Electron J Gen Med 2019;16(6):em166

ISSN:2516-3507
OPEN ACCESS Original Article https://doi.org/10.29333/ejgm/112271

Development and validation of a simple and sensitive

HPLC method for the determination of liquid form of
therapeutic substances
Suhaib Ibrahim Alkhamaisah1, Kareem M. Younes2, Abduzhappar Gaipov3, Mohamad Aljofan4

ABSTRACT
Background: Caffeine is the most consumed psychostimulant in the world that is found in numerous foods and drinks with coffee and tea have the
highest concentration of caffeine per weight. Caffeine has a number of reported physiological benefits and clinical uses such as coma recovery. Caffeine
can prove dangerous and its consumption, the amount in foods and drinks should be closely monitored, by the Food and Drug Administration.
Material and Methods: The availability of sensitive, reliable, and simple testing methods with rapid turnover time is essential for testing any
laboratories. In this manuscript, we describe the development and validation of a simple, sensitive and economical high performance liquid
chromatographic (HPLC) method with ultraviolet detection for the quantification of the amount of caffeine in different beverages.
Results: HPLC method was validated and tested to measure caffeine contents in different beverages that showed high precision, reliability, and
sensitivity that makes it suitable for routine measurement of caffeine as well as other therapeutic or chemicals in liquid forms.
Conclusions: The proposed method recovered caffeine without the need for any extraction step for recovering the caffeine from the formulation
excipients matrices, therefore decreased the degree of error, time for estimation of caffeine and the overall cost of the analysis.

Keywords: caffeine, coffee, extraction, HPLC, PDA

INTRODUCTION
Caffeine (1,3,7-tryethylxanthine) is known as a purine alkaloide that occurs naturally in many plants particularly in
Coffea arabica, Coffea robusta and Camellia sinensis (Figure 1) (1). Caffeine has a psychoactive effect and it appeared to
exert most of its biological activity through the antagonism of the adenosine receptor, which is an endogenous inhibitory
neuromodelator that promotes feeling of drowsiness (2,3). Caffeine is the most consumed pharmacological agent
worldwide that is traditionally used to improve mental alertness, concentration, fatigue, and athletic performance (4,5).
Clinically, it is widely used for the treatment of neurasthenia and the recovery of coma (6,7). Other reported uses include
weight loss (8), improved glucose tolerance (9) and lower risk of type II diabetes (10), reduced risk for incidence of
Parkinson’s disease and improvement in Parkinson’s symptoms, and reduced risk for cancer at several sites (11). Caffeine
is playing an important role in inhibition reparation of DNA double-strand breaks in HeLa cells and ataxia telangiectasia
mutated and ataxia telangiectasia mutated-related in DNA damage response pathway in breast cancer cell lines (12,13).
Previously, we demonstrated that caffeine can inhibit viral infection and replication in vitro (14,15).
Furthermore, caffeine decreases the secretion of the pro-inflammatory cytokines interleukin-1 receptor antagonist
and interleukin-10 receptor by cancer cell-stimulated peripheral blood mononuclear cells (16). Also, it is considered as
chemopreventive and anti-inflammatory agent where it is protective agent against cirrhosis and hepatic fibrosis, two
chronic inflammatory diseases that can lead to liver cancer development (17,18).

1
Department of Pharmaceutical Chemistry; College of Pharmacy, Jerash University, Correspondence: Mohamad Aljofan
the Hashemite Kingdom of Jordan. Department of Biomedical Science, Nazarbayev University School of Medicine, Nur-
2
Department of Pharmaceutical Chemistry; College of Pharmacy, Hail University, Sultan, Kazakhstan.
Hail, Saudi Arabia.
3
Department of Clinical Science, Nazarbayev University School of Medicine, Nur- E-mail: [email protected]
Sultan, Kazakhstan.
4
Department of Biomedical Science, Nazarbayev University School of Medicine, Nur-
Sultan, Kazakhstan.

