molecules

Article
Quantification of Caffeine and Chlorogenic Acid in Green and
Roasted Coffee Samples Using HPLC-DAD and Evaluation of
the Effect of Degree of Roasting on Their Levels
Shady Awwad 1, * , Reem Issa 2, *, Lilian Alnsour 2 , Dima Albals 3 and Idrees Al-Momani 4

1 Department of Pharmaceutical Chemistry and Pharmacognosy, Applied Science Private University,

Amman 11931, Jordan
2 Department of Pharmaceutical Sciences, Pharmacological and Diagnostic Research Center (PDRC), Faculty of
Pharmacy, Al-Ahliyya Amman University, Amman 19328, Jordan; l.alnsour@ammanu.edu.jo
3 Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Yarmouk University,
Irbid 21163, Jordan; dimabals@yu.edu.jo
4 Department of Chemistry, Faculty of Science, Yarmouk University, Irbid 21163, Jordan; imomani@yu.edu.jo
* Correspondence: sh_awwad@asu.edu.jo (S.A.); r.issa@ammanu.edu.jo (R.I.)

Abstract: Chlorogenic acid and caffeine are among the important components in coffee beans,
determining the taste and aroma. In addition, phenols and antioxidants content possess vital health
values. The main aim of this study is to determine the levels of caffeine and chlorogenic acid in
several coffee samples of different origins and degrees of roasting. The coffee samples were extracted





 using hot water. The levels of caffeine and chlorogenic acid were quantified using high-performance
liquid chromatography (HPLC) equipped with a diode array detector, a reverse phase system, and an
Citation: Awwad, S.; Issa, R.;
ODS column (C18). Total phenol and antioxidant contents were previously determined for the same
Alnsour, L.; Albals, D.; Al-Momani, I.
samples. The results showed that the highest content of caffeine was found in the medium roasted
Quantification of Caffeine and
Chlorogenic Acid in Green and
coffee (203.63 mg/L), and the highest content of chlorogenic acid content was found in the green
Roasted Coffee Samples Using coffee (543.23 mg/L). The results demonstrated a negative correlation between the chlorogenic acid
HPLC-DAD and Evaluation of the levels with the degree of roasting, while it showed a positive correlation between the caffeine levels
Effect of Degree of Roasting on Their with the degree of roasting till a certain point where the levels dropped in the dark roasted coffee.
Levels. Molecules 2021, 26, 7502. The origin of coffee samples did not show any effect on any of the measured variables. Antioxidant
https://doi.org/10.3390/ effects of coffee samples were largely determined by chlorogenic acid content.
molecules26247502

Keywords: coffee; caffeine; chlorogenic acid; high-performance liquid chromatography (HPLC);

Academic Editor: De-An Guo quantification; extraction

Received: 29 October 2021

Accepted: 7 December 2021
Published: 11 December 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
Chlorogenic acids (CGAs), a group of phenolic esters of caffeic and ferulic acids,
with regard to jurisdictional claims in
are found in many plants, fruits, and vegetables. They are reported to be found in high
published maps and institutional affil- concentrations in coffee, as high as 70–350 mg per cup of coffee [1]. More than 80 different
iations. CGAs were detected in green coffee beans, with 5-caffeoylquinic acid being the most
abundant [2].
The biological importance of CGAs was studied throughout the years, demonstrating
high antiviral, antidiabetic, antioxidant, and neuroprotective potential [1–3]. A study
Copyright: © 2021 by the authors.
by Zuo et al. [1] recently reported a significant anti-hepatitis B virus activity of CGA,
Licensee MDPI, Basel, Switzerland.
caffeic acid, and quinic acid; while Tunnicliffe et al. [3] have reported a direct effect of
This article is an open access article
CGAs on slowing intestinal glucose absorption and inhibiting hepatic glucose output, thus
distributed under the terms and having an important role in regulating glucose metabolism. CGAs also exhibit antioxidant
conditions of the Creative Commons and antiaging effects by enhancing cell proliferation, lowering lipid peroxidation, and
Attribution (CC BY) license (https:// accelerating wound healing, according to Chiang et al. [4].
creativecommons.org/licenses/by/ A recent study by Tang et al. [5] demonstrated a strong correlation between antioxi-
4.0/). dant capacity and total phenolic compounds in the Pitahaya fruit peel, highlighting the

Molecules 2021, 26, 7502. https://doi.org/10.3390/molecules26247502 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 7502 2 of 9

contribution of each phenolic compound (including CGAs) to overall antioxidant capacity.

