Evaluation of adsorption capacity of acetaminophen on activated charcoal dosage forms available in Nigeria by in vitro adsorption studies and scanning electron microscopy

Purpose: To investigate varying dosage forms of activated charcoal obtained from community pharmacy outlets in Nigeria for their adsorption capacity when challenged with acetaminophen. Methods: Equilibruim kinetics of acetaminophen adsorption onto activated charcoal surface was determined via batch studies at different adsorbent:adsorbate ratios. The isotherm adsorption experiment was carried out at 37 C and langmuir isotherm models were utilized to describe the equilibrium kinetics data with characterization of adsorption site and porosity elucidated via scanning electron microscopy. Results: A preponderance of microporosity was observed on the surface of the powder activated charcoal. Maximum adsorption capacity (MAC) of 299.78 mg/g was obtained using activated charcoal powder (276.11; 321.09) at 95 % confidence interval (CI), allowing for maximum adsorption of acetaminophen at pH 1.2. Mixed pore structures, which were not clearly established, were observed with both the tablets and capsules with MAC of 280.54 mg/g (273.22; 290.08) and 140.01 mg/g (135.32; 153.99), respectively at pH 1.2. MAC data at pH 6.5 showed little variation from those obtained at pH 1.2 (p < 0.05 95 % CI). Conclusion: The presence of excipients in the tablets and capsules caused a retardation in adsorption via intraparticle diffusion especially at basal micropore sites on the activated charcoal surface. Activated charcoal powder is superior to other dosage forms for use in countering acetaminophen poisoning.


INTRODUCTION
Acetaminophen overdose is usually managed utilizing activated charcoal.The ease of administration and its relative safety when compared with other gastrointestinal decontamination methods makes it desirable for drug overdose management [1,2].Large reductions in drug absorption occur when activated charcoal is administered soon after drug ingestion [2].Some workers evaluated the adsorption of fluoroquinolones on some pharmaceutical adsorbents; it was observed that activated charcoal had a superior adsorption capacity to bentonite and kaolin [3][4][5].
Acetaminophen an analgesic agent is a leading cause of acute liver failure in the industrialized countries [6,7] .Acetaminophen poisoning has also been reported in Nigeria in pediatric cases as well leading to deaths in more than 40 children [8,9], and in adults who have ingested more than 3 brands of medications for pains which all contain varying concentrations of acetaminophen [10], leading to liver damage except where immediate treatment is given via activated charcoal slurry or the acetyl cysteine antidote [11][12][13].
The nature of activated charcoal dosage form and its ability to adsorb potential poisons depends on the total pore volume, pore size distribution , prevalence of a certain pore size regime and the behavior of admolecules in these pore regimes [14][15][16][17].It is imperative that the final dosage form of activated charcoal must have pores which have affinity for the adsorbate without interference from excipients [17,18].
In this study varying dosage forms of activated charcoal obatined from community pharmacy outlets were investigated for their adsorption capacity when challenged with acetaminophen intoxication in an in vitro model at varying pH.Previous studies by Panthee and Lohani evaluated intoxication at pH 3.4 and pH 7.2 [18].The present study, however, seeks to evaluate adsorption kinetics at pH which mimicks a fasted state of the gastric and intestinal compartments using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).SEM studies will be utilized to elucidate the areas of drug absoprtion unto the surface of the varying dosage forms of activated charcoal marketed in Nigeria.Maximum adsorption capacity (MAC) data obtained via the adsoprtion isotherm model which best describes their adsorption behaviour will then aid in providing information on the effectiveness of the various dosage forms to be used especially in emergency medical situations.

EXPERIMENTAL Materials
The adsorbate used all through was acetaminophen, pure standard (Sigma Aldrich ® USA), Acetaminophen tablets (Innovator product) Panadol ® manufactured by GlaxoSmithKline (GSK) Nigeria with NAFDAC registration number 04-0205 was purchased from a registered retail pharmacy outlet in Lagos, Nigeria.
The adsorbents comprised of three dosage forms of activated charcoal, shown in Table 1, sourced via random sampling from community pharmacies in Lagos, Nigeria and were used within their shelf life.The varying characteristics of the dosage forms including the quantities %w/w of activated charcoal were provided by the manufacturer.
Potassium dihydrogen phosphate (Thomas Baker ® UK), sodium bicarbonate (Sigma Aldrich ® USA), Sodium hydroxide (Thomas Baker ® UK), Methanol (Sigma Aldrich ® USA), Hydrochloric Acid (Sigma Aldrich ® USA) and LC grade water (Omnisolv®,) was purchased from EMD Millipore Corp. (Billerica, MA, USA).All solvents and reagents used were of analytical grade and the dissolution media used were always freshly prepared.

