WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 3, Issue 8, 516-524.
Research Article
ISSN 2278 – 4357
DEVELOPMENT AND VALIDATION OF HPTLC METHOD FOR
DETERMINATION OF CAFFEINE IN FOOD, BEVERAGE AND
MEDICINAL PREPARATIONS
Ankit Raghuwanshi1, Jinu John2*, C.T. Aravindakumar2
1
2
Hislop School of Biotechnology, Hislop College, Nagpur
Inter University Instrumentation Centre, Mahatma Gandhi University, Kottayam, Kerala
Article Received on
18 May 2014,
Revised on 15 June
2014,
Accepted on 07 July 2014
ABSTRACT
A simple, precise and accurate HPTLC method was developed for the
determination of caffeine in different coffee products, beverage and
pharmaceutical products. Caffeine from different samples such as
*Correspondence for Author
natural coffee bean, locally marketed coffee powder, instant coffee
Dr. Jinu john
mix, Cola drink and tablets was extracted with dichloromethane.
Inter University
Analysis was performed on silica gel G 60 F254 HPTLC plates with
Instrumentation Centre,
chloroform and methanol in the proportion of 25:1 (v/v) as mobile
Mahatma Gandhi University,
Kottayam, Kerala-686560
phase. Samples were applied with Linomat V under nitrogen gas flow.
Caffeine gave a clear band with an Rf value 0.24±0.02. The
densitometric analysis was performed CAMAG TLC scanner 4 at 274 nm. The linear
regression analysis of data for the calibration curve showed good linearity over a
concentration range of 50 ng to 200 ng/spot with a regression value of 0.99953 and standard
deviation of 1.59%. The regression equation of the calibration curve was Y=687.946 +
24.936 * X. The limit of detection and limit of quantification of caffeine was 0.198 ng spot-1
and 0.599 ng spot-1. Using the developed method, the concentration of caffeine in complex
matrix containing samples such as marketed coffee powder, soft drink and tablet was
determined.
Key words: Caffeine, HPTLC method, beverage and pharmaceutical preparation.
1. INTRODUCTION
Caffeine is a pseudoalkaloid secondary metabolite present in different plant species including
tea, coffee and cola. It was first isolated by the German chemist Friedrich Ferdinand Runge in
www.wjpps.com
Vol 3, Issue 8, 2014.
516
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
1819. It is produced commercially both by extraction from natural sources and synthetic
procedures. The majority of caffeine produced is used in the beverage industry. Caffeine is
also used therapeutically. Caffeine is an addictive stimulant and is regulated by the Food and
Drug Administration (FDA). In humans, it stimulates the central nervous system, heart rate,
and respiration, has psychotropic (mood altering) properties, and acts as a mild diuretic.
Caffeine is being utilized as flavour-enhancer in most of the beverage (soft drinks), coffee
and tea products. So, its analytical estimation/determination methods become more important
for its regulation.
Figure 1: Structure of caffeine (1,3,7-trimethylxanthine)
Various analytical techniques are available now for the estimation of caffeine such as
chromatographic techniques: Planar chromatography coupled with electropray ionization
mass spectrometry using stable isotope dilution analysis,[1] HPTLC densitometry method,[2]
high-performance liquid chromatography (HPLC) with spectrophotometric and amperometric
detection,[3-5] Ion chromatography,[6] GC-MS method,[7] capillary electrophoresis[8] and
UV/Vis. Spectrophotometry.[9] Spectrophotometer method is fast, simple, accurate,
reproducible and inexpensive procedure as compared to other methods; however, it is not
possible to determine caffeine directly in samples with complex matrices by conventional
UV-Vis absorption measurement due to the spectral overlap of UV absorbing substances in
the sample. High performance thin-layer chromatography (HPTLC) has many advantages
when compared to other chromatographic such as several samples can be run simultaneously
using a small quantity of mobile phase, thus reducing the time and cost per analysis.
Simultaneous chromatography of samples and standards, under identical conditions, leads to
the excellent analytical accuracy and precision of analytical output compared to sequential
injection of the samples and standards in HPLC.
The aim of this study was to develop an economic, accurate, simple, rapid HPTLC method
for the analysis of caffeine in samples containing complex matrices of different nature. The
method has been validated as per International Conference on Harmonization (ICH) guide
www.wjpps.com
Vol 3, Issue 8, 2014.