Results have shown that CGA, one of the major phenolic compounds found, did not con-
tribute significantly to the antioxidant activity as an individual compound. However, the
copresence of CGA with other compounds in the extract may have an antagonistic effect,
which may explain the low contribution of CGA to overall antioxidant activity in wild rice
samples [6]. In another study done on Lonicerae Japonicae Flos, a Chinese traditional herb, it
was found that CGA possesses strong antioxidant activity, which was correlated to total
phenolic content. Moreover, the main contributor was CGA [7].
In addition, the degree of roasting and geographical origin of coffee beans were shown
to affect the CGA content. The higher the roasting degree, the lower the CGA content as it
is decomposed with exposure to heat [8].
Caffeine is another important component of coffee beans, which belongs to the
methylxanthine class. It is a bioactive compound that has CNS stimulatory effects, causing
alertness and mood enhancement. A single cup of coffee may contain between 95–330 mg
of caffeine. Most evidence suggests that caffeine intake of 400 mg per day or less has benefi-
cial effects on the body. For example, it may lower the risk of Parkinson’s and Alzheimer’s
disease, as well as lowering the risk of type 2 diabetes [9].
The concentration of caffeine in coffee varies depending on plant species, genetic traits,
agricultural practices, storage conditions, roasting degree, and brewing method [9]. Fuller
and Rao [9] claim that cold brew coffee made with medium roast had higher concentrations
of caffeine and 3-CGA than their dark roast counterparts. Moreover, cold brew coarse grind
samples had a higher concentration of caffeine than hot brew samples. Alqarni et al. [10]
confirm that dark roast coffee has lower caffeine and antioxidant capacity compared to raw
coffee.
The present work aimed to determine the CGA and caffeine content in Coffea arabica
samples consumed in Jordan, of various geographical origins and degrees of roasting, and
to compare the outcomes with total phenolic and antioxidant contents of the samples, to
determine if there is any correlation present.

2. Results
2.1. Caffeine and CGA Content in Coffee Beans Based on Roasting Degree
In this study, coffee samples were extracted and analyzed to assess the levels of
caffeine and CGA using HPLC-DAD. The experimental results for the caffeine and CGAs
analysis in coffee samples are presented in Supplementary Table S1. The HPLC-DAD
chromatograms for the caffeine and CGAs (standard solution and coffee extract samples)
are also presented in the same Supplementary Figures S1–S4).
The concentrations of caffeine and CGA in percentage (C, %) and mg/L in green and
roasted coffee beans based on roasting degree are reported in Table 1. The table shows
that green coffee beans contain the highest CGA, but the lowest caffeine content. The CGA
content is shown to decrease with increasing roasting degree. The caffeine content was
increased from green beans through medium roasted coffee beans. On the contrary, dark
coffee beans were shown to contain the lowest content of CGA, with a moderate content
of caffeine. The statistical analysis for all coffee samples of different roasting degrees
(green, light, medium, and dark) indicated that no significant difference (p ≥ 0.05) in the
concentrations of caffeine in the coffee samples was detected. On the other hand, the results
of the CGAs concentrations showed a significant difference (p < 0.05) for the four roasting
degrees of coffee samples.

