INTRODUCTION:

Caffeine (C₈H₁₀N₄O₂) is the common name for trimethylxanthine (systematic name is 1, 3, 7-trimethylxanthine or 3, 7-dihydro-1, 3, 7-trimethyl-1H-purine-2, 6-dione).It is an addictive stimulant. In humans, it stimulates the central nervous system, heart rate, and respiration, has psychotropic (mood altering) properties, and acts as a mild diuretic. Caffeine may be used for the short-term relief of fatigue or drowsiness. It is absorbed and passes quickly into the brain¹.Caffeine is official in USP and BP and their monograph revealed that RP- HPLC method methods were described for its estimation^{2, 3} . Caffeine has been determined in combination with other drugs using UV-spectrophotometer⁴, High-Performance Liquid Chromatography (HPLC)^5-10, Gas chromatography^11-13, NIRS ^{8, 14} and Mass spectrometry¹² in pharmaceutical preparations. However, most of the reported methods involve troublesome mobile phase (buffers) and difficult detection methods such as mass detectors.

In this study, efforts were made on the development of a simple and easy HPLC method using water and methanol as mobile phase with UV detection at 272 nm. The method was optimized and validated as per the guidelines of the International Conference on Harmonisation (ICH) ¹⁵.

MATERIAL AND METHODS:

Active drug and reagents

Standard Caffeine powder was kindly supplied by Incepta Pharmaceutical Ltd (Bangladesh) and was used as the reference standard. All chemicals and reagents were of analytical or pharmaceutical grade. Excipients such as maize starch, Avicel PH 101, sodium starch glycolate, Povidone K-30, Magnesium stearate and purified talc were of pharmaceutical grade.

Instrumentation and chromatographic condition

An integrated high performance liquid chromatography system (Shimadzu) was used for this experiment. A C18 L1, pH resistant (4.5 mm x 250 nm: 5µm) column (Luna, Phenomenex) was used. The detecter was set at 272 nm and the run time was 20 minutes at a flow rate of 1 ml/ minute at room temperature.

Selection of mobile phase

Initially water (distilled and demineralized) and methanol (HPLC grade) were used at 40:60, 50:50 and 60:40 ration as mobile phase. Finally water and methanol were selected as the mobile phase at a ratio of 60:40 at ambient temperature using flow rate of 1.0 ml/min and run time was set for 8 minutes.

Linearity study

Caffeine (20mg) was weighed accurately and taken in a 100 ml volumetric flask and water was added to dissolve. Then enough water was added to make the final volume to 100 ml to get the stock solution (Cs). For linearity study, five aliquots in the range of 3 to 7 ml of Cs were taken and diluted to 50 ml to obtain different concentrations within the range 12-28 µg/ml and used for the calibration plot.

Intra-day precision study

Accurately weighed tablet powder, equivalent to 20 mg Caffeine, was transferred into a 100 ml volumetric flask. An amount of water (50 ml) was added, shaken for 15 min and diluted to the 100 ml mark with same solvent. Aliquots (4, 5 and 6 ml) of this solution was taken and respectively diluted to 50 ml with the water to obtain three concentrations of 16, 20 and 24 µg/ml respectively. Sample was measured in HPLC and the mean, standard deviation and RSD were calculated for each sample.

Inter-day precision study

The above samples were analyzed again as the following day for inter-day precision study and the mean, standard deviation and RSD were calculated.

Accuracy study

This study was carried out using 80%, 100%, 120% pure Caffeine and preformulated granules of common excipients including maize starch, Avicel PH 101, Sodium starch glycolate, Povidone K-30, Magnesium stearate and purified talc etc. 120 mg granules was then transferred in to three 100 ml volumetric flasks and added 80%, 100%, 120% Caffeine active respectively into there and diluted to the 100 ml mark with water and then filtered. The solutions were analyzed by HPLC at 272 nm. for the content of Caffeine using the proposed method with a standard solution (20 µg/ml of pure Caffeine). All analyses were carried out in triplicate.

Specificity in the presence of excipients

This test was carried out using only excipients. Placebo granules devoid of the pure Caffeine were prepared, the solutions was analyzed by HPLC at 272 nm.

Limit of detection (LOD) and Limit of quantification (LOQ)

Limit of detection (LOD) and Limit of quantification (LOQ) for the assay were calculated using the following equations ¹⁶

LOD = 3.3 ×S₀/ b and LOQ = 10 ×S₀/ b

Where S₀and b are the standard deviation and the slope of the calibration line.