517
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
lines and the method could be suitable for routine analysis in beverages and pharmaceutical
industry quality control.
2. MATERIAL AND METHODS
2.1 Standard preparation: Pure caffeine was purchased from Merck was prepared by
dissolving 1 mg pure caffeine in 10 ml of methanol. To obtain homogeneous solution, it was
then sonicated for 20 minutes at 600 C. The solution was stored at 4°C in dark. All the
solvents and chemicals used were of analytical grade purchased from Merck, Mumbai.
2.2. Sample Preparation
The three coffee products (Nescafe classic, Bru intant, local coffee powder), beverage (Cola
drink) and IMOL Plus tablet were purchased from local market, Kottayam, Kerala, India.
Coffee beans were collected from the campus premises. Coffee beans were dried and
powdered without any treatment or deshelling. 10 mg of each sample was accurately weighed
and dispensed in 10 ml of distilled water and sonicated for 20 min. 10 ml of the coca cola
sample was kept for degassing by sonication for 1 hour and left overnight for complete
degassing. For the extraction of the caffeine, equal volume of dichloromethane was added in
a separating funnel containing sample. Intermittent shaking was given for 1 hour and the
lower layer of each sample was collected in separate glass tube, this process was repeated 3
times to ensure the complete caffeine extraction. The final volume of sample was made to 10
ml by evaporating dichloromethane. All the samples were stored in dark at 4°C.
2.3. Chromatographic conditions
Aluminum-backed HPTLC plates pre-coated with silica gel 60 F254 (10 cm x 10 cm) from
Merck was used as stationary phase. Samples were applied to the HPTLC plate using the
spray-on technique of CAMAG LINOMAT V sample applicator under nitrogen gas flow to
get a range of concentrations by varying the volume per spot. TLC plates were activated in an
oven.
Preliminary screening and optimization of solvent system and other chromatographic
conditions were done by trial and error method for better band resolution and accuracy.
Before sample application, the plates were washed in methanol and activated by drying in an
oven. Spots of were applied with a band length of 6 mm at a distance of 7.8 mm between
each track. Samples were applied at speed of 150 nl/sec. with nitrogen as spray gas using
CAMAG analytical syringe of 100 µl capacity. Chromatogram was developed in Camag
www.wjpps.com
Vol 3, Issue 8, 2014.
518
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Twin Through chamber (10 cm x 10 cm) with 10 ml of mobile phase, chloroform: methanol
(25:1, v/v). Application positions were at least 10 mm from the sides and 10 mm from the
bottom of the plates. Mobile phase components were mixed prior to use and the development
chamber was left for saturation with mobile phase vapour for 20 min before each run by
placing filter paper moistened with the solvent system in twin trough chamber. Development
of the plate was carried out by the ascending technique to a migration distance of 80 mm.
Then the plates were dried with a hair drier. Detection and densitometric scanning was
performed by Camag Scanner 4 at absorption mode at 274 nm with the slit dimension 5 x 0.3
mm. The sample track scanning speed was 20 mm/sec and spot spectrum scanning speed was
100 nm/sec. Densitometry was carried out with CAMAG TLC Scanner 4, fitted with winCATS planar chromatography manager software (version 1.4.8.2012). All the analyses were
carried out at controlled room temperature of 25±3 ºC.
2.4. Method Validation
The developed method was validated for different parameters (linearity and range,
specificity, precision, accuracy, limit of detection and limit of quantification) as per the
International Conference on Harmonization (ICH) guide lines.[10-11]
2.5. Determination of caffeine in different samples
Using the developed method concentration of caffeine in different samples were measured by
spotting specific volume of prepared sample in duplicate and the average area of the specific
band was measured by densitometric measurement. From calibration curve made out of the
chromatogram prepared on the same plate, the concentration of the sample was determined
by extrapolating the values. The actual concentration was then calculated by considering the
dilution factor.
3. RESULT AND DISCUSSION
3.1 Optimization of mobile phase and chromatographic conditions
Mobile phase and chromatographic conditions were optimized to get a precise accurate and
reproducible method. Chloroform and methanol (25:1 v/v) ratio was found to be the most
suitable mobile phase, which showed good band resolution with the Rf value of 0.24±0.02.