2.2. Caffeine and CGA Content in Coffee Beans from Different Geographical Origin
The concentrations of caffeine and CGA in percentage (C, %) in green and roasted
coffee beans from different countries are shown in Figure 1. It revealed that they is no
correlation with the geographical origin and the content of these components.
 Roasting CGA (Average ± SD) Caffeine (Average ± SD)
Coffee Type
Temperature (°C) (mg/L) (%) (mg/L) (%)
Green Coffee (n =11) --- 543.23 ± 8.916 a 5.43 ± 0.089 a 166.72 ± 5.08 1.67 ± 0.051
Light Roast (n = 14) 155–165 270.93 ± 10.759 a 2.71 ± 0.108 a 196.35 ± 6.67 1.96 ± 0.067
Molecules 2021, 26, 7502 3 of 9
Medium Roast (n= 11) 175–185 187.45 ± 9.05 a 1.87 ± 0.091 a 203.63 ± 3.158 2.03 ± 0.032
Dark Roast (n = 16) 205–215 90.53 ± 12.97 a 0.91 ± 0.130 a 189.85 ± 5.81 1.90 ± 0.058
Values followed by the letter a are statistically significant (p < 0.05).
Table 1. The average concentration (mg/L) for caffeine and chlorogenic acid (CGA) content in coffee beans (n = 52) with
2.2. Caffeine
different roasting degrees, regardless and CGA Content
of geographical origin. in Coffee Beans from Different Geographical Origin
The concentrations of caffeine and CGA in percentage (C, %) in green and roasted
Roasting CGA (Average ± SD) Caffeine (Average ± SD)
Coffee Type coffee beans◦
from different countries are shown in Figure 1. It revealed that they is no
Temperature ( C) (mg/L) (%) the content of(mg/L)
correlation with the geographical origin and these components.(%)
± 8.916 a 5.43 ± a
Green Coffee (n =11) The
— statistical543.23
analysis showed that different
0.089 coffee bean±origins
166.72 5.08 have1.67no±significant
0.051
Light Roast (n = 14) difference
155–165 (p ≥ 0.05) on the concentrations
270.93 ± 10.759 a of caffeine
2.71 ± 0.108 a in the coffee samples,
196.35 ± 6.67 except for cof-
1.96 ± 0.067
fee samples originating from Brazil and Columbia (p < 0.05). Similarly, no significant dif-
Medium Roast (n= 11) 175–185 187.45 ± 9.05 a 1.87 ± 0.091 a 203.63 ± 3.158 2.03 ± 0.032
ference (p ≥ 0.05) on the concentrations
a
of CGAs a
in the same coffee samples was found,
90.53 ± 12.97 ± 0.130
Dark Roast (n = 16) 205–215
except for coffee samples originating0.91
from India±(p5.81
Ethiopia and189.85 < 0.05). 1.90 ± 0.058
Values followed by the letter a are statistically significant (p < 0.05).

C, %
5.0 Caffeine CGA
c

4.0

3.0
b
2.0 a, b 4.08 c

2.54 2.80 2.54

1.0 2.031.91 2.14 2.36
1.90 1.60 1.72
1.48

0.0
All Brazil Colombia Ethiopia India Kenya
Country
Figure 1. The caffeine and CGA content (C, %) in coffee beans from different geographical origin (different roasting degrees).
Figure 1. The caffeine and CGA content (C, %) in coffee beans from different geographical origin (different roasting de-
More details
grees). Moreconcerning the number
details concerning the of samples
number offor each origin
samples are origin
for each available
arein the Supplementary
available Table S1. Bars
in the Supplementary labeled
Table by
S1. Bars
the letters a,b,c
labeled by theare statistically
letters significant (p
a,b,c are statistically < 0.05). (p < 0.05).
significant

The
Thestatistical analysisextracted
levels of caffeine showed from
that different
the coffeecoffee bean
samples originsahave
showed no significant
positive correlation
difference (p ≥ 0.05) on the concentrations of caffeine in the coffee samples, except
with the degree of roasting temperature, but it fell short in the dark roast coffee as indi- for
coffee
cated samples
in Figureoriginating from Brazil
2. In this figure, and Columbia
the levels of caffeine(p increased,
< 0.05). Similarly, no significant
as the temperature of
difference (p ≥ 0.05) on the concentrations of CGAs in the same coffee samples was found,
roasting increased, reaching its highest level in the medium roast coffee as can be seen in
except for coffee samples originating from Ethiopia and India (p < 0.05).
different types of coffee origins.
The levels of caffeine extracted from the coffee samples showed a positive correlation
with the degree of roasting temperature, but it fell short in the dark roast coffee as indicated
in Figure 2. In this figure, the levels of caffeine increased, as the temperature of roasting
increased, reaching its highest level in the medium roast coffee as can be seen in different
types of coffee origins.
However, the levels of CGAs extracted from the coffee samples showed a negative
correlation with the degree of roasting as indicated in Figure 3. In this figure, the levels of
CGAs decreased, as the roasting degree increased in all different types of coffee origins.
 C, %
Molecules 2021, 26, x FOR PEER REVIEW
Molecules 2021, 26, 7502 Brazil (a) Kenya 4 of 10
4 of 9
2.5

