Open Chemistry 2023; 21: 20230131
Research Article
Saad M. Al-Baqami, Torki A. AlZughabi, Mohammad A. Almostady, Nourah A. Alotaibi,
Amal Almostadi, Tahreer M. AlRaddadi, Saleh O. Bahaffi*
Determination of caffeine content in coffee
drinks prepared in some coffee shops in the
local market in Jeddah City, Saudi Arabia
https://doi.org/10.1515/chem-2023-0131
received July 6, 2023; accepted September 15, 2023
Abstract: The current study was aimed to develop a low
cost HPLC method for determination of caffeine content in
coffee samples in some coffee shops in Jeddah local market.
Caffeine was extracted with water and separated through
ZORBAX Eclipse XDB C-18 column using methanol/water
(40:60) as mobile phase. The average peak response time
for caffeine was 2.747 min with a relative standard devia-
tion of 1.061. The proposed assay was able to detect caffeine
in the concentration range from 5 to 45 ppm with limits of
detection (LOD = 3σ/b) and quantification (LOQ = 10σ/b) of
1.086 and 3.619 ppm using two-channel UV detector set at
274 and 264 nm with correlation coefficient (R2) of 0.9996,
respectively. The assay was adopted for the determination
caffeine content in 30 coffee samples collected from eight
brand coffee shops and from local market. Each sample
was injected in triplicate (n = 3) and the average and con-
fidence limit of the mean were calculated. The concentra-
tion of caffeine in coffee brand shops varied from 513.82 ±
12.91 to 8080.05 ± 3.06 ppm, whereas in beans and instant
coffee its content was diverse from 360.18 ± 86.72 to 1306.98
± 27.31 ppm. These results were validated using Student’s
t-test at 95% confidence level.
Keywords: caffeine content, coffee shops, coffee drink,
HPLC, limits of detection and quantifications

* Corresponding author: Saleh O. Bahaffi, Chemistry Department,
Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia,
e-mail: sbahaffi@kau.edu.sa
Saad M. Al-Baqami, Tahreer M. AlRaddadi: Chemistry Department,
Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
Torki A. AlZughabi: Department of Medical Laboratory Sciences, Faculty
of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi
Arabia; King Fahd Medical Research Center (KFMRC), King Abdulaziz
University, Jeddah, Saudi Arabia
Mohammad A. Almostady, Nourah A. Alotaibi, Amal Almostadi: King
Fahd Medical Research Center (KFMRC), King Abdulaziz University,
Jeddah, Saudi Arabia
Open Access. © 2023 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License.
1 Introduction
Coffee is one of the greatest common beverages in the
world [1,2]. The most widely cultivated species of coffee
are Coffea arabica and Coffea robusta, which supply almost
all of the world’s consumption [2]. The world production of
green coffee in 2021 was 175.35 million (60 kg/bags) and
more than 17.89 million tons of coffee beans in Saudi
Arabia has been imported with a value of $127.18 million
[3]. Nowadays, there is a growth in coffee shops and high
consumption in coffee drink in Saudi Arabia; however,
there is great alarm on the impact of having coffee for
the public health [4,5]. Caffeine (1,3,7-trimethylxanthine)
is an alkaloid that occurs naturally in the leaves, seeds,
fruit, tea, coffee, cocoa, kola trees, and more than 60 other
plants [6]. Caffeine drug is used to provide a boost of
energy or a feeling of heightened alertness and is often
used to stay awake longer [7–9]. A pleasant stimulant feeling
is obtained by consuming low dose of caffine [10].
Caffeine consumption at high levels have been a con-
cern in various disorders such as increase of gastric-acid
secretion, kidney malfunction, heart disease (cardiac
arrhythmia), and disturbances of the central nervous
system such as seizures and delirium [11,12]. On the other
hand, extraordinary consumption of caffeine leads to
rhabdomyolysis which damages muscle fiber and enter
the blood stream, increase the incidence of bladder and
stomach cancer, increases blood pressure, aggravates
diabetes, and damages the lining of the stomach [12,13].
Thus, there is a great request for precise determination
of caffeine compounds in soft drinks and energy drinks
for assurance of food safety, and quality control is
mandatory.
Recently, numerous techniques have been reported
for trace determination of caffeine in various types and
drinks of coffee samples including voltammetry [14], NIR
spectroscopy [15], UV/visible spectrometry [16], chromato-
graphic methods [17], gas chromatography (GC)-FID [18],
2  Saad M. Al-Baqami et al.
GC-NP detector [19], GC-mass spectrometry [20], and high
performance liquid chromatography (HPLC) [21–23].
In today’s era, there is a increase in coffee shops and
high consumption in coffee drink in Saudi Arabia; there-
fore, the current study was designed to (i) precisely
assess a low cost HPLC method for caffeine trace deter-
mination using HPLC in coffee water extract, (ii) sorting
of 30 commercial coffee samples available in the various
commercial coffee drink brands available in the local
coffee shops in Jeddah city, KSA with respect to caffeine
in the coffee extract in boiled water, and finally (iii)
studying the relationship between the average content
of caffeine in coffee samples with the prices in Saudi
Arabian market.
2 Experimental
2.1 Materials and methods
Caffeine anhydrous ≥99.0% HPLC grade was purchased
from Fluke Chem. Division, San Diego, CA, USA. Methanol
for HPLC ≥99.9% grade was provided from Sigma-Aldrich
Chemicals Private Limited, Bangalore, India. All measure-
ments were performed in triplicate. The sample was fil-
tered through Whatman No. 1 filter paper, and then again
filtered through Millipore 0.45 µm before injection in the
HPLC instrument.
Figure 1: Calibration curve for caffeine determination.
2.2 Instrumentation
HPLC analysis was performed using UFLC Shimadzu at
King Fahad Medical Research Center equipped with con-
trol processing unit 20A, solvent delivery pump LC 20 AD,
degassing unit 20As, photodiode array detector, pump
mode: low pressure gradient LC 20 DA at a flow rate of
1.0 mL/min. pressure max 15 MPa. UV–VIS detector SPD-
20A equipped with deuterium lamp D2, wavelength length
1 at 274 nm, and wave length 2 at 264 nm were used.
Autosampler SIL20AC sample Rack 1.5 mL 70 vials, oven
CTO-20A, temperature adjusted at 25°C, and temperature
control cell SPD-20A were also used. A ZORBAX Eclipse XDB
C-18 analytical column of 4.6 × 150 mm 5 μm (Agilent 5301
Stevens Creek Blvd. Santa Clara, CA, USA) was used at a
flow rate of 1.0 mL/min. Milli-Q plus system (Millipore,
Bedford, MA, USA) water system was used for providing
ultra-pure water for the preparation of the solutions.
2.3 Sampling
Thirty coffee samples were collected from the local market
of Jeddah city and classified as follows: eight different
brand shops (24 sample), and six samples Harare, Lagmti,
Kholani, Katamano, Edition signature blend, and Cofiq
Prive were purchased from Al Rashid store for dates and
coffee, Abdullah Alsulymani Street, Jeddah, KSA. Coffee
brands were taken in cups, cooled, and filtered before
 Caffeine content in coffee drinks prepared in coffee shops in the local market  3

