Development and Validation of HPTLC Method for Simultaneous Estimation of Paracetamol, Ibuprofen and Caffeine in Bulk and Pharmaceutical Dosage Form

Anuruddha R. Chabukswar¹*, Vishakha G.Thakur¹, Deepali L. Dharam², Mansi H. Shah¹, Bhanudas S. Kuchekar¹, Shailesh N. Sharma¹

¹Maharashtra Academy of Engineering and Educational Research’s, Maharashtra Institute of Pharmacy, MIT Campus, Paud Road, Kothrud, Pune - 411038, Maharashtra, India.

²P. E. Society’s Modern College of Pharmacy, Nigdi, Pune - 411044, Maharashtra, India

*Corresponding Author E-mail: anichem18@gmail.com

ABSTRACT:

A fast, simple, precise and accurate high performance thin layer chromatographic method was developed and validated for the simultaneous estimation of paracetamol, ibuprofen and caffeine in combined tablet dosage form. The method employed pre-coated silica gel 60F₂₅₄ plate as stationary phase and a mixture of ethyl acetate: glacial acetic acid (9.5: 0.5 v/v) as mobile phase. The plate was scanned and quantified at 205 nm. The two drugs were satisfactorily resolved with Rf 0.48 for paracetamol, 0.75 for ibuprofen and 0.17 for caffeine respectively. Linearity was found to be in the range of 300-1100 ng/band for paracetamol, ibuprofen and caffeine respectively. Intra-day and inter-day precision in %RSD were found to be 1.21-1.62% for paracetamol, 1.16-1.50% for ibuprofen and 1.29-1.64% for caffeine respectively. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation, specificity, robustness parameters as per ICH guidelines. The correlation coefficient of paracetamol, ibuprofen and caffeine were found to be 0.9995, 0.9991 and 0.9992 respectively. The average percentage recovery of paracetamol, ibuprofen and caffeine was found to be 100.25%, 99.67% and 99.63% respectively. The proposed HPTLC method has potential applications for routine simultaneous analysis of paracetamol, ibuprofen and caffeine in combined marketed tablet dosage form.

KEYWORDS: Paracetamol, Ibuprofen, Caffeine, HPTLC, Validation.

INTRODUCTION:

Paracetamol (PARA), chemically, 4-hydroxyacetanilide/ N-(4-Hydroxyphenyl)acetamide, molecular formula C₈H₉NO₂, molecular weight 151.2 with 4g daily divided dose^(1-2). It is analgesic and antipyretic. It is indicated for the relief of minor aches and pains, cold and flu remedies. It inhibits cyclooxygenase(COX) enzyme.

Fig.1: Chemical structure of Paracetamol

Ibuprofen (IBU), chemically, (RS)-2-(4-isobutylphenyl) propionic acid/ (2RS)-2-(4-(2- Methyl propyl) phenyl)propanoic acid, molecular formula C₁₃H₁₈O₂, molecular weight 206.3 with 600mg to 1.2g daily divided dose^(1-2). It is used for relief of symptoms of arthritis, primary dysmenorrheal, fever and as an analgesic. The main mechanism of action is the inhibition of cyclooxygenase(COX).

Fig.2: Chemical structure of Ibuprofen

Caffeine (CAFF), chemically, 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione/ 1,3,7-Trimethyl-3,7-dihydro-1H-purine-2,6-dione, molecular formula C₈H₁₀N₄O₂, molecular weight 194.2 with 300 to 600mg dose^(1-2). The main mechanism of action is non-selective antagonism of adenosine receptors. It is commonly used as central nervous stimulant.

Fig. 3: Chemical structure of Caffeine

In the treatment of patients, fixed-dose combination treatments such as PARA/IBU/CAFF have advantages over monotherapy like increased efficacy, reduced side effects and lower costs. Many analytical methods have been described in the literature for the determination of paracetamol, ibuprofen & caffeine individually and in combination with other drugs^(4-15). Literature survey reveals that, there is no HPTLC method reported for the simultaneous estimation of these drugs in combined dosage form. Fixed dose combination containing PARA (325 mg), IBU (400 mg) and CAFF (25mg) is available in tablet dosage form in the market. The present study developed an HPTLC method for the simultaneous determination of three active ingredients PARA, IBU and CAFF in tablet dosage form and optimized and validated the method as per the ICH guidelines.

MATERIAL AND METHODS:

Working standards of paracetamol, ibuprofen and caffeine was a gift sample from Cipla Pvt Ltd. Fixed dose combination tablets (Brand name: IMOL PLUS) containing 325mg of paracetamol, 400mg of ibuprofen and 25mg of caffeine was purchased from local pharmacy shop. All chemicals and reagents used were of analytical grade and purchased from Merck Chemicals, Mumbai, India.