Adsorption kinetics studies
Twenty tablets of acetaminophen were crushed in a clean and dry porcelain mortar and sieved with the aid of a sieve 100 mesh.900 ml stock solution of acetaminophen (0.5 % w/v) in the varying dissolution media was prepared.This was introduced into each dissolution vessel, and the vessel maintained at 37.0 ± 0.2 ºC operated at 100 rpm.
Langmuir isotherm models were utilized to describe the equilibrium kinetics data, via adsorbent: adsorbate ratios being varied from 1:1 through a 10 fold ratio of adsorbent to 1 ratio ofadsorbate.Into the respective dissolution vessels varying quantities of activated charcoal (either as tablets, capsules or as the powder) were introduced in the required adsorbent:adsorbate ratio already predetermined.At specific time intervals, aliquots were withdrawn, filtered thrice, and suitably diluted before being analyzed at 244 nm using a Distek ® Dissolution system 21000 UV-visible spectrophotometer [20].Activated charcoal in the different dosage forms were subsequently introduced into the dissolution vessels in the absence of acetaminophen, aliquots were withdrawn from the dissolution medium, filtered thrice, before being analyzed at 244 nm.This did not give any reading and as such it was concluded that the excipients in the dosage forms did not interfere with results obtained.

Adsorption equilibrium studies
Utilizing the different dosage forms of the activated charcoal, 500 mg of activated charcoal were weighed out and introduced into the bottles containing 500 ml of either SGF or SIF after which the bottles were fastened and sealed.Rotation at 30 rpm was initiated for 30 min using a rotor (Model 103906 motor, LW Scientific ® ) at 37 o C after which the bottles were allowed to settle for 3 hours while still maintained at 37 o C. 5 ml aliquot of the supernatant in each bottle was sampled, after centrifugation at 2000 rpm for 2 min.Studies were repeated at 30 min intervals in twelve replicates after which acetaminophen concentration was determined using the UV spectrophotometer at 244 nm.

Adsorption analysis
Acetaminophen adsorption was calculated at equilibrium as shown in Eq. 1 Qe = {(Co -Ce)V}/m…………………….(1) where Qe is the amount of acetaminophen adsorbed per gram of the adsorbent at equilibrium (mg/g); Initial (Co) and final equilibrium (Ce) concentration of acetaminophen in solution in mg/L; V (L), volume of solution and the amount of adsorbent in grams is m.MAC of the activated charcoal samples were calculated using Langmuir adsorption isotherm as shown in Eq 2.

Scanning electron microscopy (SEM)
Thirty minutes after commencement of the adsorption equilibrium studies 1 ml sample was obtained from the tubes and a minute amount was placed on a stub labeled and viewed using a Quanta 400F SEM FEI USA instrument.This was used to elucidate the areas of drug adsoprtion onto the surface of the varying dosage forms of activated charcoal using a JEOL JSM-6360LA Oxford instrument Software XT microscope control systems.The varying areas of adsorption were observed in both SGF and SIF.

Determination of surface area
Using the method described by Ilomuanya et al [20] surface area was determined Brunauer, Emmett and Teller (BET) method.Ten grams of the commercial samples were vortexed in distilled water thrice to remove the excipients on the surface of the charcoal and subsequently dried at 60 o C. Each of three activated carbon samples (0.2 g) were weighed and degassed under vacuum (300mmHg) until the pressure was stable at 6×10 -6 torr.and was put in the sample tube and left to constant heating at 250 o C for 10 h, after which surface area analysis was performed.

Statistical analysis
The data from this study was analyzed using Microsoft Excel sheet and SPSS Inc. version 11.0 Chicago Illinois.A comparison of the standard with mean values was evaluated via ANOVA at 95 % confidence interval (p < 0.05).

