Preparing a New Chromatographic support from Local Syrian Clay and using it in Thin Layer Chromatography for Simultaneous Determination of Paracetamol, Caffeine and Aspirin in Tablets

 

Amir Alhaj Sakur¹*, Firas Mannaa¹, Mahamed Yahia Zein Eddin²

¹Department of Analytical and Food Chemistry, Faculty of Pharmacy, University of Aleppo, Syria.

²Department of Chemistry, Faculty of Sciences, University of Aleppo, Syria.

 

ABSTRACT:

A new chromatographic support was prepared from Local Syrian Clay (Bentonite), using thermal and acid treated Clay (B_500AW) for utilizing it in thin layer chromatography (0.25mm thickness) to separate and determine of Paracetamol, Caffeine, and Aspirin in raw material and in tablets. The separation carried out using mobile phase consisted of Cyclohexane-Chloroform- Methanol - acetic acid (14:5: 0.25:0.75) v/v. The specific surface area of treated bentonite was 45m²/g. Quantification was carried out densitometerically at λ = 250nm for Paracetamol, λ = 275nm for Caffeine, and at λ = 200 nm for Aspirin. The retardation factors (R_f) of Paracetamol, Caffeine, and Aspirin were 0.10, 0.21, and 0.40 respectively. Calibration curves were obtained in the concentration ranges of 5.0-40.0µg/spot, 2.0-16.0µg/spot and 3.0-24.0µg/spot for standard solutions of Paracetamol, Caffeine and Aspirin respectively. The New Prepared Chromatographic Thin Layers were successfully applied for analysis of commercial dosage forms (tablets) containing the drugs with average recovery 98.33 –101.83% with RSD not more than 3.86%.

 

 

 

1. INTRODUCTION:

Local syrian clay (Bentonite) is rocky clay which consists of 47% SiO₂, 14.4% Al₂O₃ and some other oxides such as Fe₂O₃, MgO, CaO, Na₂O and others^{1- 4}. Bentonite has large pore volumes and high specific surface area. The thermal treatment causes decreasing of its specific surface area with increasing in the temperature of thermal treatment^1-4. Bentonite clays used in many industrial products and processes, drilling fluids, a certain lubricating grease⁵, and it can used as chromatographic supports in gas chromatography to separate different of chemical mixtures after grafting with different methods^6-10. Bentonite used as stationary phase in thin layer chromatography to separate some metal ions, and some drugs^11-14.

 

Paracetamol (Acetaminophen; N-Acetyl–p–aminophenol): is a para-aminophenol derivative, has analgesic and antipyretic properties and weak anti-inflammatory activity^{15, 16}.

 

Aspirin (Acetylsalicylic Acid; Salicylic Acid Acetate.): is a salicylate NSAID. Aspirin and other salicylates have analgesic, anti-inflammatory, and antipyretic properties; they act as inhibitors of the enzyme cyclo-oxygenase, which results in the direct inhibition of the biosynthesis of prostaglandins and thromboxanes from arachidonic acid. Aspirin used for the relief of mild to moderate pain such as headache, dysmenorrhoea, myalgias, and dental pain^15-16.

 

Caffeine (Methyltheobromine): is a methylxanthine that, like theophylline, inhibits the enzyme phosphodiesterase and has an antagonistic effect at central adenosine receptors. It is a stimulant of the CNS. Caffeine facilitates the performance of muscular work and increases the total work that can be performed by a muscle^15-16.

Due to different mechanisms of action that provide additive or synergistic efficacy. Paracetamol, aspirin, and caffeine have been extensively used as antipyretic and analgesic drugs. They are frequently prescribed in admixture with each other or in combination with other drugs^15-16.

 

Fig. 1: structures of Paracetamol, aspirin and caffeine:

 

The literature survey reveals several analytical methods for determination of each of the three components either alone or in combination with other drugs. At present, there is no specific Compendia or monograph for the simultaneous assay of aspirin, Paracetamol, and caffeine, in USP, and British Pharmacopoeia^17,18. Several methods for simultaneous determination of paracetamol, Aspirin and caffeine have been recently reported such as High performance liquid chromatography (HPLC)^19-23, Spectrophotometric techniques^24-29. Voltammetric techniques^30,31.

 

NIR transmittance spectroscopic analysis³², Fourier transform infrared spectrometry³³, spectrofluorometric technique^34,35, FT-Raman spectroscopy³⁶, Thin layer chromatography with a fluorescence plate reader³⁷, Thin layer chromatography (HPTLC) with a densitometry³⁸, and capillary electrophoresis techniques³⁹.

 

The aim of this study was to prepare a new chromatographic support from local Syrian clay (B_{500, AW}) and using it in thin layer chromatography to develop a validate analytical procedure for simultaneous determination of Paracetamol, Aspirin, and Caffeine in a tablet. The developed analytical procedure successfully used, for routine analysis of Paracetamol, Aspirin, and Caffeine in dosage form, without any interference with involved excipients.