Instruments:

Camag HPTLC system with TLC scanner 3, WinCATS Software V 4.0 and Linomat V automatic sample applicator used for analysis. Precoated silica gel 60F₂₅₄ aluminium sheets (20×10 cm, 200µm thick, E-Merck, Germany) were used as stationary phase. Pre-washing of plate was done with methanol and then it was activated by keeping in an oven at115^oC for 10 minutes. Drug solutions of paracetamol, ibuprofen and caffeine were spotted in the form of bands of width 8mm with a Hamilton 100 µl sample syringe. A constant application rate of 0.1µl/s was used and the space between two bands was 10mm. The slit dimension was kept at 6mm × 0.3mm and the scanning speed was 20mm/s. Plate development was carried out in a 20cm × 10cm twin trough glass chamber saturated with mobile phase. Scanning was performed in the absorbance mode of 205nm.The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 190 to 400nm.

Preparation of standard stock solutions:

Standard stock solutions with a concentration of 1000 μg/ml were prepared in methanol for paracetamol, ibuprofen and caffeine respectively. From the standard stock solutions 1 ml of each standard stock solutions was diluted to 10 ml by methanol to prepare diluted standard solutions of 100 μg/ml of paracetamol, ibuprofen and caffeine respectively.

Optimization of the HPTLC method:

To carry out HPTLC analysis the TLC procedure was optimized. Drug solutions of paracetamol, ibuprofen and caffeine were spotted on to TLC plates and run in different solvent systems. Finally the mobile phase consisting of ethyl acetate: glacial acetic acid (9.5: 0.5 v/v) was found to be optimum, resulting in an Rf 0.48, 0.75 and 0.17 for paracetamol, ibuprofen nad caffeine respectively (Fig 7). The optimized chamber saturation time for mobile phase was 20 min at room temperature. Migration distance was allowed to run up to a distance of 8 cm. Subsequent to the development; TLC plate was dried in the current of air with the help of an air-dryer. Scanning was performed in the absorbance mode of 205nm.

Calibration curves for paracetamol, ibuprofen and caffeine:

Aliquots of 0.3, 0.5, 0.7, 0.9, 1.1 µg/ml of standard solutions of paracetamol, ibuprofen and caffeine were applied on the TLC plate. TLC plate was dried, developed and analyzed photometrically as described earlier. The standard calibration curve was generated using regression analysis.

Fig. 4: Calibration curves for paracetamol, ibuprofen and caffeine.

Method Validation:

The method of analysis was validated as per the recommendations of ICH guidelines. The method was validated for following parameters

Linearity and range:

A stock solution containing 100µg/ml for paracetamol, ibuprofen and caffeine were prepared in methanol. Different volumes of this solution were applied to the plate resulting in application of 300-1100 ng/band for paracetamol, ibuprofen and caffeine to the plate. Each concentration was applied six times to the plate and the plate was developed as described above. Peak areas were plotted against corresponding concentrations to furnish the calibration plot.

Precision:

The precision of the method was verified by replicate analysis of homogenous samples of tablet powder at different time intervals on the same day (Intra-day precision) and on second day (Inter-day precision). The results are reported in terms relative standard deviation.

Limit of detection and limit of quantitation:

To determine the limits of detection (LOD) and quantitation (LOQ), solutions of lower concentrations were used. LOD and LOQ were calculated using the equations LOD = 3.3 × SD/S and LOQ = 10 × SD/S, where SD is the standard deviation of the y-intercept (n = 3) taken as a measure of noise, and S is the slope of the corresponding calibration plot.

Specificity:

The specificity of the method was determined by analyzing standard drug and test samples. The spot for paracetamol, ibuprofen and caffeine in the samples was confirmed by comparing the Rf and spectrum of the spot with that of a standard. It was observed that the excipients present in the formulation did not interfere with the peak of paracetamol, ibuprofen and caffeine.

Accuracy:

The accuracy of the method was determined by calculating percentage recovery of paracetamol, ibuprofen and caffeine, by applying the method to drug sample to which known amount of paracetamol, ibuprofen and caffeine corresponding to 80%, 100% and 120% of label claim was been added (standard addition method). At each level of the amount six determinations were performed and the results obtained were compared. The observations are reported in (Table 2).

Analysis of paracetamol, ibuprofen and caffeine in marketed formulation:

To determine the concentration of paracetamol, ibuprofen and caffeine in tablets, the contents of 20 tablets were weighed, their mean weight determined, and they were finely powdered. The powder equivalent to 820mg was extracted with methanol. To ensure complete extraction of the drug, it was sonicated for 45min, and the volume was made upto 100ml. Then, 1ml was taken and volume made upto 10ml by methanol so that the final solution contains 325µg/ml of paracetamol, 400 µg/ml of ibuprofen and 25 µg/ml of caffeine respectively. The resultant solution was applied on the TLC plate in triplicate.The chromatograms were run as described in the “HPTLC instrumentation” section. The spots were resolved into three peaks in the chromatogram (Fig 7). The content was calculated from the peak areas recorded and the observations reported in (Table 1).

