INTRODUCTION:

Biological source: It is the dried ripe seeds of Coffea arabica Linn.

Family: Xanthine alkaloids (Rubiaceae)

Chemical Formula: C₈H₁₀N₄O₂

Structure:

Fig. 1: Caffeine (1,3,7- Trimethylpurine-2,6-dione)

A purine alkaloid, caffeine (1,3,7-trimethylxanthine) is mostly found in a variety of drinks. Therefore, it is crucial to measure the amount of caffeine included in different foods. Caffeine is an ingredient in the most popular drinks consumed worldwide, including tea, coffee, and soft drinks. Soft drink brands differ in how much caffeine they contain, although the US Food and Drug Administration (FDA) limits the amount to no more than 6mg/oz of fluid or 200mg/L. As a result, caffeine analysis is necessary to guarantee that beverages contain the right amount of caffeine and to comply with legal requirements¹.

Establishing a more accurate, straightforward, quick, and affordable analytical method is crucial for monitoring caffeine levels in food and beverages so that its physiological effects on the human body's metabolism can be studied. These effects include raising blood pressure, stimulating the central nervous system, and controlling the quality of food^2,3. The excessive use of caffeine can also be unsafe⁴ Caffeine is an odorless, white solid that has the form of needles or powder. Caffeine has a bitter taste. The molar mass of caffeine is 194.19g/mol. Caffeine is slightly soluble in water due to its moderate polarity. Caffeine is a natural central nervous system stimulant, having the effects of reducing drowsiness and recovering alertness. Since it is widely consumed by humans, caffeine is considered the most frequently used psychoactive substance in the world³. A moderate caffeine intake from tea appeared to improve mental performance⁴. The International Olympic Committee (IOC) defined caffeine as a drug and abuse is indicated when athletes have urine caffeine concentrations higher than 12μg/mL⁵.

MATERIALS AND METHODS:

Materials:

Samples of green tea (Tetley Tea) and soft drinks (sting) were purchased from the local market. The green tea sample of Tetley was in the form of a tea bag.

All the chemical used in analytical grade. The chemical like chloroform, Caffeine, sodium carbonate anhydrous, de-ionised water are used.

For the UV/Visible study the spectrophotometer model Double Beam Spectrophotometer AU- 2702 Systronics (India) limited is used.

Methodology:

Preparation of Stock solution (Caffeine):

A 100ppm standard caffeine stock solution is prepared. Several standards of caffeine were prepared from the stock solution ranging from 2 to 30ppm. The standards are scanned from 200 to 400nm in the UV spectrum, yielding a maximum absorbance at 276nm.

Preparation of tea samples:

A 250mL beaker was filled with 500mg of dried, crushed, and sieved tea. 100mL of boiling distilled water was then added. Rather of the 40⁰C indicated in the prior method, the solution was left on a boiling water bath for five minutes at a temperature of 90 to 100⁰C. Next, the solution was filtered⁷. To precipitate the tannins, the solution was combined with anhydrous sodium carbonate. The earlier research disregarded this phase.^5,6 After another filtering to get rid of the tannins, 10ml of the solution is extracted at least three times using 10ml of chloroform. After extraction all the fractions were combined and the fraction after dilution is analyzed with the spectrophotometer. The absorbances of the prepared solutions were measured at 276nm. The % of Caffeine content was calculated by using formula:

Test Absorbance × 5 × Potency

% Caffeine = ––––––––––––––––––––––––––––

Standard Absorbance × 100

Preparation of soft drink sample:

Transfer approximately 5 milliliters (after degassing) of a suitable soft drink sample (Sting) into a 125milliliter separatory funnel. Instead of using carbon tetrachloride, as Tautua A et al.⁸ had previously done in his study, add 10ml of distilled water, 1ml of 20% aqueous Na2CO3, and then 10ml of chloroform (since caffeine is more soluble in it). After five minutes of vigorous shaking, transfer the lower, non-aqueous layer to the volumetric flask. All layers are mixed and examined following three extractions.

Analytical Method Development and Validation:

The measurement of caffeine was done using a UV spectroscopic technique. By comparing the devised method to USP and ICH criteria, its specificity, linearity, accuracy, recovery, robustness, robustness, and LOD/LOQ were evaluated.

1. Range/Linearity:

The stock solution was diluted to create solutions with varying concentrations between 10 ppm and 50ppm. For solutions with varying concentrations at λmax, absorbance was recorded, and a calibration curve was created by graphing concentration on the X-axis versus absorbance on the Y-axis.

2. Precision:

Using a UV spectrophotometer, the composition of six samples from the same batch was examined.

2.1. Intraday Precision: The percentage Relative Standard Deviation (%RSD) for the Analysis within a day (24 hours), at different time intervals, was calculated in order to determine the intraday precision.

2.2. Interday Precision: Relative Standard Deviation (RSD) was computed for the study of six replicates over the course of three consecutive days in order to determine interday precision.

3. Accuracy:

Separately, 10 milliliters of the 80%, 100%, and 120% standard solution were extracted in order to conduct an accuracy investigation. Every concentration was made twice, and each was examined three times.

