Comparative UV-Spectroscopic analysis of different local brands of black tea available in

Maharashtra region (India) for the presence of Caffeine

Authors: Harshal Ashok Pawar 1,*, Vishakha Ashok Malusare 2, Pramila Rajaram Shinde 2

Affiliation:
1
Assistant Professor and HOD [Quality Assurance], Dr. L. H. Hiranandani College of Pharmacy,

Ulhasnagar-421003, Maharashtra, India

2
Research Scholar [M. Pharm.], Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar-421003,

Maharashtra, India

*Corresponding Author:

Dr. Harshal Ashok Pawar,

Assistant Professor and Head of Department [Quality Assurance],

Dr. L. H. Hiranandani College of Pharmacy,

Smt. CHM Campus, Opp. Ulhasnagar Railway Station,

Ulhasnagar-421003, Maharashtra, India

Contact No. : +91-8097148638

Email: harshal.dlhhcop@gmail.com
Abstract:
Caffeine is a well-known stimulant which present in tea leaves. Caffeine has drawn more attention
due to its physiological effects beyond that of its stimulatory effect. Consumers are interested in
knowing the exact amounts of caffeine existing in different brands of tea powder available in the
market. However, limited data exist, especially for store brands. The objective of the present
research work was to estimate and compare caffeine content of different local brands of black tea
available in Maharashtra region (India) using validated UV-Spectroscopic method. The caffeine
content of various brands of tea powder used in Maharashtra region (India) was found in the range
of 2.51 % to 3.19 %.
Key words: Tea, Camellia sinensis, UV- Spectroscopy, Caffeine.

1. INTRODUCTION
Caffeine is a naturally occurring alkaloid which is found in the leaves, seeds and fruits of over 63
plants species worldwide. Its physiological effects on many body systems have been reported by
researchers, including the central nervous, cardiovascular, gastrointestinal, respiratory, and renal
systems1.
Tea currently is the hot topic in both nutritional and therapeutic research worldwide. This is not so
because tea is the most preferred drink after water, but because of the presence of crucial
therapeutic compounds in tea which are more bio-stable and direct acting than those found in other
plants. The activities of these compounds are so all pervading that they are virtually broad spectrum
in their actions. Besides, the natural integration of aromatic and therapeutic compounds in tea is a
rather unique attribute. The leaves and leaf buds of Camellia sinensis are used to produce tea2.
The most common sources of caffeine are coffee, cocoa beans, cola nuts and tea leaves and the
worldwide consumption of products derived from these natural materials means that caffeine is
one of the most popular and commonly consumed drugs in the world.
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 194.19 g/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.
Caffeine has drawn more attention due to its physiological effects beyond that of its stimulatory
effect. Consumers are interested in knowing the exact amounts of caffeine existing in different
brands of tea powder available in the market. However, limited data exist, especially for store
brands. Hence the present research work was undertaken to judge the quality of differentiate local
brands of black tea available in Maharashtra region (India) based on their caffeine content using
in-house developed and validated UV-Spectroscopic technique.

2. MATERIALS & METHODS

2.1 Materials
Five different brands of black tea (Quality, Taj, Society, Wagh-Bakari and Red label) were
purchased from local market (Mumbai, Maharashtra region, India). Caffeine anhydrous LR
(F13A/1013/2101/13) was purchased from sd Fine Chem. Ltd., Mumbai (India). All the
chemicals and reagent used were of analytical reagent grade.

2.2 Preparation of Caffeine Stock solution

Accurately weighed about 100 mg of Caffeine and transferred to 100 ml volumetric flask.
Dissolved in dichloromethane and made the volume up to the mark using dichloromethane (DCM)
so as to get concentration of 1000 mcg/ml (Stock solution).

2.3 Determination of Analytical Wavelength (λ max)

A solution of concentration 10 mcg/ml was prepared from caffeine stock solution. The maximum
absorbance (λ max) was found out by scanning 10 mcg/ml solution of Caffeine prepared in DCM
between 200nm to 400nm using DCM as a blank in UV spectrophotometer (Shimadzu 1800).

2.4 Preparation of test solution

100 mg of tea powder was boiled in 100ml water at 100 ͦ C in water bath for 20minutes. The mixture
was cooled and filtered. Caffeine was isolated from aqueous extract using DCM. 10ml of aqueous
extract and 15ml of DCM were transferred to 50ml volumetric flask, lead was closed and the
mixture was shaken on rotary shaker for 30minutes at 100 rpm. The DCM layer was separated and
diluted 1ml of this solution to 10ml using DCM. The absorbances of the prepared solutions were
measured at λmax.

Calculation formula
𝑇𝑒𝑠𝑡 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 5 𝑥 𝑃𝑜𝑡𝑒𝑛𝑐𝑦
% Caffeine =
𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 100

Analytical Method Development and Validation

UV-spectroscopic method was used for estimation of Caffeine in tea samples. The developed
method was validated by determining the specificity, linearity, precision, recovery, and solution
stability according to USP and ICH guidelines.

Specificity of the method

The maximum absorbance (λ max) value obtained for test solution was compared with that of
standard. Secondly the specificity was determined by subjecting the sample solution to accelerated
degradation by heat (100 °C) for 72 h in order to verify that none of the degradation products
interfered with the quantification of the caffeine.

