physiological solutions: Interaction with MFI-type zeolite, Microporous and Mesoporous Materials (2017), doi: 10.1016/j.micromeso.2017.06.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Adsorption isotherms of paracetamol

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Adsorptive elimination of paracetamol from physiological solutions: interaction with MFI-type zeolite

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Laurence Torteta, Emanuelle Lignerb, William Blanluetc, Pierre Noguezd, Claire Marichalb, Oliver Schäf*a and Jean-Louis Paillaud*b

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Université Aix-Marseille, Equipe Interfaces entre Phases Condensées et Transport (InPaCT), Laboratoire Matériaux Divisés, Interfaces, Réactivité, Electrochimie (MADIREL), UMR CNRS 7246, Centre de Saint Jérôme, 13397 Marseille Cedex 20, France b Université de Haute Alsace, Equipe Matériaux à Porosité Contrôlée (MPC), Institut de Science des Matériaux de Mulhouse (IS2M), UMR CNRS 7361, 3 bis rue Alfred Werner, F68093 Mulhouse, France c Clinique de l’Union, Service Anesthésiologie, boulevard Ratalens - BP 24336, 31240 Saint Jean, France d Cogito Energie Santé, 6 Quai des Chartrons, 33000 Bordeaux, France

Corresponding authors: [email protected], [email protected]

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Abstract: Paracetamol (acetaminophen (ATP)) at toxic concentrations to human is successfully adsorbed onto MFI-type zeolite ZSM-5 (Si/Al = 30) stepwise under conditions approaching human serum composition. While the speed of adsorption in physiological serum at 37°C is fast enough to be accomplished in a 4 hours standard dialysis session, maximum adsorption levels at equilibrium concentration is reduced when the solution becomes more complex. In pure water, at the highest equilibrium concentrations, the adsorption is almost doubled. Thermogravimetric measurements confirm paracetamol adsorption inside the micropores. Rietveld-analysis on powder X-ray diffraction data proves that after adsorption, paracetamol is located at the intersection of the straight and zigzag channel of the ZSM-5 zeolite. 1H-MAS-NMR experiments performed on ZSM-5 zeolite after paracetamol adsorption confirm the absence of interaction between paracetamol molecules and water molecules and/or between the paracetamol molecules themselves. In conclusion, elimination of paracetamol at toxic concentrations in human serum (by ultrafiltration of blood) is a smart way to eliminate selectively such a molecule by physisorption without further interference onto other biochemical equilibria.

Keywords: paracetamol removal, ZSM-5 zeolite, solid state NMR, Rietveld analysis

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1. Introduction

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Paracetamol (PC alternatively named N-acetyl-p-aminophenol, APAP, also called acetaminophen (ATP in the United States, see Scheme 1) was introduced into medical practice in the United States in 1955 and in the United Kingdom in 1956. It is an effective and now widely available analgesic and antipyretic [1] because it is cheap and easily accessible over the counter in many countries. However, paracetamol is one of the most commonly ingested drugs in self-poisoning. In excess it predominantly causes hepatic toxicity. Indeed, poisoning was first described in humans in Scotland in 1966 [2-3]. Subsequently, the mechanism of paracetamol toxicity, due to the production of a reactive benzoquinoneimine (N-acetyl-para-benzoquinoneimine) metabolite, was understood after the work of Mitchell et al. in the United States [4-5]. The reported dose of paracetamol ingested is not strictly considered as the potentially toxic dose because paracetamol concentration reaches its maximum blood concentration within 30-60 minutes after ingestion, being subsequently metabolized and the metabolite accumulated in the liver. Acute liver failure usually starts after 24 hours while in parallel renal failure becomes evident around the third day [6-7]. Paracetamol intoxication can be treated in the very initial state with activated charcoal to reduce paracetamol absorption, however, it is mainly effective if given within 1 hour after ingesting paracetamol, and its efficacy is greatly reduced thereafter [8]. The management of paracetamol poisoning was revolutionized after the use of acetylcysteine in the 1970s. Antidote N-acetylcysteine (NAC) acts to replenish glutathione storage [9-10]. Timely risk assessment to determine the need to administer the NAC is a crucial aspect of managing paracetamol poisoning [8,11], but a clear correlation between reported paracetamol dose ingested and requirement for antidotal treatment across all reported dose ranges have not demonstrated a consistent relationship [12-15]. The protocol used, consists of three weight-related infusions and requires almost 24 hours stay in hospital. It is associated with adverse events in treated patients, particularly anaphylactoid reactions and vomiting.

