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). It is commonly used as central nervous stimulant. The main mechanism of action is non-selective antagonism of adenosine receptors.

Fig. 3: Chemical structure of Caffeine

The present study developed ratio-derivative 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⁽³⁾. Dose combination treatments such as PARA/IBU/CAFF have advantages of increased efficacy, reduced side effects and lower costs. Several UV, HPLC, HPTLC methods have been described in the literature for the determination of paracetamol, ibuprofen and caffeine individually and in combination with other drugs^(4-16). However, there is no ratio-derivative 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.

MATERIAL AND METHODS:

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

Instrumentation:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. Electronic balance (Model Shimadzu AUW-220D) was used for weighing.

Preparation of Standard Stock Solutions and Calibration Curve:

Standard stock solutions of pure drug containing 1000 μg/ml of PARA, IBU and CAFF were prepared separately in methanol. Standard stock solutions were further diluted with methanol to get working standard solutions of analytes in the concentration range of 6.5-32.5 μg/ml, 8-40 μg/ml and 0.5-2.5 μg/ml of PARA, IBU and CAFF, respectively and scanned in the range of 200-400 nm. For ratio derivative first derivative amplitudes (at interval 1.2 and filter size 9) of ratio spectra were measured at 213.89 nm, 213.14 nm and 268.46 nm for PARA, IBU and CAFF respectively. First derivative amplitudes of ratio spectra and concentrations were used to construct calibration curve.

Analysis of Marketed formulation:

Twenty tablets were weighed and triturated and a quantity of tablet powder equivalent to 2.5 mg of CAFF (i.e. 32.5 mg of PARA and 40 mg of IBU) was weighed and dissolved in the 100 ml of methanol with the aid of ultrasonication for 30 min and solution was filtered through Whattman paper No. 41. Filter paper was washed with same solvent, adding washings to the volumetric flask and volume was made up to the mark with methanol. The solution was suitably diluted further with methanol to get desired concentration.

Theoretical Aspects:

Ratio Derivative:

The method involves dividing the spectrum of mixture by the standardized spectra of the divisor to get first derivative of ratio spectra which was independent of concentration of divisor (Fig.4).The ratio spectra of different PARA standards at increasing concentrations were obtained by dividing spectrum of mixtures with the stored spectrum of the standard solution of IBU+CAFF (24μg/ml+21.5 μg/ml) divisor as shown in (Fig 5) and of IBU standards at increasing concentrations were obtained by dividing spectrum of mixtures with the stored spectrum of the standard solution of PARA+CAFF (19.5μg/ml+1.5 μg/ml) divisor as shown in (Fig 6) and CAFF standards at increasing concentrations were obtained by dividing spectrum of mixtures with the stored spectrum of the standard solution of PARA+IBU (19.5 μg/ml+24 μg/ml) divisor as shown in (Fig 7). Amplitude of PARA, IBU and CAFF were measured at 213.89 nm, 213.14 nm and 268.46 nm for PARA, IBU and CAFF. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs over the selected concentration range. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The concentrations of PARA (C_PARA), IBU (C_IBU) and CAFF (C_CAFF) in solution of tablets was calculated by using equations 1, 2, 3, respectively.

At 213.89 nm, C_PARA = [A_{PARA + IBU +CAFF}/ A_IBU+ A_CAFF] − intercept(c) /slope (m) eq.. (1)

At 213.14 nm, C_IBU = [A_{PARA + IBU +CAFF}/ A_PARA+ A_CAFF] − intercept(c) /slope (m) eq.... (2)

At 268.46 nm, C_CAFF = [A_{PARA + IBU +CAFF}/ A_PARA+ A_IBU] − intercept(c) /slope (m) eq. (3)

Fig.4: Overlain spectra of Paracetamol (19.5 μg/mL), Ibuprofen (24 μg/mL), Caffeine (1.5 μg/mL) and standard mixture in methanol.

Fig 5: First derivative of ratio spectra of 6.5, 13, 19.5, 26, 32.5 µg/ml of PARA when 24µg/ml + 1.5 µg/ml of IBU and CAFF respectively, used as divisor.