4. Ruggedness:

Ruggedness was studied to measure the reproducibility of method under variation in condition other than laboratory.

5. Robustness:

UV spectrophotometer was set to 276nm ± 2nm, and test samples were analyzed in triplicate using UV spectroscopy to examine robustness.

6. LOD and LOQ:

The lowest concentration of the analyte in a sample that can be identified by an analytical process is known as its limit of detection; this concentration need not be measured to an exact value

LOD = 3.3* SD/Slope

The lowest concentration of the analyte in a sample that can be detected as an exact number with appropriate accuracy and precision is known as the limit of quantification for an analytical method.^9,10

LOQ = 10*SD/Slope

RESULTS AND DISCUSSION:

Selection of wavelength:

10 ppm Solution was prepared from stock solution and scanned over a UV wavelength (200-400nm), The observed λmax is Presented in Fig. No. 2.

Fig. No. 2: UV Spectrum of Caffeine.

1. Linearity:

The absorbance should not exceed greater than 1.8, hence the range was selected between 10-50ppm.

The Correlation coefficient is greater than 0.999 which indicates linearity of the method.

Green tea (Tetley):

Table No1: Result of Linearity of Green Tea(Tetley)

Conc.	Absorbance	Mean	0.8758
10	0.291	SD	0.4623
20	0.583	RSD	0.5278
30	0.875	%RSD	52.7892
40	1.171	CC	0.9999
50	1.459	Intercept	-0.0014
		Slope	0.02924
		Eqⁿ of line	y=0.0097x-0.0054

Fig. 3: Calibration curve of green tea (Tetley)

Linearity of Soft Drink (Sting):

Table No. 2: Linearity Soft drink (Sting)

Conc.	Absorbance	Mean	0.7066
10	0.235	SD	0.3695
20	0.476	RSD	0.5229
30	0.711	%RSD	52.29
40	0.939	CC	0.9999
50	1.172	Intercept	0.0055
		Slope	0.02337
		Eqⁿ of line	y=0.0097x-0.0054

Fig. 4: Calibration Curve of Soft Drink (Sting)’

2. Precision: %RSD values were found well within 2% limit, indicating that the current method is repeatable.

2.1 Intraday Precision:

Table No: 3 Result of Intraday Precision Tetley

Sr. No.	Time	Conc.	Absorbance
1.	0	20	0.583	Mean	0.5826
2.	2	20	0.589	SD	0.0036
3.	4	20	0.581	RSD	0.0062
4.	8	20	0.579	%RSD	0.0620
5.	12	20	0.584
6.	24	20	0.580

2.2 Interday Precision:

Interday precision of Green Tea (Tetley):

Table No 5: resul of Interday precision of Green Tea (Tetley)

Sr. No.	Time	Conc.	Absorbance
			Day1	Day2	Day3		Day1	Day2	Day3
1.	0	20	0.583	0.580	0.584	Mean	0.5826	0.5828	0.5883
2.	2	20	0.589	0.591	0.588	SD	0.0036	0.0040	0.0027
3.	4	20	0.581	0.582	0.580	RSD	0.0062	0.0069	0.0046
4.	8	20	0.579	0.581	0.582	%RSD	0.0620	0.6983	0.4681
5.	12	20	0.584	0.582	0.585
6.	24	20	0.580	0.581	0.583

Intraday precision of Soft drink (Sting):

Table No: 6 Result of Interday precision of Soft drink (Sting)

Sr. No	Time	Conc.	Absorbance
			Day1	Day2	Day3		Day1	Day2	Day3
1.	0	20	0.483	0.480	0.484	Mean	0.4826	0.4828	0.4836
2.	2	20	0.489	0.491	0.488	SD	0.0036	0.0040	0.0027
3.	4	20	0.481	0.482	0.480	RSD	0.0074	0.0084	0.0056
4.	8	20	0.479	0.481	0.482	%RSD	0.7489	0.8429	0.5649
5.	12	20	0.484	0.482	0.485
6.	24	20	0.480	0.481	0.483

3. Accuracy: The % RSD was less than 2% which indicates the method is valid.

Accuracy of Green Tea:

Table No. 7: Result of accuracy of Green Tea (Tetley)

% Level	Absorbance	Mean	SD	RSD	%RSD	Cumulative %RSD
80%	0.578					0.60
	0.569	0.5766	0.0070	0.0123	1.2302
	0.583
100%	0.589
	0.593	0.5906	0.0020	0.0035	0.3524
	0.590
120%	0.595
	0.586	0.5866	0.0080	0.0136	1.3671
	0.579

Accuracy of Soft drink (Sting):

Table No. 8: Result of accuracy of Soft drink (Sting)

% Level	Absorbance	Mean	SD	RSD	%RSD	Cumulative %RSD
80%	0.474					1.41
	0.465	0.4740	0.0090	0.0189	1.8987
	0.483
100%	0.489
	0.493	0.4793	0.0020	0.0042	0.4236
	0.456
120%	0.466
	0.493	0.4743	0.0090	0.0191	1.9129
	0.456