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Many patients treated today are unlikely to be at high risk for major hepatoxicity. This led to the development of antidotes that were designed to replace the naturally occurring antioxidant glutathione consumed by binding and neutralizing N-acetyl-parabenzoquinoneimine. Availability of glutathione is, however, influenced by environmental factors, particularly nutritional status. Production of N-acetyl-para-benzoquinoneimine from paracetamol is potentially inducible, and also can be inhibited by acute ingestion of ethanol. These different factors might be difficult to assess in poisoned patients [8,16]. An alternative method for paracetamol removal from blood could be dialysis in the early stage of intoxication. However, paracetamol is known to be bound to albumin at a high degree in blood preventing its efficient transport across a dialysis membrane. Albumin constitutes more than 50% of the total plasma proteins and is the principal vector of transport in blood because it presents binding properties with a great number of ligands such as fatty acids, metal cations, some uremic toxins [17] and drugs such as paracetamol. Instead, an adsorption process could remove paracetamol. Zeolites whose pore size corresponds to that of 2

ACCEPTED MANUSCRIPT paracetamol molecules can adsorb the paracetamol in their micropores. Indeed, the molecular length, width and thickness of paracetamol are about 8, 5 and 3.5 Å, respectively [18]. Such a molecular size is compatible with 10-membered ring zeolites like ZSM-5 whose the pore diameters of the two kind of channels for this MFI topology are about 5.1 Å × 5.5 Å and 5.3 Å × 5.6 Å [19].

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Zeolites are insoluble inorganic adsorbents in plasma, they have the potential to displace the free paracetamol/albumin bound paracetamol equilibrium relative to the zeolite-bound paracetamol without disrupting other biochemical equilibria[20].

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In order to simplify, a potential adsorption procedure should use the same set-up as used in dialysis with a paracetamol selective zeolite adsorbent instead of a classical dialysis membrane. The main advantage of paracetamol removal from blood by adsorption onto a zeolite is that it is a purely physical, non-chemical elimination method that can be processed as a complementary approach. Thus, as a softer method, and regardless of the amounts of paracetamol ingested, it is applicable instantly while waiting for blood test results. A “Zeolite protocol” could offer clinicians and patients the possibility for better targeting of therapy, fewer adverse effects, and shorter hospital stay.

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In this paper, adsorption kinetics and isotherms of paracetamol were obtained from physiological saline solutions and ZSM-5 zeolite as adsorbent. Adsorption kinetics was performed using a rather toxic, but not too unrealistically high paracetamol concentration corresponding to 1.2 g of paracetamol per liter of blood, which corresponds to 7.94 mmol per liter of physiological salt solution (D-PBS). Adsorption isotherms in pure water and artificial serum were performed in order to compare. In addition, in order to prove adsorptive interaction with the zeolite host, a structural study on a paracetamol loaded zeolite sample (paracetamol in pure water) was accomplished from multinuclear solid state NMR and Rietveld analysis.

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2. Experimental

2.1. Materials and Methods

Paracetamol and albumin from bovine serum (bovine serum albumin (BSA), minimum 96%, electrophoresis molecular weight: 67 kDa) were purchased from Sigma-Aldrich. Physiological solution used was Dulbecco phosphate buffered saline (D-PBS, pH 7.4) purchased from Invitrogen. D-PBS composition was: NaCl (8 g/L), CaCl2 (0.1 g/L), MgCl2 (0.1 g/L), KCl (0.2 g/L), KH2PO4 (0.2 g/L) and NaH2PO4 (2.16 g/L). Physiological salt solution containing bovine serum albumin ((BSA): artificial serum), was prepared by adding 50g/L BSA to D-PBS. Water was purified with a Milli-Q® integral water purification system. The Al-containing MFI-type zeolite (ZSM-5) was synthesized in a fluoride medium following the method described earlier by Guth et al. [21]. Tetrapropylammonium bromide (≥99.0%, Aldrich), NH4F (≥99.99%, Aldrich) were dissolved in distilled water. AlF3·3H2O 3

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(purum, 93.5%, Aldrich) was added and the mixture stirred for 15 minutes. Then, this combination was slowly poured onto the silica source (precipitated silica, Merk) and mixed with a magnetic stirrer during 1 hour. The resulting gel of composition 1SiO2 : 0.1TPABr : 0.5NH4F : 0.033Al2O3 : 50H2O was hydrothermally treated at 200°C for 5 days in a PTFElined steel autoclaves. The recovered solids were washed with hot distilled water and dried overnight at 40°C. The pH of the starting gel and the mother liquor after hydrothermal treatment was 7. In order to release the porosity, the sample was calcined under air at 520 °C for 6 h in a furnace using a heating rate of 0.2 °C/min. The chemical composition of the zeolite before and after adsorption experiment was determined by elemental analysis from wavelength dispersive X-ray fluorescence spectroscopy (PHILIPS MagiX apparatus). The microporous volume of the zeolite was determined from nitrogen adsorption isotherms obtained at -196 °C (liquid nitrogen) on a Micromeritics ASAP 2010 porosimeter using the tplot method.