Chromatogram developed on HPTLC silica gel layers containing fluorescent indicator,
produced crisp and dark bands of caffeine on a bright green background, when viewed under
UV light (254 nm). Better results were obtained when saturation time was kept 20 minutes at
room temperature 25±3 0C.
www.wjpps.com
Vol 3, Issue 8, 2014.
519
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
3.2 caffeine calibration curve
The linearity of the caffeine calibration plot was determined by spotting increasing amount of
caffeine standard solution, range starting from 50 ng to 200 ng/spot. The method showed
good linearity in this range (Fig. 1) with a linear regression equation of Y=687.946 + 24.936
* X (r2= 0.99953; sdv= 1.59%).
Figure 1: Calibaration curve of caffiene shows linear relation
3.3. Validation of method
3.3.1. Precision: The precision was expressed in terms of percentage relative standard
deviation of the developed HPTLC method (Table 1). The intra-day and inter-day precision
(% RSD levels) were found to be less than 1.0 in all cases, which indicated that there were
no significant variations in the analysis of cholesterol at these concentrations.
Table1: Precision of the HPTLC method developed for caffeine determination
Sample
Concentration
(ng/spot)
100
150
200
* mean of three replicates
caffeine
www.wjpps.com
Intra-day
Inter-day
Accuracy
Precision Accuracy Precision
(%)*
(% RSD)
(%)*
(% RSD)
99.58 ± 0.69
0.18
99.03 ± 0.52
0.20
98.14 ± 1.11
0.34
99.91 ± 0.38
0.12
99.77 ± 1. 09
0.33
98.17 ± 1.30
0.45
Vol 3, Issue 8, 2014.
520
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
3.3.2. Limit of detection and limit of quantification
The standard solutions were applied as the above mentioned method, representing 10–50 ng
caffeine per spot. The regression equation of the calibration curve for caffiene was Y =
278.511 + 32.679*X, and the correlation coefficient (r) was 0.99964, when the peak area was
plotted against concentration. The limit of detection (LOD) and limit of quantification (LOQ)
were calculated using the equation, LOD = 3.3 × N/B and LOQ = 10 × N/B, where N is the
standard deviation of the peak area taken as a measure of the noise and B is the slope of the
corresponding calibration curve. The LOD for caffeine was found to be 0.198 ng and the
LOQ was 0.599 ng. These values are comparatively better to that of previous reported
analytical methods.[2]
3.3.3. Selectivity and specificity
HPTLC chromatogram was developed for the determination of selectivity and specificity
with solvent blank and complex matrix containing samples with standard caffeine. Caffeine
extract of Coca cola sample was used for the determination of selectivity and specificity.
Sample showed well separated and clear peak with an average Rf value of 0.24±0.02 (Fig. 3).
Thus the method was considered to be specific.
Figure 2: Densitogram at 274 nm showing the specificity of Caffeine (Rf= 0.24 ± 0.02) in
Coca cola sample
www.wjpps.com
Vol 3, Issue 8, 2014.
521
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
3.3.4. Accuracy and Recovery
Determination of accuracy was done by standard spiking method. It was calculated by
comparing the determined concentration of spiked samples to the theoretical concentrations.
Sample was spiked by the addition of the known amount of caffeine at different concentration
level in to the sample. For each concentration spotting was repeated in three different tracks.
The mean percentage recovery for each concentration was calculated at each concentration
level and reported with its standard deviation. Results showed very good recovery of caffeine
in spiked samples (Table 2).
Table 2: Recovery of caffeine showing the accuracy of the method
Spiking Caffeine
level
added (ng)
0
1
50
2
100
Caffeine found (ng)
(Mean ± SD, n=3)
254.37 ± 1.03
303.76 ± 1.28
352.87 ± 1.65
% Recovery
(Mean ± SD, n=3)
99.79 ± 0.42
99.57 ± 0.46
3.3.5 Determination of Caffeine in samples
Extracted caffeine fraction of all samples gave sharp and well defined peak with similar Rf
value of 0.24±0.02. Concentration of caffeine in different samples was determined by the
developed method and is given in Table 3. Concentration of caffeine in IMOL Plus tablet was
found to be almost matching with the labeled quantity (25mg).