Assay of content of Caffeine in selected marketed brands

This was carried out using the developed and validated method as follows-

Sample preparation

Accurately weighed tablet powder, equivalent to 20 mg Caffeine, was transferred into a 100 ml volumetric flask. An amount of water (50 ml) was added, shaken for 15 min and diluted to the 100 ml mark with same solvent. It was then filtered and 5ml of it was further diluted to 50ml to obtain sample solution, then assayed for content of Caffeine using the proposed method with a solution containing 20 µg/ml of pure Caffeine prepared from C_s as standard for comparison. All analyses were carried out in triplicate.

Reference standard preparation

5 ml of Cs was diluted to 50 ml of water to obtain a 20 µg/ml Caffeine reference standard solution. The sample and reference solutions were analyzed by HPLC at 272 nm. The content of Caffeine in the marketed brands was determined using the following equation-

Content of Caffeine (%) per tablet = (As/Ast) × (Wst/100 × 5/50) × (100/Ws × 50/5) × W × P/100

Where, As = area of the peak of generic sample solution,

Ast = area of the peak of reference Caffeine standard solution,

Wst = weight of reference Caffeine powder (mg)

Ws = weight of generic powder sample (mg)

W = average weight of tablet (mg)

P = Potency of standard Caffeine hydrochloride

Statistical analysis

Where applicable, results were expressed as mean ± SD and analyzed statistically.

Table 1. Absorbance and corresponding concentrations of standard Caffeine solution

No of Observations	Concentration (µg/ml)	Area of the peak
1	12	743987
2	16	1069804
3	20	1451788
4	24	1763308
5	28	2106581

RESULT AND DISCUSSION:

The linearity parameter (Table 1 & Figure 1) and the corresponding regression data, indicated excellent linear relationship (R²= 0.9992) over the working concentration range (12 -28 µg/ml). Table 2 and Table 3 presents respectively the intra-and inter-day precision of the new method, confirming adequate sample stability and method reliability over a 24 h period. This is because for the three selected concentrations within the linearity range, the observed RSDs were all < 2.0 %.

Figure 1: Calibration curve for Caffeine

Table 2: Intra-day precision study of Caffeine

Intra-day Precision study( n=3 replicates)
Declared Conc. (µg/ml)	Calculated concentration (µg/ml)			Mean ± SD	RSD	Average Potency
Declared Conc. (µg/ml)	1	2	3	Mean ± SD	RSD	Average Potency
16	15.8	15.9	15.7	15.8±0.10	0.63%	98.75%
20	20.1	19.8	19.8	19.9±0.17	0.87%	99.50%
24	23.8	23.9	23.8	23.8±0.05	0.24%	99.29%

Table 3: Inter-day precision study of Caffeine

Inter-day Precision study( n=3 replicates)
Declared Conc. (µg/ml)	Calculated concentration (µg/ml)			Mean ± SD	RSD	Average Potency
Declared Conc. (µg/ml)	1	2	3	Mean ± SD	RSD	Average Potency
16	15.8	15.8	15.7	15.76±0.057	0.36%	98.50%
20	20.1	19.8	19.8	19.90±0.17	0.87%	99.50%
24	23.8	23.9	23.8	23.83±0.057	0.24%	99.29%

Table 4: Accuracy study of Caffeine

Accuracy study ( n=3)
Dosage form	Labeled claim	Amount added (%)	Area of peak	% Recovered
Pre formulated granules	500 mg	80	1096083±0.12	98.6
		100	1381916±0.58	99.38
		120	1693813±1.05	100.02

Table 5: Assay of Caffeine in marketed tablets

Assay of Caffeine in marketed tablets (n=3)
Formulation	Labeled claim	Amount found ±SD	Assay	RSD
Brand 1	65 mg	64.720 ± 0.195	99.57%	0.0395
Brand 2	65 mg	65.637 ± 0.096	100.98%	0.0196
Brand 3	65 mg	98.133 ± 0.098	104.82%	0.0199
Brand 4	65 mg	65.845 ± 0.150	101.30%	0.0302

The result of Accuracy (Table 4) was within the range of ICH guideline. The % accuracy indicated non-interference from excipients of formulation. The results of analysis of 5 marketed brands were good and shown in Table 5. The limit of detection (LOD) and Limit of quantification (LOQ) were calculated as 0.152 µg/ml and 0.461µg/ml respectively.

CONCLUSION:

The results and the statistical parameters demonstrate that the proposed HPLC method is simple, rapid, selective, accurate, precise and highly sensitive. Therefore, it can be used for the determination of Caffeine either in bulk or in their corresponding dosage forms or in a combination dosage form with paracetamol without interference from commonly used excipients and related substances whose λmax are not close to 272 nm. The proposed method is simple and do not involve laborious time-consuming sample preparation. So this HPLC method can be used in the quality control department.

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Received on 12.02.2012 Accepted on 28.02.2012

Asian J. Pharm. Ana. 2(1): Jan.-Mar. 2012; Page 01-04