2.2.1. Kinetic study

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2.2. Adsorption experiments

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The adsorption experiments were performed in static conditions using the solution depletion method (equation 1). For each experiment, 20 mg samples of the adsorbent (ZSM-5, ms) were added to a 1 ml of D-PBS - paracetamol solution (V) 7.94 mmol/L (C0). The suspension was shaken automatically at 37°C for different times t. After the defined time t, the suspension was centrifuged for 10 minutes in order to separate liquid and solid phases. In consequence, minimum time of exposition was 10 minutes. The paracetamol concentration in solution at time t was measured in solution from the UV absorbance at 220 nm and is called Ct. A HPLC integrated UV-visible spectrophotometer (Agilent Technologies Inc., USA, Series 1200) was used for these measurements in order to minimize quantities of chemicals used. The limit of detection is about 2 µmol·L−1. The amount of paracetamol adsorbed at time t (qt) was calculated using the equation (1): qt = (C0 – Ct)V/ms

(1)

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The paracetamol concentration in solution decreases with time until equilibrium is reached due to the adsorption onto the solid phase. At equilibrium, the process is supposed to be in a dynamical state (rate of the forward process is equal to the rate of the backward process of adsorption). At equilibrium time the corresponding concentration of solute in the solution is the equilibrium concentration Ce. The reproducibility of the measurement of the absorbed quantity is in all cases of the order 5% in the concentration ranges studied. 2.2.2. Isotherms From the preliminary kinetic studies, it was deduced that 12 hours were completely sufficient to achieve equilibrium (at times corresponding to the time of a dialysis session of 4 hours about 95% of paracetamol equilibrium concentration value is reached). Isotherm experiments are made at 37°C for various initial paracetamol concentrations under equilibrium conditions. 4

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qe = (C0 – Ce)V/ms

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As for the kinetic experiments, for each experiment, 20 mg samples of the adsorbent ZSM-5 (ms) were added to a 1 ml solution (V) having an initial paracetamol concentration ranging from 5 µmol·L−1 to 7.94 mmol/L (C0). The suspension was shaken automatically at 37°C for 12 hours. After that, the suspension was centrifuged in order to separate liquid and solid phases. The paracetamol concentration in solution at equilibrium (Ce) was measured from the UV absorbance at 220 nm as described above for the kinetics study. For each initial concentration (C0), the amount of paracetamol adsorbed at equilibrium (qe) was calculated using the equation (2):

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The reproducibility of these depletion method measurements was better than 5% within the studied range of concentrations. 2.3. Paracetamol adsorption for the structural study

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1g of calcined ZSM-5 powder was introduced in 50 mL of an aqueous solution of paracetamol (Ci = 7.94 mmol/L, pH ≈ 5.8) at 37°C during 24h. The powder was subsequently separated from the liquid by centrifugation and dried under room temperature and atmosphere without any special drying procedure. The chemical analysis from X-ray fluorescence spectroscopy proved a total absence of sodium in the product indicating a cation exchange process. In this case, a classical exchange of Na+ by H+ in zeolites is involved due to the acidic pH of the suspension. For this sample, hereafter named PC@ZSM-5, the determined amount of loaded paracetamol from UV-visible (according to the above described depletion method) is about 0.3 mmol/g of zeolite. 2.4. Thermogravimetric study

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Thermogravimetric analysis (TGA) experiments were performed on the starting calcined ZSM-5 and exposed at 25°C to a water saturated atmosphere during 4 days (ZSM5(30)). This non-loaded sample was analyzed using a TGA Q500 from TA Instruments, USA. This was done in order to compare the results to a sample treated the same way, previously loaded with paracetamol (PC@ZSM-5). TGA program for sample ZSM-5(30) was: ramp of 5 °C/min until 800 °C + isotherm 300 min at 800 °C under air (start at 25.8 °C) and for (PC@ZSM-5): ramp of 5 °C/min until 800 °C + isotherm 300 min at 800°C under air (start at 24.3°C). 2.5. X-Ray diffraction