Received: 30 Jul 2019, Accepted: 11 Sep 2019

© 2019 by the authors; licensee Modestum Ltd., UK. This article is an open access article distributed under the terms and conditions
of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).
Electronic Journal of General Medicine
Alkhamaisah et al. / HPLC and Caffein

O CH3
H3C
N N

O N N
CH3
Figure 1: Chemical structure of Caffeine

Approximately, caffeine is found in the leaves, seeds, or fruits of over 63 plant species (6) with the highest contents
per source was found in coffee beans (19). However, the amount of recoverable caffeine from the different sources is
influenced by a number of factors including preparation processes such as fermentation and roasting (20). Therefore,
accurate measurement of the recoverable amounts will enable us to increase the availability of the natural occurring
substance.
Therefore, we developed and validated a sensitive high performance liquid chromatographic (HPLC) method with
ultraviolet detection to measure the amount of caffeine in different beverages and to compare the caffeine yields from
each preparation.
The developed method, which is amenable to use to measure other substances in liquid including biologics, employs
RP- Thermo C18 column, (4.6 x150 mm, 5µm id) with flow rate of 1.0 ml/min using PDA detector at 254nm and.

METHODS
Apparatus
The HPLC chromatograms were obtained using a Shimadzu instrument, Model LC-10 ADVP, equipped with a variable
wavelength UV-visible detector, Model SPD-10 AD VP, Degasser Model DGU-12 A and a 20-µl volume Rheodyne injector.
RP-Thermo C18 (5µm, 250mm x 4.6mm I.D) column was used as a stationary phase.
Materials
Samples
Caffeine standard (B.N: 08209B5-012) was purchased from Aldrich, Germany. Its purity was found to be 99.0 ± 0.26,
according to its official procedures (21). Caffeine containing products such as green/ brown/ black coffee, green/ black/
tea, and soft/ energy drinks.
Chemicals
All chemicals and reagents were of pure analytical grade. De-ionized water, Acetonitrile, Methanol (E-Merck,
Darmstadt, Germany) was of HPLC grade.
Caffeine stock and working solutions
Accurately weighed 100 mg of caffeine powder was transferred into 100-ml measuring flask. 50.0 ml of de-ionized
water was added to the flask, sonicated for a while to dissolve the drug. The flask was made up to 100 ml with de-ionized
water to get a final concentration of 1000 µg/ml. Caffeine working standard solution was prepared by accurately
transferring 25-ml of the stock solution (1000 µg/ml) into 100-ml measuring flask and then was diluted with water to a
final concentration of 250 µg/ ml.
Chromatographic conditions
The stationary phase used is C-18 RP-Thermo, 5µm, 250 mm x 4.6 mm column. A low gradient mobile phase
containing water, methanol and acetonitrile in a ratio of 84:1:15 (v/v/v) was filtered using 0.45 µm filter paper. The flow
rate was 1.0 ml/min and the detection was done using PDA detector at 254 nm. The mobile phase was degassed for
about 15 min by sonication and samples of 20µl were injected into the HPLC system where a sharp peak was obtained.

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Figure 2: Caffeine calibration curve

Table 1: Linearity and regression characteristics of standard caffeine

Parameter Caffeine
Concentration range (µg / ml) 0.122 – 125
Regression equation Y = 25686 X + 21332
Slope 25686
Intercept 21332
Correlation coefficient (R2) 0.9995
N 8
LOD (µg / ml) 0.0091
LOQ (µg / ml) 0.0303

Procedures
Construction of calibration curve
Five different concentrations that ranged from 7.813 to 125 µg/ml were prepared by accurately measuring different
volumes of working caffeine standard solutions (250 µg/ml). The prepared series of caffeine were injected into the
chromatographic system and the peak area for each concentration was recorded. The relation between the concentration
and the peak area was plotted and the regression equation was calculated (Figure 2).
Sample Analyses
Sample preparation represents one of the most important steps in analysis (22), thus samples from solid sources
(coffee beans or tea leaves) were carefully prepared as follows: a 100 g of green, brown, black or instant coffee, and 10
g of black tea were accurately weighed and finely powdered. Caffeine was extracted by boiling with water for 10 minutes,
then transferred into a separating funnel, and placed on shaker with 25 ml of Dichloromethane twice. Organic phase was
collected in round bottom flask and condensed using rotary evaporator. Preparation of liquid samples from were
performed by separately measuring 100-ml of soft and energy drinks and directly transferring them into a separating
funnel. We carried out an extraction with 25-ml of dichloromethane twice and collected the organic phase in round
bottom flask. The residue obtained from both solid and liquid sources were then transferred into 100-ml measuring flask
and completed to volume with de-ionized water.
Method Validation and Parameters
The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH)
parameters like system suitability, linearity and range, precision, accuracy, LOD and robustness (23). The linearity of the
method was studied by injecting the concentrations of the standard solution prepared in the mobile phase in the range
of (0.122-125) µg/mL for caffeine; in triplicate into the HPLC system keeping the injection volume constant. The peak
areas were plotted against the corresponding concentrations to obtain the calibration curves (Table 1).
The sensitivity of the proposed method was estimated in terms of Limit of Detection (LOD) and Limit of Quantification
(LOQ) (24). LOD = 3 SD/S and LOQ = 10 SD /S, where S.D. is the standard deviation of y-intercept and S is the slope of
the line (25) as shown in Table 1. Also, we tested the accuracy of the method by preparing recovery samples containing