4. Ruggudness: The % RSD for the sample was less than 2% shows the Ruggedness of the method

Ruggedness of Green Tea (Tetley):

Table No. 9: Result of Ruggedness of Green Tea (Tetley)

Sr. No.	Parameter.
1.	Analyte	X	Y
2.	System	A	B
3.	Day	Monday	Tuesday
4.	Time	Morning	Afternoon	Mean	SD	RSD	%RSD
5.	Conc.	20	20	0.5846	0.0056	0.0097	0.9725
6.	Absorbance	0.583	0.579	0.5843	0.0075	0.0129	1.2958
		0.580	0.593
		0.591	0.581

Ruggedness of Soft drink (Sting)

Table No. 10: Result of Ruggedness of Soft drink (Sting).

Sr. No.	Parameter
1.	Analyte	X	Y
2.	System	A	B
3.	Day	Monday	Tuesday
4.	Time	Morning	Afternoon	Mean	SD	RSD	%RSD
5.	Conc.	20	20	0.4846	0.0056	0.0117	1.1732
6.	Absorbance	0.483	0.479	0.4843	0.0075	0.0156	1.5633
		0.480	0.493
		0.491	0.481

5. Robustness: The % RSD was less than 2% which indicates the method is valid.

Robustness of Green Tea (Tetley):

Table No. 11: Result of Robustness of Green Tea (Tetley) Wavelength (-2nm)

Sample No.	Concentration	%Caffeine Content
	(Ppm)	At 274nm	At278nm
1.	30	0.801	0.865
2.	30	0.881	0.871
3.	30	0.871	0.879
Mean		0.8510	0.8716
SD		0.0435	0.0057
RSD		0.0112	0.0065
%RSD		1.1220	0.6589

Robustness of Soft drink (Sting):

Table No. 12: Result of robustness of Soft Drink (Sting)

Sample No.	Concentration	%Caffeine Content
	(Ppm)	At 274nm	At278nm
1.	30	0.701	0.701
2.	30	0.781	0.706
3.	30	0.721	0.698
Mean		0.7343	0.7016
SD		0.0416	0.0032
RSD		0.0166	0.0047
%RSD		1.6695	0.4702

6. LOD and LOQ:

Soft Drink (Sting):

Table No. 13: Result of LOD of Tetley

Parameter	Concentration.	Absorbance.
LOD	30	0.869
	30	0.889
	30	0.857
LOQ	30	0.867
	30	0.875
	30	0.891
Mean	0.8716	0.8776
SD	0.0161	0.0122
RSD	0.0185	0.0139
%RSD	1.8545	1.3923

1. LOD = 3.6 ×SD/Slope LOD= 0.5506

2. LOQ =10 ×SD/Slope LOQ =0.2924

Green Tea (Tetley):

Table No. 14: Result of LOD of Soft Drink (Sting)

Parameter	Concentration.	Absorbance.
LOD	30	0.729
	30	0.719
	30	0.757
LOQ	30	0.697
	30	0.715
	30	0.731
Mean	0.7350	0.7143
SD	0.0196	0.0170
RSD	0.0207	0.0208
%RSD	2.07	2.08

1. LOD= 3.6×SD/Slope LOD = 0.8386

2. LOQ= 10×SD/Mean LOQ= 0.7274

Marketed Sample Analysis:

The local brand of Green Tea (Tetley)and Soft drink (Sting) available in Market (India) was analyzed for caffeine content using current in-house developed and validated UV-spectroscopic technique.

According to https://www.tetleyusa.com >Tetley the caffeine present in Tetley Green Tea is 0.6 % (i.e. 15mg per 250gm) and https://m.indiamart.com Soft drink (Sting) is 2.88% (i.e. 72/250ml), the result of the research was found to be Tetley 0.4% and Sting 2.5which is nearly similar.

The results of caffeine content obtained for brand (Parivar) is summarized in table no.15

Table No. 15: Result of Caffeine content under different brands

Sr. No.	Category	Name brand	Caffeine Content
1.	Green Tea.	Tetley Tea	0.4%
2.	Soft Drink.	Sting	2.5%

CONCLUSION:

We used UV-Visible spectrophotometry to modify the approach, which may have contributed to the greater caffeine level we found in the local green tea (Tetley) and soft drink (Sting) samples.

The UV visible spectrophotometric method is a sensitive, accurate, and reliable technique that was used in this work to quantitatively analyze the caffeine contents in green tea and soft drinks. When compared to HPLC and other analytical procedures, it is also a less expensive and time-consuming method.

The suggested UV spectrophotometric approach is easy to use, quick, precise, and accurate, as shown by the results and statistical parameters.

The research shows the caffeine content in Tetley is 0.4% and sting is 2.5% which is in range of promising limit by the company Tetley (i.e., 0.6%) and Sting (i.e. 2.88%.

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Received on 15.06.2024 Modified on 18.07.2024

Asian J. Pharm. Ana. 2024; 14(3):155-160.

DOI: 10.52711/2231-5675.2024.00028