A small amount of paracetamol loaded ZSM-5 (PC@ZSM-5), was transferred into a glass capillary (Mark-tube made of special glass, no. 14, outside diameter 0.3 mm, Hilgenberg Gmbh) for powder XRD (PXRD) analysis. Three scans were collected between 7° and 90° (2θ) on a STOE STADI-P diffractometer in Debye−Scherrer geometry, equipped with a linear position-sensitive detector (PSD, 6° in 2θ) and employing Ge monochromated Cu Kα1 radiation (λ = 1.5406 Å) (step 0.01°, time acquisition per PSD step (0.1°), 86 s). The three scans were averaged into a single one for better statistics on the collected data. The resulting 5

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powder X-ray diffraction patterns show well-resolved diffraction peaks which confirm a good crystallinity. As expected for a ZSM-5 with a Si/Al molar ratio of about 30 [22-23], all diffraction peaks were ticked and indexed [24] with an orthorhombic unit cell and refined in space group Pnma (No. 62) with a = 20.085(3) Å, b = 19.932(4) Å, c = 13.413(2) Å, V = 5370.0(14) Å3, and FOM(30) = 54.0. From the PXRD data, no indication allowed us to choice a different space group such as Pna21 or P212121 [23]. Therefore, the structure of synthetic ZSM-5 zeolite described in Pnma space group published by van Koningsveld et al. was used as a starting model for the Rietveld refinement [25-26] which was performed with the GSAS package [27] and EXPGUI as an interface [28]. In a first step, a Lebail refinement [29] allowed us to determine the background and profile parameters. Then, the first stage of the Rietveld analysis, after scale factor determination, Fourier difference maps shows electronic density residues inside the straight channels and also, in a less extend, inside the zig-zag channels. At this point, we decide to place one paracetamol molecule in the straight channel parallel to the b-axis through molecular modeling using the Cerius2 software [30] and two independent oxygen atoms representing adsorbed water molecules in the zigzag channels. The starting structure of paracetamol used in the present work was the one extract from the hemiadduct of paracetamol with piperazine [31]. The paracetamol molecule was treated as a rigid body, only its position and factor occupancy were refined. Successive Fourier maps revealed the presence of several additional independent crystallographic sites of water molecules. During the Rietveld refinement process, all atoms were refined isotropically and soft restraints were placed on framework bond lengths and angles (Si―O = 1.61(2) Å and O―T―O = 109.5(20)°). When the refinement became stable and in order to improve it, the rigid body constraint was removed and an additional set of soft restraints were added to maintain a proper structure of the paracetamol molecule. The final Rietveld refinement gave excellent reliability factors; crystal as well as Rietveld refinement parameters are listed in Table 1. In the Supporting Information are listed atomic parameters and selected bond distances and angles (Tables A1-3). Further details of the crystal structure investigation (cif file) can be also obtained from the Fachinformationszentrum Karlsruhe, 76344 EggensteinLeopoldshafen, Germany, (fax: ++49 7247 808 666; e-mail: [email protected], http://www.fiz-karlsruhe.de/request_for_deposited_data.html) on quoting the CSD number 432575. 2.6. Solid state NMR study

For the 1H decoupled 29Si MAS (Magic Angle Spinning) Nuclear Magnetic Resonance (NMR) study, the paracetamol loaded ZSM-5 (PC@ZSM-5) was packed in a 7mm diameter cylindrical zirconia rotor and spun at a spinning frequency of 4 kHz. The spectrum was recorded at ambient temperature (21 °C) on a Bruker Avance II 300 spectrometer (B0 = 7.1 T) at 59.6 MHz with a π/6 pulse duration of 1.8 µs and a recycle delay of 80 s. 1

H and 27Al MAS NMR spectra, 1H 2D Double Quantum-Single Quantum MAS NMR spectra and 1H-13C CPMAS NMR spectra of the same sample were recorded on a Bruker AVANCE II 400WB spectrometer (B0 = 9.4 T) operating at 400.18 MHz for 1H, 104.27 MHz for 27Al and 100.6 MHz for 13C. Samples were packed in a 2.5 mm (or 4 mm) cylindrical 6

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zirconia rotor and spun at a spinning frequency of 30 kHz (or 12 kHz) for 1H (or 13C and 27Al) experiments. 1H experiments were performed with a π/2 1H pulse duration of 2 µs. Two rotor cycles of BABA dipolar recoupling were used for both excitation and reconversion of double quantum coherences [32]. 1H-13C CPMAS NMR spectrum was recorded with 4 µs corresponding to the 1H π/2 pulse duration, 1 ms contact time and 5 s of recycling delay according to the 1H T1. 27Al MAS NMR spectrum was recorded with a π/12 pulse duration of 0.5 µs and 0.5 s of recycling delay. Si, 1H and 13C chemical shifts are relative to TetraMethylSilane (TMS, 0 ppm) whereas 27Al chemical shifts are relative to an aqueous solution of aluminum nitrate (Al(NO3)3). Decompositions of the NMR spectra to extract the proportion of the corresponding species were performed with the Dmfit software [33].