Table 1: Estimation of Paracetamol, Ibuprofen And Caffeine From Tablet Formulation:

Formulation	Label claim mg/tablet	Amount found in mg	%Label claim* mean ± SD
Paracetamol	325	324.97	99.3±0.18
Ibuprofen	400	399.94	99.2±0.02
Caffeine	25	24.82	99.5±0.15

*Average value ± SD of six determinations, SD is standard deviation.

Table 2: Recovery Study of Paracetamol, Ibuprofen And Caffeine:

Formulation	Label claim mg/ tablet	Amount added in (mg)	Total amount added in(mg)	Amount Recovered (mg) ± % RSD	% Recovery
Paracetamol	325	260	585	585.22±1.03	101.25%
	325	325	650	649.93±1.04	99.66%
	325	390	715	716.96±0.68	99.84%
Ibuprofen	400	320	720	718.43±1.03	99.71%
	400	400	800	801.99±1.35	99.99%
	400	480	880	881.56±0.97	99.31%
Caffeine	25	20	45	45.45±0.85	99.14%
	25	25	50	49.93±1.06	99.96%
	25	30	55	56.57±1.69	99.80%

*Average value ± SD of six determinations, SD is standard deviation and % RSD is relative standard deviation.

RESULTS AND DISCUSSION:

The mobile phase consisting of ethyl acetate: glacial acetic acid (9.5: 0.5 v/v) gave R_f values of 0.48, 0.75 and 0.17 for paracetamol, ibuprofen and caffeine respectively. UV overlain spectra of paracetamol, ibuprofen and caffeine showed that all three drugs absorbed appreciably at 205 nm. The result of validation studies on simultaneous estimation of paracetamol, ibuprofen and caffeine are given below.

Linearity:

The linearity was found to be in the range of 300 ng/band to 1100 ng/band for paracetamol, ibuprofen and caffeine respectively. The correlation coefficient of paracetamol, ibuprofen and caffeine was 0.9995, 0.9991 and 0.9992 respectively.

Precision;

The results of the repeatability and intermediate precision experiments depicts that the developed method was found to be precise as the % RSD values for repeatability and intermediate precision studies were < 2%, as recommended by ICH guidelines.

LOD and LOQ:

LOD and LOQ were found to be 0.003 µg/ml and 0.01µg/ml for paracetamol, 0.43 µg/ml and 1.30 µg/ml for ibuprofen and 0.036 µg/ml and 0.10 µg/ml for caffeine respectively.

Accuracy:

The percentage recovery was found to be 100.25%, 99.67% and 99.63% for paracetamol, ibuprofen and caffeine respectively, ensuring that the method is accurate.

Specificity:

Specificity of the method was well demonstrated by efficient separation of all three drugs by the solvent system.

The developed HPTLC technique is simple, precise, specific and accurate, and statistical analysis proved that the method is reproducible and selective for the analysis of paracetamol, ibuprofen and caffeine simultaneously in bulk drug and tablet formulation.

Average value ± SD of six determination, SD is standard deviation and %RSD is relative standard deviation

CONCLUSION:

HPTLC plays a major role in terms of quality assurance. The developed HPTLC technique is simple, precise, specific and accurate. The proposed HPTLC method is less expensive, more flexible and has no interferences from the excipients. Statistical analysis proves that the method is suitable for the analysis of paracetamol, ibuprofen and caffeine as bulk drugs and in pharmaceutical formulations.

ACKNOWLEGDEMENT:

The authors are grateful to Management of MAEER’s Maharashtra Institute of Pharmacy, Pune, India for providing necessary facilities to carry out the work. The authors are thankful to Anchrom Enterprises (I) Pvt. Ltd., Mulund (E), Mumbai, India for their kind support and co-operation during HPTLC analysis.

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Received on 30.07.2012 Modified on 22.08.2012

Research J. Pharm. and Tech. 5(9): September 2012; Page 1218-1222

Parameter	Result
Parameter	Paracetamol	Ibuprofen	Caffeine
Linearity range(ng/band)	300-1100	300-1100	300-1100
Correlation coefficient (r)	0.9995	0.9991	0.9992
LOD µg/ml	0.003	0.430	0.036
LOQ µ/ml	0.010	1.304	0.109
Precision (%RSD)
Intra-day	1.2186	1.1646	1.2924
Inter-day	1.6217	1.5057	1.6444
Reproducibity (%RSD)	1.6685	1.6636	1.6287