Sorption of Phenobarbital and Amobarbital on Na-montmorillonite

In order to characterize the sorption kinetics of phenobarbital sodium and amobarbital sodium on natural Na – montmorillonite, the influence of the initial drug concentration and clay particles sizes was investigated. Equilibrium isotherms have been measured and analyzed using a Langmuir isotherm model. The affinity of drugs sorbed onto clay varied in the following descending order: phenobarbital sodium > amobarbital sodium, respectively 11.2 and 9.1 mg/g. Also, sorption of phenobarbital sodium and amobarbital sodium on Na-montmorillonite was studied as a function of pH. The results show that adsorption of the drugs is highest below the pHZPC due the neutrality of the drugs which adsorbs via an attraction of the positively charged surface sites at lower pH by weak van der Waals forces.

Phenobarbital sodium (5-ethyl-4,6-dioxo-5-phenyl-1H-pyrimidin-2-olate sodium) is a barbituric acid derivative that acts as a nonselective central nervous system depressant. It binds to gammaaminobutyric acid A (GABAA)-sensitive ion channels found in the central nervous system, where they allow an influx of chloride into cell membranes and, subsequently, hyperpolarize the postsynaptic neurons [11]. The drug is a first-line agent of choice for treatment of neonatal seizures [12].
Toxic effects of overexposure may include prolonged coma, cardiovascular depression with hypotension and shock leading to renal failure, hypothermia and pyrexia. Death may occur due to respiratory and circulatory failure [13].
Amobarbital (formerly known as amylobarbitone or amytal sodium) chemically designated as 5ethyl-5-(3-methylbutyl)-4,6-dioxo-1H-pyrimidin-2-olate sodium is a drug that is a barbiturate derivative. Amobarbital works by depressing the brain's sensory cortex, which decreases motor activity and changes cerebellar function, resulting in drowsiness, sedation and hypnosis. If amobarbital is taken for extended periods of time, physical and psychological dependence can develop.
An overdose on amobarbital can vary in severity but often includes the following symptoms: depression of the central nervous system, irregular breathing, decreased urine output, irregular heartbeat, hypotension, coma, shock, hypothermia, ceased electrical activity in brain. [11]. Therefore, we have investigated Na-montmorillonite as an antidote for phenobarbital and amobarbital overdose.

Materials and methods
The montmorillonite used in this study was a natural Na-montmorillonite supplied by I.C.P.M MINESA -S.A. Cluj Napoca.
Mineral clay has 99% of the clay particles < 10 μm and a cation exchange capacity (CEC) of about 0.92 meq/g. The drugs used in the present work were phenobarbital sodium and amobarbital sodium. Information regarding the two drugs are presented in Table 1. The stock phenobarbital sodium and amobarbital sodium solutions was prepared by dissolving the adsorbate in 500 mL of simulated intestinal fluid.

Clay characterization methods
In order to analyze the chemical composition of mineral clay X-ray fluorescence (XRF) technique was used. XRF were performed using a JASCO FP-6500 Analyser.
A Shimadzu XRD 6000 diffractometer with Ni filtered Cu Kα radiation (λ = 1.5406 Å) was used in order to determine X-ray diffraction (XRD) pattern of natural Na-montmorillonite. A Perkin-Elmer Spectrum 100 spectrometer with ATR was used to perform investigation of clay samples by the FTIR method. Bands were recorded as the absorbance function, within the wave number range 4000-400 cm -1 .
Scanning electron microscopy images (SEM) were obtained using a HITACHI S2600N. Surface area, pore volume, pore size distribution were determined by us in previous study [14][15][16] using standard N2 adsorption techniques [17]. Total pore volume was calculated from amount of adsorbed N2 at P/Po 0.95. [18]. The micropore volume was determined by Dubinin-Radushkevich method [19]. Mesopore volume was calculated from the amount of N2 adsorbed between relative pressures P/Po 0.40-0.95. We considered 35.0 cm 3 /mol the molar volume of liquid nitrogen [20].
pH at zero point of change (pHzpc) was calculated using pH-drift method [21]. The pHzpc was determined as the point where the curve pH final vs pH initial intersects the line pH initial = pH final [21].

Simulated intestinal fluid preparation
Simulated intestinal fluid (SIF) without pancreatin was prepared according to the United States Pharmacopeea, [22]. Monopotasic KH2PO4 (6.8g) was dissolved into 50 mL of water. To this 190.0 mL of 0.2N NaOH and water were added to make 1000 mL (pH adjusted to 7.0 ± 0.1 with 0.2N NaOH).