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3. Results and Discussion 3.1. ZSM-5 zeolite

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Adsorption

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From chemical analysis, the chemical formula per unit cell of our anhydrous ZSM-5 is |Na3.1| [Si92.9Al3.1O192], the corresponding Si/Al molar ratio being ≈ 30. This parent zeolite is then called ZSM-5(30), its equivalent specific surface area and pore volume values are around 240 m2/g and 0.11 cm3/g, while the external specific area is about 1.5 m2/g as expected for such a ZSM-5 zeolite [34].

3.2.1 Kinetics of adsorption

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Figure 1 shows the amount of paracetamol in D-PBS (physiological salt solution, phy) adsorbed onto zeolite ZSM-5(30) as a function of time. The adsorbed paracetamol increases with time until equilibrium is achieved. It is associated with the decrease of the paracetamol concentration in solution due to the adsorption of paracetamol onto the adsorbents (quantity qads, external surface or/and pores). In fact, the time necessary to reach 90% of the equilibrium concentration is about 90 minutes. Equilibrium concentration Ce is reached after 5 hours. Two kinetic models for sorption from liquid solution, the first- and second-order models, were derived and used in the literature independently of any assumption or specialization of process conditions [35]. In our case the pseudo-second order model, equation (3), was chosen because it fits the kinetic data much better than the first order model using the nonlinear method [36]. q = k2qe2t(1 + keqet)

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In equation (3) k2 is a dimensionless constant, t the time and qe the maximum quantity of adsorbent at saturation. On Figure 1, the full red line indicates the best kinetic fit, which is

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3.2.2. Adsorption isotherms

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Earlier experiments [17] of our group have shown that the presence of BSA in D-PBS does not significantly influence the kinetics of adsorption of a partially protein boundmolecule (differences are in the error range of 5%). This is why the same behavior is expected in the present case. Thus, these results can be extrapolated for an artificial serum (D-PBS + BSA).

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The isotherms of paracetamol adsorption from aqueous solutions (aq), physiological salt solutions (phy) and physiological salt solutions containing bovine serum albumin (BSA) in physiological concentrations onto zeolite ZSM-5(30) are plotted in Figure 2. The adsorption isotherm corresponds to the amount of paracetamol adsorbed per gram of zeolite ZSM-5(30) adsorbent as a function of the equilibrium concentration. The full lines indicate simple Langmuir fits [37], assuming here that only one isolated adsorption site for paracetamol within ZSM-5(30) micropores exists (see section 3.4.). As expected, the capacity of adsorption diminishes as the solution becomes more complex [17]. In pure aqueous solution, the MFI lattice adsorbs only neutral paracetamol molecules. In physiological salt solution (DPBS), additional cation-exchange takes place. Finally, in artificial serum, BSA molecules covering the zeolites external surface are too big to enter the channel system but are partially hindering paracetamol molecules to access the adsorption sites within the channel [17]. Furthermore, paracetamol is partially bound to protein. It is only the free fraction of paracetamol that is able to enter within the ZSM-5(30) pore system. This process displaces the initial BSA-paracetamol- equilibrium with respect to the zeolite, but both remain in competition. Such a competitive process together with the BSA coverage of the zeolite external surface leads, at the end, to an effective reduction of the paracetamol quantity adsorbed into the ZSM-5(30) pore system.

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While the p-cresol molecule, which has similar shape, adsorbs with the almost same characteristics as paracetamol onto ZSM-5(30), it also adsorbs with up to 0.6 mmol/g onto pure silica MFI (silicalite 1) [38]. Paracetamol adsorption on silicalite-1 has been found to be negligible under the same conditions in our study probably due to the higher hydrophobicity of silicalite-1. 3.3. Thermogravimetric analysis

The thermogravimetric curves of hydrated ZSM-5(30) and paracetamol loaded ZSM-5(30) (PC@ZSM-5) (see section 2.3.) are plotted on Figure 3. The TGA curves show that zeolitic water in paracetamol free ZSM-5(30) is desorbed stepwise, and the sample is water-free above 200°C. After 600 °C, a weak continuous weight loss until 800 °C of about 0.35 % is visible due to an additional dehydroxylation reaction that corresponds to the desorption of water resulting from silanols condensation. When these results are compared to the paracetamol loaded sample (PC@ZSM-5), it can be seen that after water removal of 4.7 % 8