Adsorption equilibrium
Langmuir adsorption isotherms were utilized in describing the adsorption behavior of the adsorbent unto the adsorbate.Langmuir's adsorption is based on 4 major assumptions, that only a monolayer is formed, that the adsorbent and adsorbate species are none interacting, adsorption occurs uniformly across specific homogenous sites inside the pore structure within the adsorbent leading to saturation occurring after which point no other adsorption can occur.Eq 2 is the Langmuir equation where K is the Langmuir constant which corresponds to the heat energy of adsorption.The maximum adsorption capacity MAC which elucidates the formation of a monolayer coverage (as seen with the initial inflection point in Figure 1 and Figure 2 was calculated from Qm. Figures 1, Figure 2 and   Figure 3 show Langmuir adsorption isotherm of paracetamol by capsules, tablets and powder activated charcoal samples.There was little variation reflected at the two pH utilized in the study, however Qe values for pH 6.5 were slightly higher but not statistically significant with p < 0.05.

Adsorption kinetics
The solid (activated charcoal) -solution interface where adsorption takes place is controlled by the rate of uptake of acetaminophen as the solute, the time that it is retained on the surface of the adsorbent is also dependent on the acetaminophen uptake rate, and this is described by the adsorption kinetics of acetaminophen unto the varying forms of activated charcoal evaluated.
The pseudo first order model was utilized to fit in acetaminophen adsorption unto varying activated charcoal dosage forms as shown in Table 2. Eq 3 represents Qe and Qt (mg/g) as the amount of acetaminophen adsorbed in one gram of the adsorbent at equilibrium at time t (minutes) with k 1 being the pseudo first order kinetic rate constant.
Intraparticle diffusion model was used to analyze the data where k p (mg/m/min 0.5 ) represents the intra-particle diffusion constant (Eq 4).This model looks at adsorption as beginning first with a rapid adsorption phase followed by a more gradual adsorption process where the intra particle diffusion occurring controls and drives the adsorption process as reflected in Table 2. Qt = k p t 1/2 + C ……………………………… (4) Maximum adsorption capacity derived from Eq 2, of the three formulations at the pH 1.2 and 6.5 is reflected in Table 2. Significant difference in MAC data between the dosage forms were evident as shown in Table 2 with activated charcoal powder having the highest MAC value at pH 6, this difference wasn't seen in the use of varying media (p > 0.05).
Scanning electron microscopic studies revealed that adsorption of acetaminophen occurred on the microporous pores of the powder (Figure 4C and F), with increased adsorption densities seen in 3F when pH 6.5 was utilized, this is in consonance with MAC values obtained.It was evident that the presence of excipients in the capsule formulation greatly hampered adsorption unto available sites (Figure 4A and D), surface coverage was minimal, the tablet formulation showed more acetaminophen adsorption (Figure 4C and E) than the capsule and this was also buttressed by MAC data in Table 2.