Sorption kinetic of phenobarbital sodium and amobarbital sodium Sorption for different initial drug concentrations
Sorption experiments were carried out for three different initial concentrations of drugs (approximately 6, 12, and 18 g/L) at 37.0  0.1 o C in glass vessels immersed in a thermostatic water bath. Three grams of Na-montmorillonite were contacted with 15 mL of phenobarbital and amobarbital, respectively, SIF solutions.
The suspensions pH was maintained at 6.8  0.05 by adding small volumes of 10 -2 M NaOH or 10 -2 M HNO3 solutions. The pH was monitored by a pH electrode connected to a pH meter (Agilent 3200P). The electrode was calibrated with buffers (Merk, titrisol) at 20 o C.
A vigorous stirring of the suspension was ensured by a rotating magnetic bar. At given times, 5-mL samples were withdrawn from the reaction vessel and immediately centrifuged. The phenobarbital sodium and amobarbital sodium concentrations, both before the addition of Namontmorillonite and after contacting with this mineral clay, were determined with a high-performance liquid chromatography (HPLC) system. The HPLC system included the following equipment: liquid pump Model LC-10AS, integrator Model CR501, variable wavelength UV-VIS detector Model SPD-10A, auto-injector Model SIL-10A, and system controller Model SCL-10A (all from Shimadzu Scientific Instruments).
The amount of adsorbed drug was calculated by means of the difference between the initial and the final solution concentration.

Sorption on different Na-montmorillonite particle sizes
Sorption studies were carried out similarly as described before but for three different particle sizes respectively < 2 μm, (4-6) μm and (8-10) μm. The initial concentration of drugs in suspension was approximately 18 mg/mL.

Effect of pH on phenobarbital sodium and amobarbital sodium sorption
Three grams of natural Na-montmorillonite were contacted for 2 h (till equilibrium attainment) with 15 mL of phenobarbital and amobarbital SIF solutions with drug initial concentrations of 18 mg/mL. The experiments were performed at 37.0  0.1 o C in glass vessels immersed in a thermostatic water bath. The pH was varied from about 6 to 9.5 and adjusted by adding small volumes of 10 -2 M NaOH or https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (3)

Sorption equilibrium of phenobarbital sodium and amobarbital sodium
Three grams of Na-montmorillonite were contacted for 2 h with 15 mL of phenobarbital and amobarbital SIF solutions with drug concentrations ranging from 2 to 20 mg/mL.
The pH of the drug solutions was 6.8±0.05. Isotherms for drugs sorption on Na-montmorillonite were determined at 37  0.1 o C in glass vessels immersed in a thermostatic water bath.
After equilibrium attainment, clays and solutions were separated by centrifugation (Universal 32 Hettich) at 10000 rpm for 30 min.
The concentrations of phenobarbital sodium and amobarbital sodium in the supernatant were determined with the HPLC system described before. The amounts of adsorbed drugs were calculated as the difference between initial and final solutions concentrations.
All measurements were run in duplicate. The reported values represent the average values.

3.Results and discussions Characterization of the raw clay XRF characterization
The chemical composition of the minerals present in the clay have been studied. The data presented in Table 2 show that the clay is composed of silica, aluminum, sodium, iron, potassium and calcium oxides in major quantities and other elements in trace amounts.

FTIR characterization
In order to investigate the surface characteristics of bentonite vibrational spectrum was investigated using FTIR spectroscopy technique. The Na-montmorillonite spectrum obtained by the FTIR technique is presented in Figure 1.

SEM analysis
The scanning electron microscope (SEM) is suited for studying clays because it affords a magnified, three-dimensional view of the unmodified (natural) clay surface with great depth of focus. Figure 2 presents SEM micrographs of raw clay and indicates the presence of diverse size particles with a diameter of the range of 1.5 µm.
SEM examination of the studied sample showed the predominance of the clay mineral montmorillonite, which was verified by XRD. A Na-montmorillonite is more open-textured or fluffy, presumably because the Na + interlayer ion is more easily hydrated.