Fig 6: First derivative of ratio spectra of 8, 16, 24, 32, 40 µg/ml of IBU when 19.5µg/ml + 1.5 µg/ml of PARA and CAFF respectively, used as divisor.

Fig 7: First derivative of ratio spectra of 0.5, 1, 1.5, 2, 2.5 µg/ml of CAFF when 19.5µg/ml + 24 µg/ml of PARA and IBU respectively, used as divisor.

Table 1: Optical characteristics of the proposed method and result of precision and formulation analysis

Parameter		PARA	IBU	CAFF
Wavelength (nm)		213.89	213.14	268.46
Beer’s Law Range (μg/ml)		6.5-32.5	8-40	0.5-2.5
Regression Equation*	Slope	0.0095	0.0074	0.0113
	Intercept	0.0102	0.0103	0.0013
Correlation Coefficient (r²)		0.996	0.995	0.999
Precision (n= 3)	Repeatability	0.82	0.64	0.76
	Intra-day	0.77	0.89	0.83
	Inter-day	0.98	0.65	0.94
	Analyst	0.72	0.78	0.69
Formulation analysis (% assay, %RSD), n=6		99.32 ± 0.45	100.97 ± 0.57	98.12 ± 0.73
Limit of Detection (LOD) (μg/ml)		0.098	0.129	0.048
Limit of Quantitation (LOQ) (μg/ml)		0.299	0.397	0.146

Recovery Studies:

The accuracy of the proposed method was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50 %, 100 % and 150 %) within the range of linearity for all three drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 13 μg/mL, 16 μg/mL and 1 μg/mL of PARA, IBU and CAFF, respectively.

Solution Stability:

Method stability was checked by analyzing solutions kept in fridge and at room temperature. Solutions at room temperature were stable for 12 hours and solutions in fridge were stable for 7 days (% RSD < 2).

Table 2: Result of recovery studies by using formulation

Recovery Level	Base level Amount (µg/ml)			Amount Spiked (µg/ml)			% Mean Recovery % RSD (n=3)
Recovery Level	PARA	IBU	CAFF	PARA	IBU	CAFF	PARA	IBU	CAFF
50 %	13	16	1	6.5	8	0.5	99.38 ,1.23	101.37,0.85	98.20,0.56
100 %	13	16	1	13	16	1	98.72, 1.27	98.91, 1.12	98.45,0.47
150 %	13	16	1	19.5	20	1.5	100.13,0.87	100.16,0.91	98.98,0.74

RSD = Relative Standard Deviation, Y* = mX + c, where Y is the absorbance and X the concentration in micrograms per millilitre

Precision of the Method:

Method repeatability was determined by three times repetitions of assay procedure. For intra-day precision method was repeated 3 times in a day and the average % RSD was determined. Similarly, the method was repeated on three different days for inter-day precision and average % RSD was determined (Table 1). Precision of analyst was determined by repeating study by another analyst working in the laboratory.

RESULTS AND DISCUSSION:

Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed. Mixtures of increasing concentrations in ratio PARA:IBU:CAFF (13:16:1) were prepared and scanned. Three separate standard mixture PARA+IBU (19.5+24µg/mL), IBU+CAFF (24+1.5 µg/mL), PARA+CAFF (19.5+1.5 µg/mL) were prepared and used as a divisor. A critical evaluation of proposed method was performed by statistical analysis of data where slope, intercept, correlation-coefficient are shown in Table 1. As per the ICH guidelines, the method validation parameters checked were linear, accurate and precise. Beer-Lambert’s law is obeyed in the concentration range of 6.5-32.5μg/mL, 8-40μg/mL and 0.5-2.5μg/mL for PARA, IBU and CAFF respectively. Correlation-coefficient was greater than 0.990 for all three drugs. The proposed methods were evaluated for the assay of commercially available tablets containing PARA, IBU and CAFF. The results of formulation analysis are presented in (Table 1). Recovery was found in the range of 98.22-100.57% for PARA, 98.34-101.75% for IBU and 98.15-99.25% for CAFF method (Table 2). The accuracy is evident from the data as results are close to 100 % and standard deviation is low.