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up to 240°C, paracetamol is removed by decomposition and combustion in a two-step process. A first weight loss of about 0.8 % occurs between 240°C and about 350°C then a second one of 3.3 % between 350 and 800 °C. For PC@ZSM-5, the dehydroxylation reaction is less visible because of a different chemical composition resulting in a possible overlapping of all thermal phenomena i.e., decomposition-combustion of the organic moieties and an earlier dehydroxylation before about 650 °C in absence of Na cations that stabilize the zeolitic framework in the parent ZSM-5(30) [39]. Water and paracetamol (PC) desorption temperatures and quantities are due to desorption from the microporous channel system. Indeed, when paracetamol is not confined in micropores, it decomposes almost totally in onestep between 220 and 350 °C under similar condition (except a fastest heating rate (10 °C)/min) [40]. Only the confinement inside the micropores of the zeolites can explain such a difference. Furthermore, knowing the size of the paracetamol molecule (given above) and the specific external surface of the zeolite powder (1.5 m2/g), with the assumption that the adsorption phenomenon is limited to one monolayer of molecules oriented perpendicular to the surface, it is possible to determine maximum theoretical adsorbed amount of paracetamol onto the external surface of our employed ZSM-5(30), which is 1.5 mg/g. The 4.1% loss in mass of paracetamol is in good agreement with its uptake during adsorption, as determined from UV-visible (see section 2.2.2.). 3.4. X ray diffraction

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The final Rietveld plot of PC@ZSM-5 is displayed on Figure 4. From these displayed powder data, it is clear that ZSM-5(30) and a fortiori PC@ZSM-5 are well-crystallized materials. The paracetamol molecules are localized inside the straight channels (Figure 5a). The phenyl group of paracetamol is located at the intersections between the straight and zigzag channels (Figure 5b). The Rietveld refinement allowed us to localize seven independent crystallographic sites for water molecules. The paracetamol molecules are as well isolated from the water molecules as from the framework. Indeed, on Figure 6, a projection along [010] shows that among all the disordered water molecules, Ow1, Ow4 and Ow6 are located in the straight channels only when the paracetamol molecule is not present. Their site occupancy factors clearly indicate that the paracetamol molecules are isolated from the water molecules thus preventing any interactions between them. The shortest contacts between the hydrogen atoms of the paracetamol molecules and the framework oxygen atoms (Figure 7) are H8b−O19 = 2.36(2) Å and H5p−O22 = 2.36(1) Å. The remaining water molecules Ow2, Ow3, Ow5 and Ow7 are located inside the zig-zag channels. The observed shortest distance between water oxygen atoms and the framework is Ow5−O1 = 2.1(1) Å (Table A3). After the Rietveld refinement, the chemical formula of PC@ZSM-5 per unit cell is |(C8H9NO2)1.72(H2O)14.75|[Si96O192] (see Table 1 for explanation). However, the actual chemical formula must be rewritten as |(C8H9NO2)1.72(H2O)14.75|[Si92.9Al3.1H3.1O192], the protons compensating the framework negative charge. The corresponding mass percentages of water and paracetamol are 4.4 and 4.1 %, respectively. These values are in very good agreement with those of the thermogravimetric analysis (see section 3.3.) which confirms the

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From the 27Al solid state MAS NMR of PC@ZSM-5 spectrum (Figure 8), the main part of the Al species are in a tetrahedral coordination (resonance at 52.8 ppm) as expected for aluminum at T positions of a zeolitic framework [41]. From the deconvolution of the NMR spectrum, less than 3% of the aluminum species (about 1 aluminum per 11 unit cells) are in an octahedral coordination sphere which means here a very low amount of extra framework aluminums (EFAL) and justified its non-integration in the above X-rays structural study.

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Proton decoupled 29Si MAS NMR spectrum of the PC@ZSM-5 sample is presented in Figure 9. The spectrum displays at least 3 resolved resonances at -116, -112.5 and -107 ppm corresponding to 24, 62 and 14% of the total signal. The first two resonances are assigned to Q4 sites (Si(–OSi)4) whereas the last one at -107 ppm may corresponds to Si(1Al) [42]. From this decomposition and assignment of the NMR resonances, it is possible to estimate a Si/Al ratio close to 28.5 in agreement with the Si/Al ratio derived from chemical analysis. This result suggests a low amount (<1%) of silanol defects whose signal is expected around -100 ppm. H-13C CPMAS NMR spectrum of PC@ZSM-5 sample displays Figure 10 at least 5 resonances at 178.7, 157.7, 126.5, 117 and 18.2 ppm. According to ACDlab, they are assigned to the carbonyl, to the carbon bearing the alcohol group, to the other aromatic carbons and to the methyl group, respectively. It is interesting to note that the quaternary carbon linked to the NH group, is not resolved but the resonance at 126.5 ppm is slightly broader than the one at 117 ppm and consequently may account for this carbon too. In conclusion, paracetamol molecules are present in the sample in agreement with previous results. Moreover, the resonance assigned to the methyl group displays two components differing by their broadness revealing different environment and/or mobility for the methyl group. Indeed, paracetamol molecules may have two conformations due to the different dihedral angle between the aromatic ring and the amide group. One is almost flat (form I) whereas the other is slightly tilted and more flexible (form II). The coexistence of both conformations may explain the two components of the methyl resonance. Furthermore, N. Tsapatsaris et al. [43] also show that the dynamical behavior of the methyl group in paracetamol is sensitive to the local environment. Nevertheless, the PXRD study (section 3.4) shows only one crystallographic site for paracetamol. However, from powder data, it is not possible in this case to differentiate both conformations, which are certainly located at the same position inside the zeolite channels and with a low occupancy factor.