DISCUSSION
Langmuir's assumption that adsorption will occur uniformly as a monolayer unto the adsorbent surface was refuted in this study as adsorption was shown to be varied across the pore surface of the varying activated charcoal dosage forms.
A correlation between the quantities of acetaminophen adsorbed and the scanning electron micrographs was observed.There was an increased density of acetaminophen adsorption on the activated charcoal (AC) microporous surface.The powdered activated charcoal was most suited for adsorption of acetaminophen.This result is similar to that obtained by other workers [18] where both the powdered activated charcoal and the activated charcoal slurry both had MAC that were much higher than those obtained from other activated charcoal dosage forms which he evaluated.
A preponderance of microporosity was observed with the powder activated charcoal adsorption occuring on the micropore surface , the adsorption was a good fit to the langmuir isotherm, which allowed for maximum adsorption capacity (MAC) of acetaminophen at pH 1.2.A preponderance of mixed pore structures which were not clearly established was observed with both the tablets and capsules.Consequently BET surface surface area was much lower for the tablets and capsules, when compared to the powder dosage form.Scanning electron micrographs showed that adsorption did not take place on the surface of the activated charcoal because these adsorption sites were unavailable for acetaminophen adsorption.
Very high correlation was obtained using the pseudo first order kinetic model when compared with intra-particle diffusion model with R 2 values of 0.991 in pH 6.5 for the acetaminophen adsorption via powdered activated charcoal, thus reflecting high correlation coefficients.This model suggests that physisorption is the rate controlling mechanism via which adsorption takes place and this is buttressed by the close values obtained for predicted and experimental Qe for drug adsorption.
Adsorption may also have been influenced by the functional groups associated with the surface of the activated charcoal.Excipient interactions with the functional group on the surface of the AC leads to variation in MAC values and this accounts principally for the lower values obtained from capsule activated charcoal, as this was formulated with the most excipient %w/w concentration.This associated interaction lends credence to the reduced acetaminophen adsorption density observed in the SEM.Excipients utilized in the formulation of the activated charcoal capsule and tablets act as deterrents to acetaminophen adsorption as they may contribute either acidic or basic functional groups to the surface of the activated charcoal leading to an interaction between the adsorbent and adsorbate functional groups thus making the micro and macro pore surfaces unavailable for adsorption.R 2 values obtained via the intraparticle diffusion model were close to 1, which is suggests that adsorption was initially very fast but was followed by a gradual adsorption stage indicative of adsorption occurring through the intra-particle space via diffusion [19], and this process drives the adsorption phenomenon.R 2 values were lower than those obtained via pseudo first order model; thus, although intraparticle diffusion may have occurred, it was responsible for driving the overall adsorption process.
Although there was no significant difference in adsorption occurring at varying pH in consonance with previous work, it was observed that the MAC values obtained in all three dosage forms of activated charcoal for acetaminophen adsorption were higher in pH 6.5 than in pH 1.2, thus lending credence to the fact that adsorption was more pronounced in intestinal pH.Therefore, in cases of poisoning, adsorption from the small intestine would be more efficient than that occurring in the gastric region, as has been shown in similar studies [4].
Acetaminophen has a very high ease of abuse in Nigeria, first, because of ease of purchase as an over the counter medication requiring no prescription and more disturbingly, the concomitan use acetaminophen with other drugs.
In the Nigerian market, diclofenac and aspirin tablet combinations are marketed as combinations with acetaminophen and because they are available to both pharmacies and local medicine peddlers [8,9], duplication of medications containing acetaminophen may arise.Agaba et al [10] found that common analgesics regularly consume was acetaminophen in 58.1 % of respondents with, 28.9 % taking mixture of analgesic containing acetaminophen and acetaminophen abuse bordering on an overdose was present in 22.6 % of the respondents.These data make it imperative that activated charcoal formulations available in the market be such that are effective in preventing hepatotoxicity by rapid adsorption of acetaminophen in both the stomach and the small intestine.The dose of acetaminophen widely accepted as hepatotoxic is 145 mg/kg body weight [13]; thus with MAC values ranging from 139.38 to 301.92 mg/g for the varying activated charcoal dosage forms, the charcoals should be effective in countering acetaminophen overdose, especially the powdered dosage form which has the highest MAC.
Ease of administration of the powder presents a draw back for it use thus tablet formulations can be used but would require a larger dose.The activated charcoal capsule should be least preferred for the treatment of acetaminophen poison due to due to its relatively low MAC value.

CONCLUSION
The presence of excipients in the tablets and capsules causes a retardation in adsorption via intraparticle diffusion especially at the basal micropore sites on the activated charcoal surface, especially with adsorption of acetaminophen powder occuring largely via the pseudo first order kinetics.

Figure 1 :
Figure 1: Langmuir adsorption isotherm of acetaminophen using activated charcoal capsule in pH 1.2 and pH 6.5

Figure 2 :
Figure 2: Langmuir adsorption isotherm of acetaminophen using activated charcoal tablet in pH 1.2 and pH 6.5

Figure 3 :
Figure 3: Langmuir adsorption isotherm of acetaminophen using activated charcoal powder in pH 1.2 and pH 6.5

Table 1 :
Activated charcoal dosage forms utilized

Table 2 :
Kinetic model for adsorption of acetaminophen unto varying activated charcoal dosage forms

Table 3 :
Experimental values of amount of acetaminophen adsorbed per gram of the adsorbent at equilibrium in different kinetic model