ACKNOWLEDGEMENT:

The authors are grateful to Management of MAEER’s, Maharashtra Institute of Pharmacy, Pune, India for providing necessary facilities to carry out the work.

CONCLUSION:

The developed UV method is simple, precise, specific and accurate. The proposed 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 for routine determination.

REFERENCES:

1. Indian Pharmacopoeia Vol II and III. The Indian Pharmacopoeia Commission office. Ghaziabad. 2010; 6^th Edition: 953, 1479, 1859.

2. British Pharmacopoeia Vol I and II. British Pharmacopoeia Commission Office. London. 2012; 327-328, 1116-1118, 1656-1659.

3. International conference on Harmonization. Validation of Analytical Procedure. Text and Methodology Q2 (R1). ICH, IFPMA, Geneva, 2005.

4. Janhavi R. et al. Simultaneous HPTLC Determination of Paracetamol and Dexketoprofen trometol in pharmaceutical dosage. Der Pharma Chemica. 3 (3); 2011: 32-38.

5. Chitlange S. et al. Spectrophotometric Methods for Simultaneous Estimation of Dexibuprofen and Paracetamol. Asian J. Research Chem. 2 (1); 2009: 30-33.

6. Reddy P. et al. RP-HPLC Method for Simultaneous Estimation of Paracetamol and Ibuprofen in Tablets. Asian J. Research Chem. 2 (1); 2009: 70-72.

7. Hassan W. et al. Determination of Ibuprofen and Paracetamol in Binary Mixture Using Chemometric-assisted Spectrophotometric Methods. American Journal of Applied Sciences. 5 (8); 2008: 1005-1012.

8. Vyas A. et al. Simultaneous Estimation of Nabumetone and paracetamol by Vierodt’s Method in Combined Tablet Dosage Form. International Journal of ChemTech Reseach. 2 (1); 2010: 543-547.

9. Vichare V. et al. Simultaneous Spectrophotometric Determination of Paracetamol and Caffeine in Tablet Formulation. International Journal of PharmTech Research. 2 (4); 2010: 2512-2516.

10. Hajian R. et al. The Spectrometric Multicomponent Analysis of a Ternary Mixture of Ibuprofen, caffeine and Parcetamol by the Combination of Double Divisor-Ratio Spectra Derivative and H-point Standard Addition Method. E-Journal of Chemistry. 9 (3); 2012: 1153-1164.

11. Baheti K. et al. Validated Simultaneous Estimation of Parcetamol and Etoricoxib in Bulk and tablet HPTLC Method. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2 (2); 2011: 672-675.

12. Patel M. et al. The Simultaneous Estimation of Paracetamol and Tolperisone Hydrochloride in Tablet by UV Spectrophotometric Methods. Journal of Pharmaceutical Sciences and Bioscientific Research. 2 (2); 2012: 63-67.

13. Rao J. et al. Validated HPTLC method for simultaneous estimation of Rabeprazole Sodium, Paracetamol and Aceclofenac in bulk drug and formulation. Der Pharma Chemica. 3 (4); 2011: 171-179.

14. Nyola N. et al. Simultaneous Estimation of Famotidine and Ibuprofen in pure and Pharmaceutical Dosage Form by UV-Vis Spectroscopy. International Research Journal of Pharmacy. 3 (4); 2012: 277-280.

15. Khoshayand R. et al. Simultaneous spectrophotometric determination of paracetamol, ibuprofen and caffeine in pharmaceuticals by chemometric methods. Spectrochim Acta A. 70 (3); 2008: 491-499.

16. Kabra P. et al. Spectrophotometric Simultaneous Analysis of Paracetamol, Propyphenazonel and Caffeine in tablet Dosage For. International Journal of Pharmacy and Pharmaceutical Sciences. 3 (3); 2011: 170-174.

Received on 23.07.2014 Modified on 16.08.2014

Asian J. Research Chem. 7(12): December, 2014; Page 1019-1022