Table 1: Regression data and statistical parameters for caffeine cali- were injected in triplicate in an autosampler with 5.0 µL
bration curve injected in HPLC injection volume.

Correlation Slope (S) Y-intercept Linear

coefficient (R2) range (ppm)

0.9996 15,870 768.29 5–45 2.4 Recommended standard solution

injection, coffee powder was prepared in boiling water as
traditional Saudi coffee. The other three fast preparing
coffee brands were purchased from local market. All sam-
ples are filtered through Whatman No. 1 and then again
filtered using Millipore 0.45 µm filter paper before injec-
tion in the HPLC Autosampler, except Expresso coffee that
was diluted ten times before injection. All measurements
preparation
Caffeine stock standard solution of 1,000 µg/mL was pre-
pared by dissolving 0.10 g of caffeine standard (Sigma-Aldrich)
in 80 mL deionized water and sonicated for 10 min. The solu-
tion was then transferred to 100 mL volumetric flask and the
flask was filled up to the mark by deionized water. Working
standard solutions of 5, 10, 15, 20, 25, 30, 35, 40, 45 µg/mL were
prepared by serial dilution of the stock solution with

Figure 2: Chromatogram of 5 ppm caffeine at 274 nm.

Table 2: Caffeine concentration (ppm) in coffee brand shops

Number Coffee shop Concentration (ppm)

Black & V 60 Americano Espresso

1 LA Cima 937.61 ± 4.16 1030.26 ± 9.55 7701.54 ± 6.99

2 Brew 92 971.52 ± 21.18 854.49 ± 7.13 5801.12 ± 11.79
3 Cloud 9 956.54 ± 14.01 1093.71 ± 9.99 4852.15 ± 23.31
4 Starbucks 513.82 ± 12.91 819.67 ± 1.39 2488.87 ± 7.12
5 Ratio 1081.71 ± 4.20 1319.43 ± 4.78 8080.05 ± 3.06
6 Costa 725.16 ± 3.26 485.56 ± 4.50 1366.29 ± 5.16
7 Kims 821.69 ± 21.89 557.16 ± 7.38 5647.89 ± 1.68
8 Dose 723.23 ± 2.42 516.54 ± 6.46 839.686 ± 20.12
4  Saad M. Al-Baqami et al.

Figure 3: Chromatogram obtained for one of the injections of one coffee sample.

Figure 4: Caffeine concentration in coffee drinks in different brand shops.

deionized water. A blank solution was prepared as same as
the standard without caffeine in it. Each concentration was
Table 3: Caffeine concentration in coffee beans and instant coffee
injected in triplicate and an external calibration curve of the
average peak areas (n = 3) versus concentration of the stan-
dards was plotted. The results are presented in the calibration
plot (Figure 1) for standard preparations of standard caffeine.