Linearity
Solutions of different concentrations were prepared in the range of 1mcg/ml to 14mcg/ml by
diluting the stock solution. Absorbances were noted for solutions of different concentration at
λmax and calibration curve was constructed by plotting concentration on X-axis against
absorbances on Y-axis.

Method Precision / Repeatability

It was performed on six samples from the same batch and samples were analyzed for the caffeine
content using UV spectrophotometer.
Intermediate precision was evaluated to determine the effects of random events on the precision
of the analytical procedure. This was done by performing the analysis of the same batch of black
tea on different day by changing the analyst.
Recovery/Accuracy
A recovery study was carried out by extracting 10ml of 80%, 100%, and 120% of solution of
standard caffeine in water with 15 ml of DCM separately. Each concentration was prepared in
duplicate and each one was analyzed in triplicate.

Robustness
Robustness was studied by changing the wavelength of UV spectrophotometer at 273nm ± 2nm
and analyzing test sample in triplicate using UV-Spectroscopy.

Solutions Stability
To evaluate solution stability, the standard and the sample solutions were stored at room
temperature and were analyzed by UV spectrophotometer for 24 hrs at various time intervals.

Result and discussion

Method development and Optimization

During extraction of tea powder using water, many other phytochemicals such as phenolic
constituents and tannins come into aqueous extract which interfere with caffeine while quantitative
estimation using spectroscopic technique. Caffeine is more soluble in DCM as compared to water.
DCM is selective solvent for extraction of Caffeine. Hence, initially the tea powder was extracted
using water followed by DCM. To optimize the extraction time, tea powder was extracted in water
for different time interval and the caffeine contents were estimated. Extraction time was optimized
to 30 minutes at 100rpm.
The pre-determined wavelength of maximum absorption (λmax) was 273 nm for caffeine (Fig.1).
 Fig.1: UV-Spectrum of Caffeine

Lambda max : 233.6 and 275.8 tea pwder (taj)

Lambda max: 236.2 and 275.4 ( caffeine)

Linearity and range

The calibration curve obtained was evaluated by its correlation coefficient. The absorbance of
the samples in the range of 1.0–14.0 μg/mL was linear with a correlation coefficient (R2) greater
than 0.997. The LOD and LOQ were calculated as 0.88 μg/mL and 2.66 μg/mL respectively.
 0.6

0.5
y = 0.035x + 0.0042
0.4 R² = 0.9973
Absorbance
0.3

0.2

0.1

0
0 2 4 6 8 10 12 14 16
Co ncentration (in mcg/ml)

Fig. 2: Calibration curve of caffeine in DCM

Intra-day and inter-day precision

The intra-day and inter-day precision study (Table 1) of the developed method confirmed
adequate sample stability and method reliability where all the RSDs were <2%.

Table-1: Results of Precision study

% Caffeine content
Sample No.
Method Intermediate
Precision Precision
1 3.29 3.21
2 3.23 3.27
3 3.24 3.25
4 3.22 3.21
5 3.27 3.25
6 3.24 3.23
Mean 3.25 3.24
SD 0.026 0.024
 % RSD 0.81 0.75
Cumulative Mean 3.24
Cumulative SD 0.025
Cumulative % RSD 0.77

Table-2: Results of Robustness (Wavelength: ± 2nm)

Sample No.
% Caffeine content
1
3.24
2
3.29
3
3.26
Mean *
3.25
SD*
0.025
% RSD*
0.78
* Mean, SD and % RSD of 9 samples (6 of Method precision +3 of Robustness)

Recovery
Results within the range of 98.88–101.11% ensure an accurate method (Table 3) as well as
indicate non-interference with the excipients of formulation.

Table-3: Results of Recovery study

Level Actual % Recovery Mean SD % RSD

Added (in
mg)
80% 2.44 99.71
2.47 98.96
2.41 99.46
100 3.04 98.88
 3.11 100.05
3.08 99.41
120 3.68 101.11
3.61 99.78
3.63 99.59

Solution stability: Stability study's results were within the acceptance range (Table 2) and
indicated the samples stability over 24 h (short-term).Stable upto 12hr in DCM

Table-4: Results of Caffeine content of different local brands of black tea

Sr.
Sample Name (Brand) Caffeine Content (%)*
No.
1 Quality 2.75
2 Tajmahal 3.19
3 Society 2.51
4 Wagh-Bakari 3.09
5 Red Label 2.65
* Mean of 3 determinations

Conclusion
The results and the statistical parameters demonstrate that the proposed UV spectrophotometric
method is simple, rapid, specific, accurate and precise. Therefore, this method can be used for the
determination of Caffeine in tea powder without any interference with other phytoconstituents
such as phenolic compounds, tannins etc. The present investigation will be helpful to consumer to
judge the quality of various local brands of black tea used in Maharashtra region (India) based on
their caffeine content using in-house developed and validated UV-Spectroscopic technique.

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgment
Authors are very much thankful to Dr. P. S. Gide, Principal of Hyderabad Sindh National
Collegiate Boards (HSNCB), Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar, for his
continuous support, guidance, and encouragement.

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