Colombia (a)
2.0
C, %
Brazil (a) Kenya
2.5
1.5
Colombia (a)
2.0
1.0

1.5
0.5

1.0
0.0
0 1 2 3 0 1 2 3 0 1 2 3
0.5 Degree of roasting

Figure 2. The effect of degree of roasting on the concentration of caffeine in coffee beans from different sources. (0 = green,
0.0
1 = light roasting, 2 = medium roasting, 3 = dark roasting). Bars labeled by the letter a are statistically significant (p < 0.05).
0 1 2 3 0 1 2 3 0 1 2 3
Degree
However, the levels of roasting
of CGAs extracted from the coffee samples showed a negative
correlation with the degree of roasting as indicated in Figure 3. In this figure, the levels of
Figure
Figure2. 2. The
The effect
effect of
of degree
degree of
of roasting
CGAs roasting on
on the
decreased,the concentration
concentration
as
of
of caffeine
the roasting caffeinein
degree in coffee
coffeebeans
increased beans from
from different
different sources.
sources. (0
(0== green,
green,
11== light ain allstatistically
different types of coffee origins.
lightroasting,
roasting,22== medium
mediumroasting,
roasting,33== dark
darkroasting).
roasting).Bars
Barslabeled
labeledby
bythe
theletter
letter a are
are statistically significant
significant(p(p<< 0.05).
0.05).

C, % However, the levels of CGAs extracted from the coffee samples showed a negative
Colombia
correlation with the degree of roasting as indicated in Figure 3. In this figure, the levels of
7.0 Brazil Kenya
CGAs decreased, as the roasting
b degree increased in all different types of coffee origins.
6.0 a
c
C, % Colombia
5.0
7.0 Brazil Kenya
b
4.0
6.0 a
c
a b
3.0 c
5.0 b
c
2.0 a b
4.0
b c
1.0 a a
c
3.0
b
0.0 c
2.0 b
0 1 2a 3 0 1 2 3 0 1 2 3
Degree of roasting c
1.0 a
Figure
Figure 3.3. The
The effect
effect of degree of roasting
roasting on
on the
the concentration
concentration of
ofCGA
CGAin incoffee
coffeebeans
beansfrom fromdifferent
differentsources.
sources.(0(0= =green,
green,1
1==light 0.02 =ofmedium
lightroasting,
roasting,
degree of
roasting, 3 = dark roasting. Bars labeled by the letters a,b,c
2 = medium roasting, 3 = dark roasting. Bars labeled by the letters a,b,c are statistically significant (p < 0.05).
are statistically significant (p < 0.05).
0 1 2 3 0 1 2 3 0 1 2 3
Degree
2.3. Correlation between the Effect of roastingDegree with Caffeine, CGA, GAE, and
of Roasting
TEAC Contents
Figure 3. The effect of degree of roasting on theour
Results from concentration of CGA in coffee
previous unpublished beans
study from different
entitled sources. of
“Quantification (0 =total
green, 1
phenols
= light roasting, 2 = mediumand
roasting, 3 = dark roasting. Bars labeled by the letters a,b,c are statistically significant (p < 0.05).
antioxidants in Green and Roasted Coffee Samples of Different Origins and Evaluation
of the Effect of Degree of Roasting on Their Levels” using the same coffee samples were
used to investigate the effect of degree of roasting on the concentration of caffeine, CGA,
total phenols equivalent to gallic acid (GAE) and antioxidant equivalent to Trolox (TEAC)
in coffee beans from different sources. These parameters would collectively reflect the
health benefits of drinking coffee as a common beverage, and may also contribute to the
 Results from our previous unpublished study entitled “Quantification of total phe-
nols and antioxidants in Green and Roasted Coffee Samples of Different Origins and Eval-
uation of the Effect of Degree of Roasting on Their Levels” using the same coffee samples
were used to investigate the effect of degree of roasting on the concentration of caffeine,
Molecules 2021, 26, 7502 CGA, total phenols equivalent to gallic acid (GAE) and antioxidant equivalent to 5Trolox of 9
(TEAC) in coffee beans from different sources. These parameters would collectively reflect
the health benefits of drinking coffee as a common beverage, and may also contribute to
the choice
choice of of coffee
coffee type
type thatthat individuals
individuals wouldwould
make,make, in addition
in addition to its to its known
known influence
influence on
onthe
the taste of coffee.
taste of coffee.
Figure
Figure44 shows thatthe
shows that thelargest
largest effect
effect of the
of the roasting
roasting degree
degree was onwas the on the content
content of CGA of
CGA
and and
TEAC,TEAC,
which which showed
showed a sharp
a sharp reduction
reduction in content
in their their content
in greenin green
beans beans
throughthrough
the
the roasted
roasted samples.
samples. Oncontrary,
On the the contrary, theofeffect
the effect of roasting
roasting degree ondegree
caffeine oncontent
caffeine content
showed
a slow increase
showed from green
a slow increase from samples
green to light and
samples medium
to light androasted
medium samples,
roastedfollowed
samples,byfol-
a reduction
lowed with its content
by a reduction with its upon the dark
content uponroasting
the dark level. The content
roasting of GAE
level. The showed
content a
of GAE
fluctuation
showed in its content
a fluctuation among
in its contentdifferent
amongroasting degrees.
different roasting degrees.