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The resonances characteristic of paracetamol are also detected on the 1H MAS NMR spectrum of PC@ZSM-5 sample shown Figure 11 (methyl at 2.2 ppm, aromatic protons at 6.5 and 6.8 ppm and the NH group at 9 or 10 ppm). A broad resonance is also observed at 5.8 ppm that could account for both protons from water molecules and for OH groups of paracetamol. Indeed, thermal analysis data suggest the presence of about 4.5 wt % of water 10

ACCEPTED MANUSCRIPT molecules in the sample that are detected by 1H MAS NMR. Due to the low resolution of the spectrum, decomposition of the resonances is difficult and not sufficiently reliable to deduce the amount of water molecules.

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In order to focus on possible interactions between paracetamol, water and the inorganic framework through Si-OH or Al-OH groups homonuclear 2D 1H-1H NMR experiments were performed. The result on PC@ZSM-5 material is shown on Figure 12 with the 1H spectrum projection. Note that a correlation appears in this type of 1H-1H MAS NMR spectrum when at least two protons are separated by a maximum distance of about 4.5 Å [32]. 1H resonances characteristics of paracetamol molecules are present whereas neither water nor silanol/Al-OH species are detected probably because of the low amount of defects. For water molecules, the dynamic may explain the absence of intramolecular correlation because motion is known to average dipolar coupling. 1H-1H autocorrelation due to intramolecular spatial proximities between the 3 protons of the methyl group of paracetamol at 2.5 ppm is visible on the diagonal drawn in black (label A on Figure 12). 1H-1H correlations due to intramolecular spatial proximities between the aromatic protons (at C2,6 and C3,5, label D on Figure 12) or between the aromatic protons (C3,5) and the methyl groups (label B on Figure 12) or between the NH group and the methyl protons (label C on Figure 12) are evidenced. More interestingly, no intermolecular correlations are observed between either paracetamol molecules through OH or NH groups neither with water molecules. It seems that paracetamol molecules are not spatially close to each other and neither to water molecules. This result is in good agreement with the PXRD study presented in the preceding section.

4. Summary and Conclusions

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Paracetamol at toxic concentrations was successfully adsorbed onto ZSM-5 (Si/Al=30), a MFI-type zeolite in distilled water, D-PBS and artificial serum at 37°C with a higher adsorption rate in physiological serum at 37°C in agreement with a four hours dialysis session. As in previous studies, under comparable conditions, adsorption quantities are reduced the more complex the solution become, with lowest adsorption quantities in artificial serum containing BSA. For paracetamol elimination from human serum, this implies that ZSM-5 quantities have to be almost doubled. Thermogravimetric, PXRD and chemical analysis, confirmed, independently, that paracetamol adsorption (from an aqueous solution) is exclusively done into the micropores, adsorption onto the external surface of the zeolite being negligible. Moreover, in pure water, the adsorption is almost doubled at the highest equilibrium concentrations. A Rietveld study, after paracetamol adsorption, revealed that paracetamol molecules are located at the intersection of the straight and zig-zag channel system. Solid state MAS-NMR confirmed the X-rays study, i.e. the absence of interaction between paracetamol molecules and the co-adsorbed water molecules and between the paracetamol molecules themselves. Based on these results it can be concluded that elimination of paracetamol at toxic concentrations in human serum (by ultrafiltration of blood) is a smart way to eliminate 11

ACCEPTED MANUSCRIPT selectively such a molecule by physisorption without further interference onto other biochemical equilibria.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.micromeso.2017.xx.xxx.

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N. Tsapatsaris, B.A. Kolesov, J. Fischer, E.V. Boldyreva, L. Daemen, J. Eckert, H.N. Bordallo, Mol. Pharm., 11 (2014) 1032-1041.