 

2. MATERIALS AND METHODS:

2.1 Apparatus:

Surface area was measured using a Spectrophotometric method depend on adsorption of methylene Blue, Jasco V-650 dual beam UV-VIS spectrophotometer. Scanner-densitometer CS-9301PC (Shimadzu) equipped with mercury, tungsten and deuterium lamps, Camag Hand Operated TLC Coater (Switzerland), Camag UV Cabinet for assessing and marking thin layer chromatograms under UV light (Switzerland), planetary ball mill (Germany) and different size of syringe (Hamilton, Switzerland) were used.

 

2.2 Reagents and Materials:

Paracetamol (99.0%, Jiangsu, China), Aspirin (99.6%, Sinochem, China) and Caffeine (99.1%, Hebei, China) were used. Methanol, Cyclohexane, chloroform, acetic acid were of analytical grade, Merck, Germany.

 

3. PROCEDURE:

3.1 Preparation of stationary phase:

Bentonite crushed using a mortar and milled by planetary ball mill to obtain small pieces, which have diameter less than 25µm, the bentonite powder thermally treated at temperature of 500^oC for 12 hours. Further, the bentonite powder is activated with chemical reaction by Suspending 50g of bentonite in 100ml of HCl (6M) for 24 hours, to remove soluble oxides especially iron oxide. The magnetic stirrer employed to mix the solution at 300 rpm and temperature 85^oC. After that, the bentonite solution is washed several times using distilled water until the pH is neutral, and dried at 200°C for 3 h. So we obtained a new stationary phase named (B_{500, AW}).

 

3.2 Preparation of TLC plates:

For preparation of thin chromatographic layers, (10g) of treated bentonite was mixed with (0.5g) of fluorescence substance (F₂₅₄), then the mixture was added to 20mL hot water containing (0.5g) Sodium Carboxymethylcellulose as binder to obtain homogeneous slurry. The slurry was spread over glass plates (10×20cm) by [Camag Hand Operated TLC Coater) to form uniform thin layer 0.25mm thick, dried at 105°C.

 

3.3 Mobile phase:

Cyclohexane-Chloroform- Methanol - acetic acid (14:5: 0.25:0.75) v/v, were used for the development method as mobile phase.

 

3.4 Standard solutions:

Stock solutions were prepared by dissolving (2500mg) of Paracetamol, (1500mg) of Aspirin and (1000mg) of Caffeine in 30mL mixture of Chloroform and methanol (1:2), then transferred into a 50mL volumetric flask and the final volume was completed to 50mL with the same mixture of solvents. Volumes 1, 2, 3, 4, 5, 6, 7 and 8mL from the stock solution, were transferred into 10mL volumetric flasks and completed to the mark with the same mixture of solvents (these solutions contain: 5, 10, 15, 20, 25, 30, 35, and 40mg.mL^-1, for Paracetamol, 2, 4, 6, 8, 10, 12, 14 and 16mg. mL^-1, for Caffeine, and 3, 6, 9, 12, 15, 18, 21 and 24 mg.mL^-1 for Aspirin.

 

3.5 Sample preparation (Excedrin):

Twenty tablets were weighed and the average tablet weight determined (each tablet contains 250mg Paracetamol, 250mg Aspirin and 65mg Caffeine). The tablets finely powdered, and a portion of powder, equivalent to the weight of one tablet, is dissolved in 20 mL mixture of Chloroform and methanol (1:2), vigorously shaken, for a 20 min on a mechanical shaker, then filtered and transferred into a 25mL volumetric flask and the final volume was completed to 25mL, with the same mixture of solvents. This solution contains 10mg/ml Paracetamol, 10mg/ml Aspirin and 2.6mg/ml Caffeine).

 

3.6 Chromatographic conditions:

1µL of standard solutions were applicated on TLC glass plates (10cm × 20cm) pre-coated treated bentonite (F₂₅₄ with 0.25mm thickness). Mobile phase was used for development method, then the plates were dried at room temperature and quantification was carried out densitometerically at λ = 250nm for Paracetamol, λ = 275nm for Caffeine, and λ = 200nm for Aspirin. The process repeated five times for each concentration, and calibration curves were obtained in the range 5-40 µg/spot for Paracetamol, 3-24µg/spot for Aspirin and in the range 2 - 16µg/spot for Caffeine.

 

3.7 Pharmaceutical formulations (Excedrin tablets):

1 µL of solution of tablets content, were spotted on TLC glass plats for separation of (Paracetamol, Caffeine, and Aspirin) and quantification was carried out densitometerically at λ = 250 nm for Paracetamol, λ = 275 nm for Caffeine, and λ = 200nm for Aspirin. The concentrations calculated using the standard curves.

 

4. RESULTS AND DISCUSSION:

4.1 Surface area of treated bentonite:

Surface area of treated bentonite determined by the adsorption of Methylene Blue; it is found that the surface area was 45m²/g.