Figure
Figure4. The effect
4. The effectofofdegree
degree of roastingon
of roasting onthe
theconcentration
concentration of caffeine,
of caffeine, CGA,
CGA, GAEGAE
(total(total phenols)
phenols) and (antioxidants)
and TEAC TEAC (antioxi-
dants) in coffee
in coffee beansbeans
from from different
different sources.
sources.

3.3.Discussion
Discussion
Thisresearch
This researchwas
was carried
carried out
out to
to evaluate
evaluatethe
thecontents
contentsofofcaffeine
caffeineand CGAs
and CGAsusing thethe
using
HPLC-DAD method in several coffee samples of different origins, which
HPLC-DAD method in several coffee samples of different origins, which were purchased were purchased
fromthe
from the Jordanianlocal
Jordanian local market.
market. TheThe research
research waswas
alsoalso conducted
conducted to investigate
to investigate the the
influ-
influence of the degree of roasting on the levels of caffeine and CGA. The extraction and
ence of the degree of roasting on the levels of caffeine and CGA. The extraction and quan-
quantification methods proved to be simple, efficient, sensitive, accurate, precise, and linear
tification methods proved to be simple, efficient, sensitive, accurate, precise, and linear in
in the specified range.
the specified range.
The results of this research demonstrated that the medium roasted coffee beans have
The results of this research
the highest concentration demonstrated
of caffeine, that the
while the green medium
coffee has theroasted
highestcoffee beans have
concentrations
the
of highest
CGA in concentration
the studied coffeeof caffeine,
samples, while
whichthe green
agrees coffee
with has the
the results ofhighest concentrations
most literature cited.
A study by Macheiner et al. [11] investigated green coffee infusion as a source of caffeine
and CGA. The results indicated that the levels of CGAs decreases, and these compounds
will undergo thermal degradation as the temperature of roasting increases. The loss of
CGA levels during the roasting process of coffee beans has been previously reported in
several studies. It was demonstrated that the use of high temperature during the process of
roasting can lead to the breaking of the carbon-carbon bonds of the CGA structure, which
would result in thermal degradation and isomerization of the CGAs structures [12]. Results
Molecules 2021, 26, 7502 6 of 9