Specific surface area and pore size distribution
Specific surface area (SSA) of Na-montmorillonite was obtained by N2 adsorption at 77K. SSA was calculated by applying the BET method as 112.4 m 2 g -1 .
The textural characteristics of adsorbent including particle size distribution, pore diameters, pore volume, pore size distribution are presented in Table 3 and Table 4.  Table 4. Pore size distribution

Effect of pH on phenobarbital sodium and amobarbital sodium sorption
The kinetic of phenobarbital sodium and amobarbital sodium uptake by Na-montmorillonite at different initial concentrations and for different clay particle sizes is illustrated in Figures 3-6.
The amount, qt, of drugs sorbed at time t, is calculated from the mass balance equation: where: C0initial metal concentration Cifinal metal concentration Vvolume of solution Mmass of sorbent  The influence of the initial concentration of phenobarbital sodium and amobarbital sodium on the sorption rate is shown in figures 3 and 5. Increasing of the initial drug concentration one obtain the increase of the amount of drug uptake.
For all three values of the initial concentrations the drugs adsorption equilibrium was reached within 2 h of shaking. The amounts of drugs adsorbed after 5 h were not significantly different from the amounts adsorbed after 2 h. Therefore, 2 h were considered sufficient time to reach drug adsorption equilibrium.
The effect of the Na-montmorillonite particle size was investigated. As shown in figures 4 and 6 the sorption rate of drugs on mineral clay is dependent on montmorillonite particle size used. The amounts of phenobarbital sodium and amobarbital sodium on Na -montmorillonite increases while the clay particle size decreases.

Effect of pH on phenobarbital sodium and amobarbital sodium sorption
The adsorption of phenobarbital sodium and amobarbital sodium on the Na-montmorillonite is governed primarily by the pH of the bulk solution and the surface charge of the adsorbent. The pH dependent charge is located at the edge sites, where the surface hydroxyl groups can be protonated or deprotonated, depending on the pH of solution.
The pHZPC of sodium montmorillonite was determined as 7.3. The surface of the adsorbents is positively charged below the pHZPC and negative above the point. The results show that adsorption of both drugs is higher below this point (Figure 7) due the neutrality of the drug which adsorbs via an attraction of the positively charged surface sites at lower pH by weak van der Waals forces [24]. The decreases in adsorption above pHZPC could be attributed to gradual ionization of phenobarbital sodium and amobarbital sodium in alkaline medium as a result of lactam-lactim tautomerism at higher pH which is as a result of active methylene group (upon tautomerization)   between two carbonyl groups of phenobarbital or amobarbital and the presence of a diiminocarbonyl system in the tautomeric forms [25].
Removal of drugs from their SIF solutions by mineral clay was pH dependent. The minimum and maximum amounts of drugs adsorbed on Na-montmorillonite are presented in Table 5. Sorption equilibrium of phenobarbital sodium and amobarbital sodium Phenobarbital sodium and amobarbital sodium adsorption isotherms are presented in Figure 8.
The drugs sorption data were well described by the Langmuir equation: bC bC a a m   1 (2) where:   and therefore a plot of C/a vs. C will give a straight line of slope 1/am and intercept 1/bam.
Knowing values for the slope and the intercept allows one to easily calculate values of equilibrium sorption parameters am and b.
The straight line through the data is usually obtained by a linear leastsquare fitting procedure. Using this procedure the equations 4 and 5 have been obtained, for phenobarbital sodium and, respectively, amobarbital sodium adsorption on Na-montmorillonite: The equilibrium parameters for drugs sorption on Na-montmorillonite are shown in Table 6. It can be seen that phenobarbital sodium and amobarbital sodium are adsorbed on the Namontmorillonite. At lower pH, the drugs molecules are adsorbed on the mineral clay edge sites, via an attraction of the positively charged surface sites.

Conclusions
This study shows that Na-montmorillonite can be used for removal of phenobarbital sodium and amobarbital sodium. Basic findings revealed that Na-montmorillonite has a high adsorption capacity for the weakly acidic phenobarbital sodium and amobarbital sodium. The Langmuir isotherm model was able to describe the sorption of drugs on the mineral clay. Two hours is sufficient time to reach drug adsorption equilibrium.
Adsorption of both drugs is higher below the pHZPC of sodium montmorillonite due the neutrality of the drug which adsorbs via an attraction of the positively charged surface sites at lower pH by weak van der Waals forces.
In order to obtain the most benefit possible from using natural Namontmorillonite is to administer it within one hour of the ingestion of a potentially toxic amount of a poison. The amount of drug that is absorbed to the activated charcoal is dependent on the on Namontmorillonite /drug ratio, Adsorption results from Van der Waals forces and desorption of the solute (drug or poison) can occur if Na-montmorillonite is sufficiently present. The poison or the drug ingested adheres to the surface of the Na-montmorillonite and since the mineral clay cannot be absorbed through the digestive tract's membranes, it passes out of the body eliminating the poisons with it.