No Coffee brand Concentration (ppm)

1 Harare 1264.20 ± 13.21 3 Results and discussion

2 Lagamti 1306.98 ± 27.31
3 Kholani 895.37 ± 12.18 The calibration graph in Figure 1 shows a strong positive
4 Catamona 360.18 ± 86.72 correlation between the instrumental signal and the stan-
5 Edition sig. blend 583.61 ± 28.00
dard caffeine concentrations. Ten different concentrations
6 Cofique Prive 530.77 ± 33.68
from 5 to 45 µg/mL including the blank were precisely
 Caffeine content in coffee drinks prepared in coffee shops in the local market
Figure 5: Caffeine concentration (ppm) in coffee beans and instant coffee.
analyzed, which represent the available caffeine in the
coffee product. The calibration graph was plotted using
the caffeine concentration in the standard against the
detector response (peak area). The calibration plot of var-
ious caffeine concentrations was found to be linear with
excellent correlation coefficient (R2 = 0.9996). The limits of
detection (LOD) and quantification (LOQ) were calculated
based on the standard deviation of the response (Sy) of the
curve and the slope of the calibration curve (S) at levels
approximating the LOD according to the formula: LOD =
3.3σ/b and LOQ = 10σ/b, where σ is the standard deviation
of the blank and b is the slope of the calibration plot. The
standard deviation of the response was determined based
on the standard deviation of y-intercepts of regression
lines. The calculated values of LOD and LOQ were found
to be equal to 1.086 and 3.619 µg/mL, respectively, with a
sensitivity factor of 15,870. Table 1 summarizes the regres-
sion data and statistical parameters for the designed cali-
bration plot of caffeine (Figure 2).
The data of average concentrations (μg/g) of caffeine in
coffee brand shops are summarized in Table 2 and the
representative chromatograms are illustrated in Figures 3
and 4. On the other hand, caffeine concentration (µg/g) in
coffee beans and instant coffee (μg/g) is summarized in
Table 3. The distribution pattern of caffeine concentration
(µg/g) in various coffee beans and instant coffee is illustrated
in Figure 5. Caffeine contents in the collected samples varied
 5
widely according to brands’ type and the selected coffee
shops in the current study. Overall, based on the caffeine
content, the results can be categorized into two categories
and can be arranged as follows:
The first category of selected coffee shops:
LA Cima Espresso > Americano > Black & V60
Cloud Espresso > Americano > Black & V60
Starbucks Espresso > Americano > Black & V60
Ratio Espresso > Americano > Black & V60
The second category of coffee brands:
Brew 92 Espresso > Black & V60 > Americano
Costa Espresso > Black & V60 > Americano
Kims Espresso > Black & V60 > Americano
Dose Espresso > Black & V60 > Americano
The concentration of caffeine was set from high to low
Lagmti > Harari > Kholani, whereas in instant coffee, the
caffeine concentration is highest in the Edition signature
blend, followed by the Confique Prive and Catamona.
4 Conclusion and future
perspectives
In summary, the current study reported the levels of caf-
feine in various commercial products of coffee beans,
6  Saad M. Al-Baqami et al.
instant coffee, and coffee shop drinks and provides data on
the spectrum of caffeine intake levels by human. Caffeine in
coffee can enrich a diet with microelements that have antiox-
idant activity. The level of caffeine content in eight different
brand shops varied, where high levels were found in the
espresso in all coffee brand shops while Americano and Black
& V60 share the arrangement. In the traditional coffee, Lagmti
has the highest followed by Harari and Kholani, whereas in
instant coffee, the Edition signature blend has the highest con-
centration of caffeine followed by Confique Prive and Catamona
the last. Thus, there is a need for more research that would also
take into account the content of caffeine in the water used to
prepare infusions, as well as their bioavailability for humans.
Further research and exploration are merited for the possible
physical dependence potential of low doses of caffeine such as
those concentrations found in decaffeinated coffee. A detailed
study will be performed to fully assign other microelements
and their relation with caffeine in various coffee brands.
Acknowledgements: The authors would like to thank King
Fahad Medical Research Center, Main Laboratory and
Forensic Laboratory for chromatographic analysis. The
authors also thank Prof. Dr Mohammad El-Shahawi for
his assistance and guidance. They would also like to thank
AFAQ Speciality Coffee and Trad Basonbul (development
consultant) for their assistance and guidance.
Funding information: No funding was received for con-
ducting this study.
Author contributions: Saad M. Al–Baqami: formal analysis
and writing – first draft; Torki A. AlZughaibi: project co-super-
visor and writing – review and editing; Mohammad A.
Almostady, Nourah A. AlOtaibi, and Amal A. Almostadi: formal
analysis and data procuration. Tahreer M. Alraddadi: formal
analysis, validation, and data assignment. Saleh O. Bahaffi: pro-
ject administration, supervision, and writing – original draft.
Conflict of interest: No conflict of interest among the
authors and all authors agreed to the contents in the text.
Ethical approval: This article does not contain any studies
with human participants or animals performed by any of
the authors.
Data availability statement: The original contributions
presented in the study are included in the article/supple-
mentary material. Further inquiries can be directed to the
corresponding authors. The raw data supporting the con-
clusion of this article will be made available by the authors
without undue reservation.
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