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Table 2: Percentage recovery of Caffeine

Amount added (µg / ml) Amount found (µg / ml) Recovery %
26.73 26.00 97.30
Caffeine 53.94 53.00 98.30
111.00 108.61 98.00

Figure 3: HPLC Chromatogram of Caffeine

different known quantities of caffeine standard. Each sample was prepared in triplicate and then all samples were injected
into the HPLC system and the percentage recovery for the amount added was estimated.
Intra-day and Inter-day Precision
While intra-day precision were determined by estimating the % RSD of the peak area for three replicate injections of
three standard solutions in different occasions in same day, inter-day precision were estimated by assessing the % RSD
of the peak area for three replicate injections of three standard solutions in three consecutive days.
Furthermore, the specificity of the method was assessed by comparing chromatogram obtained from standard
caffeine with that from marketed solutions (26). To estimate the system’s suitability parameters, a standard solution of
caffeine was injected into the HPLC system using mobile phase solvents of different ratios (80/5/15, 85/5/10, 75/10/15,
84/1/15 and 80/1/19) and a standard solution of caffeine was injected into the HPLC system using different C8 and C18
columns, results are shown in Table 5 and 6, respectively. The effect of mobile phase flow rate variation was investigated
by injecting a standard solution of caffeine into the HPLC system using different flow rate values ranging from 0.5 to 2
ml / min, Table 7.

RESULTS AND DISCUSSION

The HPLC method with diode array detection developed for the quantitative determination of caffeine from different
liquid sources and compared to that from solid, which provided a stable retention times and a detection limit of 0.01
mg/L for a signal-to-noise ratio of 3 (Figure 3). Different columns and mobile phase ratios were tried and good results
were obtained using RP-Thermo C18 (5µm, 250 mm x 4.6 mm) column and acetonitrile: water: methanol in a ratio of
(15:1:84, v/v/v) as a mobile phase. Caffeine was detected by using PDA detector at 254 nm. The method was validated
in terms of linearity, range, specificity, precision, accuracy and robustness. The mean recovery of caffeine was 97.87% as
shown in Table 2 with a calibration curve and linearity study of caffeine showed good correlation coefficient in
concentration range of (0.122-125.0) μg/mL with an R2 value of greater than 0.9995. The detector response over a wide
range of concentrations of the analyte were plotted to obtain the calibration curve on Figure 2, and the value for R2 and
equation for the curve is shown in Table 1. Based on previously published studies, it is acceptable to use a single point
calibration in analysis of actual samples (27).
Furthermore, the accuracy of the method was confirmed by studying sample recovery using three different
concentrations for each sample in triplicates (n=3). Samples of known concentration (reference standard solutions) were

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Table 3: Precision of standard Caffeine

Amount added (µg / ml) R.S.D % (Intra-day) E%
26.73 0.022 1.93
Caffeine 53.94 0.128 1.41
111.00 0.037 0.67
Amount added (µg / ml) R.S.D % (Inter-day) E%
26.00 0.115 1.87
Caffeine 53.00 0.154 1.52
111.00 0.921 0.81

Table 4: Specificity of proposed HPLC method for Caffeine

Pure CAF / CAF preparation tR (min)
Standard CAF 3.127
Black tea 3.091
Green tea 3.146
Black Coffee 3.147
Brown Coffee 3.129
Green Coffee 3.132
Nescafe 3.125
Pepsi 3.155
Pepsi Diet 3.126
Mountain Dew 3.132
Red Bull 3.124