Table 3: Concentration caffeine determined by the developed method in different
samples
Sample name
Nescafe Classic
Bru Instant Coffee
Local coffee powder
Coffee Beans
Coca Cola
IMOL Plus Tablet
Concentration
43.30 ± 1.02 mg per gram
27.40 ± 2.31 mg per gram
19.18 ± 0.98 mg per gram
11.70 ± 1.63 mg per gram
8.22 ± 1.47 mg per 100 ml
24.78 ± 0.72 mg per tablet
CONCLUSION
The developed method has demonstrated a highly reliable validated method for the
quantification of caffeine by thin layer chromatography. The method showed several
advantages in comparison with other analytical techniques in terms of limit of detection and
quantification. Among the most relevant features were that it was cost-effective and timesaving, its versatility with regard to stationary phase and the very low detection limits – in the
www.wjpps.com
Vol 3, Issue 8, 2014.
522
John et al.
World Journal of Pharmacy and Pharmaceutical Sciences
nanogram range. At a time we can run multiple samples in a single plate. Analysis result of
pharmaceutical and beverages samples with this developed method was found to be
comparable with the label. Thus this analytical method may therefore, be recommended for
the rapid, accurate and sensitive quantification of caffeine in routine analysis.
ACKNOWLEDGEMENT
Authors are grateful to the Coordinator, DST-Purse program for providing financial support
and infrastructure facility for the research work.
REFERENCES
1.
Aranda M, Morlock G. New method for caffeine quantification by planar
chromatography coupled with electropray ionization mass spectrometry using stable
isotope dilution analysis. Rapid Commun Mass Spectrom, 2007; 21: 1297-1303.
2.
Sullivan C, Sherma J. Development and Validation of an HPTLC‐Densitometry Method
for Assay of Caffeine and Acetaminophen in Multicomponent Extra Strength Analgesic
Tablets. Journal of Liquid Chromatography and Related Technologies, 2003; 26(20):
3453-62.
3.
Bispo MS, Veloso MCC, Pinheiro HLC, et al. Simultaneous determination of caffeine,
theobromine, and theophylline by high-performance liquid chromatography. J
Chromatogr Sci, 2002; 40: 45-48.
4.
Meyer A, Ngiruwonsanga T, Henze G. Determination of adenite, caffeine, theophylline
and theobromine by HPLC with amperometric detection. Fresenius J Anal Chem, 1996;
356: 284-87.
5.
Gennaro MC, Abrigo C. Caffeine and theobromine in coffee, tea and cola-beverages:
Simultaneous determination by reversed-phase ion interaction HPLC. Fresenius J Anal
Chem, 1992; 343: 523-25.
6.
Chen QC, Wang J. Simultaneous determination of artificial sweeteners, preservatives,
caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion
chromatography. J Chromatogr A, 2002; 937: 57-64.
7.
Yang MJ, Orton ML, Pawliszyn J. Quantitative determination of caffeine in beverages
using a combined SPME-GC/MS method. J Chem Educ, 1997; 74: 1130-32.
8.
Zhao Y, Lunte CE. Determination of caffeine and its metabolites by micellar
electrokinetic capillary electrophoresis. J Chromatogr B, 1997; 688: 265-74.
www.wjpps.com
Vol 3, Issue 8, 2014.
523
John et al.
9.
World Journal of Pharmacy and Pharmaceutical Sciences
Muszalska I, Zajac M, Wrobel G, Nogowska M. UV/VIS spectrophotometric methods for
determination of caffeine and phenylephrine hydrochloride in complex pharmaceutical
preparations. Validation of the methods. Acta Pol Pharm, 2000; 57(4):247-52.
10. International Conference on Harmonization Guidance for Industry. In: Q2B Text on
Validation of Analytical Methods, Switzerland, IFPMIA, 1996; 1-8.
11. Jose K, Jayasekhar P, Jinu J. HPTLC Determination of Cilostazol in Pharmaceutical
Dosage Forms. International Journal of Advanced Research, 2014; 2(2):952-957.
www.wjpps.com
Vol 3, Issue 8, 2014.
524