 

4.2 Chromatograms processing:

The position of the spots from the mobile phase front on the chromatographic plate for different concentrations (5 to 40µg/spot) of Paracetamol, (3 to 24µg/spot) of Aspirin and (2 to 16 µg/spot) of Caffeine at λ = 200nm was shown in Fig. 2.

 

Fig. 2. TLC plate of standard concentrations 5 - 40 µg/spot, of Paracetamol, 3- 24 µg/spot Aspirin, and 2-16 µg/spot, Caffeine.

 

The chromatogram of mixture of Paracetamol, Aspirin and Caffeine (20 µg/spot for each) can be observed with three peaks at different wavelengths (λ) at 200 to 300nm (Fig. 3). The first peak area for Paracetamol increases to λ = 250nm then decrease, the second for Caffeine increase to λ = 275nm then decrease, and the third for Aspirin decrease with increasing of λ Fig. (4). It is inferred from the Figs. (3, 4) that the best wavelengths to determine of Paracetamol, Caffeine, and Aspirin were 250nm, 275nm, and 200 nm respectively.

 

The retardation factors (R_f) of Paracetamol, Caffeine, and Aspirin were 0.10, 0.21, and 0.40 respectively

 

 

Fig.3 Chromatograms of mixture of Paracetamol, Aspirin, and Caffeine at different wavelengths.

 

 

Fig.4: Effect of wavelengths (λ) on peak areas of Paracetamol, Aspirin and Caffeine by TLC densitometric method.

 

 

Quantitative evaluation:

The method being validated through precision, linearity and accuracy for the determination of different standard mixtures of Paracetamol, Aspirin and Caffeine in the range of 5.0 to 40.0µg/spot for Paracetamol, 3.0 to 24.0 µg/spot for Aspirin, and in the range of 2. 0 to 16.0 µg/spot for Caffeine using λ = 200nm, Fig. 5, Tables (1- 4).

 

Application:

The results of the validation verify the fitness of the proposed analytical procedure for the identification and quantitative determination of Paracetamol, Aspirin and Caffeine in the mixture. Commercial product (that contained of the three studied Substance was analyzed in order to estimate of their contents using λ = 200nm for Paracetamol, Aspirin and Caffeine. The pharmaceutical formulation selected for the study as the following:

 

Tablets:

Each tablet contains 250mg Paracetamol, 250mg Aspirin and 65mg Caffeine of brand Excedrin (manufactured by Unipharma Pharmaceutical Industries, Damascus-Syria).

 

The three substances in the mentioned pharmaceutical formulation simultaneously evaluated, on the same plate, reducing the time and the amount of the materials required for the analysis. The obtained results and their confidence intervals are listed in Tables (1-3) and Table (4).

 

The results are in good agreement with the results of HPLC. It can observed that the difference between the results by HPLC and the found values by this method are less than 5% in and the relative standard deviation is not exceeding ± 5%. The proposed method successfully applied to determine Paracetamol, Aspirin and Caffeine in pharmaceutical formulations.

 

 

Fig. 5. Calibration curves for determination of Paracetamol (1), Aspirin (2) and Caffeine (3) in pure forms by TLC-densitometric method at λ = 200 nm.

 

Table 1: Determination of Paracetamol in Pure form by TLC Densitometric Method Using treated Local Syrian Clay (B_500AW) as support at λ = 250 nm.

*n = 5, t = 2.776. 

 

Table 2: Determination of Aspirin in Pure form by TLC Densitometric Method Using treated Local Syrian Clay (B_500AW) as support at λ = 200 nm.

*n = 5, t = 2.776.

 

Table 3: Determination of Caffeine in pure form by TLC Densitometric Method Using treated Local Syrian Clay (B_500AW) as support at λ = 275 nm.

*n = 5, t = 2.776.

 

Table 4: analytical parameters of method

 

CONCLUSION:

In the preceding method, determination of Paracetamol, Aspirin and Caffeine in pure form and Tablets pharmaceutical formulations by TLC densitometric method using treated bentonite and mobile phase: Cyclohexane-Chloroform- Methanol - acetic acid (14:5: 0.25:0.75, v/v) has been applied.

 

Quantification was carried out densitometerically at λ = 250nm for Paracetamol, λ = 200nm for Aspirin and λ = 275nm for Caffeine. The retardation factors (Rf) of Paracetamol, Caffeine and Aspirin were 0.10, 0.21 and 0.40, respectively.

 

Calibration curves were obtained in the range of 5.0- 40.0µg/spot for Paracetamol, and were obtained in the range of 3.0- 24.0µg/spot for Aspirin, and in the range of 2.0 - 16.0µg/spot for Caffeine (at λ = 200nm) for standard solutions, and Tablets pharmaceutical formulations.

 

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Received on 17.04.2020            Modified on 14.05.2020

Accepted on 21.06.2020         © RJPT All right reserved