for caffeine in green coffee infusion reached from 113 to 188 mg/L in C. arabica infusions,
and for CGA ranged from 628 to 1040 mg/L in C. arabica infusions. Narita and Inouye [2]
reported that the highest CGA content was found in C. arabica from Brazil. It was found
highest in green coffee beans of C. arabica, ranging between 3.40–14.4% w/w dry matters.
The experimental previous studies of caffeine were mixed and the correlations between the
levels of caffeine with the degree of roasting varied from negative to constant and positive.
The majority of these studies indicated that as the degree of roasting increases, the level
of caffeine increases, reaching the maximum in the light and medium roast coffee, then
the caffeine levels start to decline in the dark roast coffee. It is anticipated that increasing
the temperature can reduce the water content in the coffee beans and therefore can help
release the volatile compounds (e.g., caffeine) from coffee; indeed, the caffeine levels were
reduced significantly compared to the light and medium roast coffee after increasing the
temperature to higher limits (dark roast) [13–15].
CGA has an important role in determining coffee beans’ quality and beverage taste
and is a key contributor to the radical scavenger activity of coffee brews [16,17]. The
previous studies demonstrated that there is a loss of CGA during roasting. The higher
the roasting degree, the lower the content of CGA [15]. As expected, in our study, a large
variability was observed in the CGA content of the coffee samples which ranged between
5.43 to 0.9% w/w (543.23 to 90.53 mg/L), in green beans throughout dark roasted beans,
which represents an approximate six-fold reduction. These findings suggest that these
coffee beans were roasted under high temperatures and moist conditions. Similarly, Budryn
et al. [16] found that the concentration of CGA in dark roasted C. arabica was 5-fold lesser
than in green beans. The reduced content of CGA by roasting was explained by previous
studies. It was found that the increased levels of free caffeic acid were attributed to the
hydrolysis of CGA during the curing of this type of specialty coffee by exposing them to
moisture during the roasting process [12,18,19].
The CGA richest coffees were those that also registered the highest TEAC. The results
were in good agreement with the previous reports about the antioxidants of coffee [20,21].
These findings indicate that only CGA (rather than the total phenolics) is responsible for
the antioxidant activity associated with coffee consumption.
It was previously found that roasting level would contribute more to changes in CGA
content than the influence of the geographical origin [22]. As expected, the subgroup with
the highest levels of CGAs among all different types of roasted coffee (green, light, medium,
and dark) was found in the Ethiopian coffee, at 4.08% (488 mg/L), while the lowest was
found in Indian coffee, at 1.48% (148 mg/L). On the other hand, the subgroup with the
highest levels of caffeine among all different types of roasted coffee (green, light, medium,
and dark) was found in the Indian coffee, at 2.54% w/w, while the lowest was found in
Columbian coffee, at 1.60% w/w.
Among the same samples, caffeine content ranged from 1.67 to 2.03% w/w (166.72 to
203.63 mg/L), in green coffee to medium roasted coffee beans, while a slight reduction
was detected in further roasting samples. Similar results were obtained in a study by
Trandafir et al. [23], which detected caffeine contents of some commercial coffees available
on the Romanian market. It found that caffeine content ranged from 1.89 to 3.05% w/w. A
large variability was observed in CGA content of the investigated coffee samples, which
ranged between 0.6 and 2.32% w/w. Farah et al. [24], reported caffeine content in regular
coffees of around 2.54–3.33% w/w, which matches the results in several studies [10,15],
while some studies showed no correlation between the caffeine content and the degree of
roasting [25].

4. Experimental Part
4.1. Materials and Reagents
Caffeine standard, anhydrous, extra pure, was purchased from AZ Chem. (Thun-
der Bay, ON, Canada); its purity was certified to be 99.8%. CGA standard, extra pure, was
purchased from Tokyo Chemical Industry CO. LTD, Japan; its purity was certified to be
Molecules 2021, 26, 7502 7 of 9

98.0%. Methanol, Chromosolv. for HPLC ≥99.9%, was purchased from Honeywell Re-
search Chemicals (Muskegon, MI, USA). Water, in compliance to the specifications of USP,
BP, and EP grades, was purchased from LABCHEM, (Zelienople, PA, USA). Acetonitrile
(HPLC grade) was purchased from ALPHA Chemika (Mumbai, India). Formic acid (96%,
ACS grade) was purchased from TEDIA (Fairfield, OH, USA).

4.2. Sample Preparation and Extraction

A total 52 samples of ground coffee beans (Coffea Arabica), which consisted of light-
roasted (14 samples), medium-roasted (11 samples), dark-roasted (16 samples), and green
coffee (11 samples) from various origins, were acquired from several stores in Jordan. The
extraction of coffee samples was performed according to the extraction procedure that
was described by Hernandez et al. [26], but with minor modifications. All coffee samples
were extracted using hot water at 75–85 ◦ C at a ratio of 1:100 (coffee-to-solvent ratio).
Next, the coffee samples were ultra-sonicated for 5 min in order to homogenize the coffee
solutions (ultrasonic bath, OVAN, Barcelona, Spain). Afterwards, the coffee samples were
centrifuged for fifteen minutes at a speed of 7900× g using a laboratory centrifuge machine
(MPW-260R, Warsaw, Poland). Then, the coffee extracts were filtered using Whatman No. 2
filter paper. Lastly, the coffee extracts were preserved in the freezer at a temperature of
−20 ◦ C until the analysis day.

4.3. Instruments and Conditions

HPLC instrumental setup comprised products of Hitachi Technologies (Tokyo, Japan).
All analyses were performed in an air-conditioned laboratory (18 ± 2 ◦ C). The measure-
ments were performed on a VWR-Hitachi Elite LaChrom system (Pump L-2130, Autosam-
pler L-2200, Column oven L-2300, UV Detector L-2455, VWR, (Tokyo, Japan), equipped
with a (30 cm × 4 mm × 5 µm) Supelcosil LC-18-DB column (SUPELCO), flow rate of
1.0 mL/min, and UV detection was performed at 274 nm for caffeine and 330 nm for CGA.
An organizer L-2000 system (Tokyo, Japan), with a diode array detector (VWR Hitachi) was
controlled by EZChrom Elite (Chromatography Data System, version 3.3.2 SP1, Scientific
Software Inc., Agilent, Santa Clara, CA, USA) software. All injections were performed
using HPLC syringe filters (0.45 µm).