Table 5: Effect of mobile phase ratio on separation of standard Caffeine

Caffeine
Mobile phase ratio
Area tR (min)
80 / 5 / 15 417645 3.265
85 / 5 / 10 242177 3.332
75 / 10 / 15 16048 3.421
84 / 1 / 15 708313 3.127
80 / 1 / 19 417645 3.233

Table 6: Effect of column type on separation of standard Caffeine

Caffeine
Column type
Area tR (min)
C8 column 117645 4.321
C 18 Lichrosphere 212388 3.311
C 18 waters 510211 3.221
C 18 Thermo 708313 3.127
C 18 Zorbax 417645 3.309

analyzed and the measured values, from the respective area counts, were compared with the true values. The results
obtained from the determination of accuracy, expressed as percentage recovery (Table 2), which shows that the method
was able to accurately quantify an estimation of caffeine and that all the results were within acceptable limit of
measurement (28). Interestingly, the evaluation of the precision that was performed using 3 different sample
preparations showed a significantly high percentage of precision (Table 3) with percentage relative standard deviation
(RSD %) was found to be less than 1, and E% less than 2 which proves that the developed method is precise and
reproducible (30).
However, the specificity was evaluated by estimating the retention time of caffeine in pure and the investigational
samples from both solid and liquid sources. There was no difference in the retention time between the standard and the
investigational samples, either solid or liquid forms (Table 4). The results of limit of detection and limit of quantification
are illustrated in Table 1.
The robustness of the method was measured via determining the effect of the mobile phase ratio variation, column
variation and flow rate variation. Both of the selected mobile phase ratio of 84: 1: 15 (v/v/v) (Table 5) and column
variation, C 18 RP-Thermo,5µm, 250 mm x 4.6 mm (Table 6), resulted in a good peak shape (Table 5), acceptable back
pressure and good resolution. Also, flow rate 1mL/min, which was selected because of less retention time resulted in a
significantly better separation of the analyte (Table 7).

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Table 7: Effect of mobile phase flow rate on separation of standard Caffeine

Caffeine
Flow rate (ml / min)
Area tR (min)
0.5 542139 3.421
0.8 644317 3.261
1.0 708313 3.127
1.5 502114 3.031
2.0 337215 2.945

Figure 4: Percentage of caffeine recovered from investigational samples

Nonetheless, the method was successfully applied to measure caffeine contents from different and commonly used
beverages including, coffee (different preparations), tea (green and black) as well as several soft drinks (Figure 4). Our
results indicated that roasting of coffee beans can significantly affect the amount of caffeine found, for example there
was a significant difference in the caffeine amount recovered between black and green (raw) coffee beans as well as
between black and green tea (Figure 4). These findings are supported by a number of previously published findings that
suggested that caffeine amount depends on the preparation and extraction of the beans (2,24). Furthermore, Angeloni
et al., reported that the chemical composition of brewed coffee depends on numerous factors: the beans, post-harvest
processing and, finally, the extraction method (31). Interestingly, there was no difference between the amount of caffeine
recovered from instant coffee and that of energy drinks and that regardless of preparation methodology used, coffee
beans (black, brown or green) were shown to have significantly more caffeine contents than energy drinks (Figure 4).
These findings are demonstrate that roasted beans have significantly higher amount of caffeine than non-roasted or raw
and that a cup of coffee could potentially have more caffeine contents than a can of energy drink.
The graph above shows the amount of caffeine recovered from the investigational samples. The results indicate that
sample preparation plays an important role that can significantly affect the amount of caffeine yielded (*) represent
significance.

CONCLUSION
The current study describes the development and validation of a reversed-phase HPLC with PDA detection method
for the determination of substances from different liquid sources. In this example we used caffeine measurements from
coffee, tea and soft drinks. This is a simple, economical method with a broad detection and quantification limits that
produced good resolution for caffeine with a short analysis time in less than 3.5 minutes.
The proposed method recovered caffeine with precision without the need for any extraction step for recovering the
caffeine from the formulation excipients matrices, therefore decreased the degree of error, time for estimation of caffeine
and the overall cost of the analysis.
Finally, the rapidity and capability of quantifying low concentrations of caffeine, makes the method suitable for variety
of analyses, including pure drug analysis and assay of formulations analysis as well as its suitability in a quality control
laboratory for routine sample analysis.

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