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ACCEPTED MANUSCRIPT Table 1: Crystal and Rietveld refinement data of PC@ZSM-5.

|(C8H9NO2)0.215 (H2O)1.84|[Si12O24]a

Space group λ (Å), CuKα1

Pnma (no. 62) 1.5406

Data collection temperature T (°C)

20

a (Å)

20.0933(1)

b (Å)

19.9259(1)

c (Å)

13.4154(1)

3

V (Å )

5371.21(4)

Z

8

Number of data points, 2θ range (°) (step (° 2θ)) Number of contributing reflections

8299, 7.00-89.99 (0.01)

Number of profile parameters

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Number of structural variables

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Chemical formula

202 15

Total number of restraints (bonds, angles, planar)

173 (140, 31, 2)

Total number of constraints

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Rp

b

wRp

b

Rexp

b

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RF2 χ

b 2

0.024 0.030

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Largest differences peak and hole (e⋅Å−3 )

0.036 0.024 0.034 0.987 0.201, -0.227

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After the Rietveld refinement, the total refined number of extra framework oxygen atoms per unit cell (Ow1-Ow7) is about 19.66 (see Table A1). However, the refinement did not take into account the scattering power of the hydrogen atoms of each water molecule. Consequently, the factual number of water molecules is about 25 % lower than the total number of refined extra framework oxygen atoms, i.e. about 14.75 water molecules per unit cell or about 1.84 per asymmetric unit as reported in the chemical formula of the Table. All T atoms have been refined as Si atoms. b The definition of these residual values are given in references [27].

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Scheme 1: Paracetamol

Figure captions:

Kinetics of paracetamol adsorption of potentially toxic concentration (7.94 mM (1.2 g/L)) in physiological salt solution (physiological serum), the error bars correspond to 5%. The red full line is the best simulation by pseudo-second-order kinetic (from equation (3) k2 = 4.46 10-4, qe = 200.6 and the coefficient of determination R = 0.981).

Figure 2:

Adsorption isotherms of paracetamol in distilled water (aq), physiological salt solution ((phy), physiological serum) and physiological salt solution containing bovine serum albumin ((BSA), artificial serum together with the Langmuir fits (full lines), the error bars correspond to 5%.

Figure 3 :

Thermogravimetric analysis of ZSM-5(30) (dotted line) and PC@ZSM-5 (full line).

Figure 4:

Rietveld plot of PC@ZSM-5 (Cukα1, λ = 1.5406 Å), experimental (×), calculated (solid red line) and background (solid green line). Vertical magenta ticks are the positions of the theoretical reflections for space group Pnma. The lowest trace (solid blue line) is the difference plot. On the inset, the low angles part (40-90°, 2 Theta) is magnified by a factor of eight.

Figure 5:

Perspectives showing the paracetamol molecule of PC@ZSM-5 inside the straight channel, (a) view down the b-axis and (b) a perpendicular projection. The framework oxygen atoms have been omitted for clarity.

Figure 6:

View down [010] of PC@ZSM-5 highlighting the relative positions of the water molecules versus the paracetamol species. The numbers under brackets are the site factor occupancies together with the uncertainties that take into account the scattering factors of the hydrogen atoms of the water molecules (see Table 1 for explanation). The framework oxygen atoms have been omitted for clarity.

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Figure 1:

Figure 7:

Shortest contacts between the paracetamol molecule of PC@ZSM-5 and the framework oxygen atoms.

Figure 8:

27

Figure 9:

1

H decoupled 29Si MAS NMR spectrum of PC@ZSM5 sample.

Figure 10:

1

H -13C CPMAS NMR spectrum of PC@ZSM5 sample.

Figure 11:

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H MAS NMR spectrum of PC@ZSM5 sample.

Figure 12:

1

H-1H DQ-SQ MAS NMR spectrum of PC@ZSM5 sample.

Al MAS NMR spectrum of PC@ZSM-5. Only the isotropic region is shown.

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Figure 1:

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ACCEPTED MANUSCRIPT Figure 3: 100 99 4.7 %

98

6.9 %

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Weight (%)

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8.8 %

95

0.8 %

94 93

0.35 %

91 90 100

200

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700

800

T(°C)

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Figure 7:

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Figure 9:

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Figure 11:

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Figure 12:

C3,5 C2,6 NH OH

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CH3

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B C D

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ACCEPTED MANUSCRIPT Highlights ► Potential use of ZSM-5 zeolite for elimination of paracetamol at toxic concentrations ► Short and long-range order proved by solid state MAS NMR and Rietveld analysis

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► No interaction between paracetamol and co-adsorbed water molecules within ZSM-5 pores