4.4. Preparation of Mobile Phase

For caffeine: The mobile phase was prepared by mixing water/methanol (60:40, v/v).
The mobile phase was degassed by ultrasonic vibrations for 20 min prior to use.
For CGA: The mobile phase was prepared by mixing 0.1% formic acid/acetonitrile
(85:15, v/v). The mobile phase was degassed by ultrasonic vibrations for 20 min prior
to use.

4.5. Standard Solutions

The standard stock solution of caffeine was prepared by transferring 0.1002 g of the
caffeine standard (equivalent to 100 mg of caffeine standard) into a 100 mL volumetric flask,
and then dissolving it in the eluent to prepare a 1000 ppm concentration. The standard stock
solution of CGA was prepared by transferring 0.1020 g of the CGA standard (equivalent to
100 mg of CGA standard) into a 100 mL volumetric flask, and then dissolving it in methanol
to prepare a 1000 ppm concentration.

4.6. Calibration Curves

For HPLC, calibration standard solutions for caffeine (10, 50, 100, 200, 500, 1000 ppm)
were prepared from the standard stock solution (1000 ppm) by appropriate dilution pro-
cesses using the mobile phase. Calibration standard solutions for CGA (10, 50, 100, 200,
500, 1000 ppm) were prepared from the standard stock solution (1000 ppm) by appropriate
dilution processes using methanol. The peak area obtained for each caffeine concentration
was plotted versus concentration, and the regression equation was computed.
Molecules 2021, 26, 7502 8 of 9

4.7. Method Development

The total run-time of the HPLC analysis for both compounds ranged from 8–9 min.
The correlation coefficients (r) were calculated from external-standard calibration curves
and the values were 0.9998 for caffeine and 0.9999 for CGA, respectively. The concentration
was found to be proportional to the area, and the response of the detector was determined
to be linear over the range of 10 to 1000 mg/L. The retention time and shape of the caffeine
and CGA peaks between the standards and samples chromatograms showed a precise
similarity and no interference was found. Consequently, the identification of the caffeine
and CGA in the coffee samples was confirmed. The content of caffeine and CGAs in the
coffee samples (mg/L) were determined using the calibration curve, and the percentage of
these bioactive compounds were calculated by taking the mass of caffeine calculated from
the calibration curve.

4.8. Statistical Analysis

The measurements of the coffee sample were performed in triplicates. The results
were expressed as mean ± standard deviation for all the replicate measurements. The data
obtained were statistically analyzed by using SPSS software (version 25, Chicago, IL, USA).
The data were also evaluated using one-way ANOVA to test the significance differences in
the mean values of caffeine and CGA levels obtained by the HPLC method.

Supplementary Materials: The following are available online. Table S1: The Average concentration
(%), SD, Min, Max, Median for caffeine and chlorogenic acid content in coffee beans (n = 52) obtained
from the Jordanian market; Figure S1: HPLC-DAD chromatogram of caffeine standard dissolved in
MeOH:H2 O [40:60] (Rt = 3.84 min, λ = 273 nm); Figure S2: HPLC-DAD chromatogram of caffeine
extract sample (Rt = 3.90 min, λ = 273 nm); Figure S3: HPLC-DAD chromatogram of CGA standard
dissolved in MeOH (Rt = 4.98 min, λ = 330 nm); Figure S4: HPLC-DAD chromatogram of CGA
extract sample (Rt = 5.09 min, λ = 330 nm).
Author Contributions: S.A.: corresponding author, samples analysis, and writing the first manuscript.
R.I.: corresponding author, methodology design, and conceptualization. L.A.: Experimental analysis,
writing—review and editing. D.A.: samples collection and resources. I.A.-M.: Data Analysis, editing
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was not funded.
Institutional Review Board Statement: This study did not require ethical approval.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful to the Applied Science Private University (ASU),
Amman, Jordan, for the full support for this research project.
Conflicts of Interest: The authors report no conflicts of interest in this work.
Sample Availability: Samples of the standard compounds are available from the authors.

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