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. 2025 Oct 23;10(43):50896–50903. doi: 10.1021/acsomega.5c03408

A New Segmented Gradient HPLC-DAD and Spectra Index Plots for Measuring Caffeine and Chlorogenic Acid in Dark and Green Coffees

Rania F Abou El-Ezz †, Khaled Meselhy †,‡,*, Samy Emara §, Asmaa Ibrahim †
PMCID: PMC12593112  PMID: 41210770

Abstract

A rapid and sensitive segmented gradient elution high-performance liquid chromatographic methodology with a diode array detector (SGE-HPLC-DAD) has been developed for quantifying important bioactive compounds, chlorogenic acid (ChGA) and caffeine (CAFF), in different coffee samples. A Luna cyano-column (5 μm, 25 × 0.46 cm) conditioned at ambient temperature (22 ± 1 °C) and a gradient elution system consisting of solvents A (1% trifluoroacetic acid in water) and B (acetonitrile) were employed. The elution process used a segmented gradient program, starting with a gradual increase in solvent B from 5% to 8% over the first 4 min. This was followed by a rapid ramp-up from 8% to 100% over 1 min (4–5 min) and then an isocratic elution at 100% B lasting 5–7 min. Thereafter, there was a linear decrease from 100% to 5% B over the next minute 7–8 min and finally an isocratic elution by a mixture of A and B (95:5, v/v) until 11 min. The flow rate was maintained at 1.5 mL/min with effluent detection at 254 nm. The SGE-HPLC-DAD method revealed linear relationships between peak areas and concentrations, with CAFF ranging from 0.4 to 1000 μg/mL and ChGA ranging from 0.6 to 1000 μg/mL. The recovery percentages for CAFF and ChGA were estimated to be between 100.97% and 101.33%, respectively. The relationship between ChGA and CAFF contents in dark and green coffees commercially available in the Egyptian market that were brewed using stainless-steel and glass cookware materials on direct heat was also investigated.

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1. Introduction

Coffee’s distinct flavor has contributed to its popularity among consumers, establishing it as one of the most desired and commonly enjoyed beverages. It is also very important from a historical, cultural, social, and economic perspective. In terms of botany, coffee is a member of the genus Coffea, which is classified under the Rubiaceae family. Commercial coffee drinks are prepared from beans such as Coffea arabica, Coffea robusta, or a combination thereof. Robusta is often from lowland Central and West African and South Asian regions (mainly from Vietnam), , whereas arabica is mostly from South America (primarily from Brazil) and upland and mountainous regions of East Africa. The quality of coffee is determined solely by the chemical composition of the roasted beans, which is influenced by the characteristics of the green beans and postharvest processing conditions.

Coffee beans contain an ingredient called chlorogenic acid (ChGA), which is known as 3-(3,4-dihydroxycinnamoyl)­quinic acid (Figure S1). In chemical terms, it is the result of the esterification of quinic acid and caffeic acid. ChGA contributes to the color, taste, and aroma of coffee beans, thereby influencing the overall quality and desirability of the beverage. ChGA is generally classified as a secondary metabolite in plants, serving as a defense mechanism against environmental stress. Furthermore, it is thought that ChGA has antioxidant qualities, playing a crucial role in protecting food, cells, and organs against oxidation, , and may have health benefits, , including positive effects on blood pressure regulation and glucose control. ,

Coffee is a rich source of purine alkaloids, which include caffeine (CAFF). CAFF, known as 1,3,7-trimethylxanthine, is derived from purine base methylxanthine (Figure S1). It is responsible for the bitter taste of coffee and can be used to assess its quality. CAFF acts as a stimulant for the central nervous system, helping coffee drinkers stay awake, reduce fatigue, and increase energy levels. Additionally, it exhibits a mild diuretic effect. However, excessive consumption of CAFF can lead to unpleasant symptoms and potentially cause excitement and anxiety in individuals. , As a result, the recommended daily coffee consumption may vary depending on each individual’s health status. CAFF content in a single cup of coffee can range from 95 to 330 mg. While CAFF remains relatively stable in solution, the concentration of CAFF in coffee products may change depending on the degree of roasting.

The significant variation in the composition and concentration of phytochemicals in coffee presents a key challenge that requires a thorough investigation. Accurately understanding the physiological and pharmacological effects of coffee necessitates a comprehensive analysis of its phytochemical profile, with particular emphasis on CAFF and ChGA. The concentrations of these bioactive components are highly influenced by the processing and preparation methods employed. In the current study, we focused on coffee preparation using the traditional Egyptian method, which is widely practiced in local cafés and allows for brewing a homemade cup in just 10 min. In this context, it is essential to examine the effect of the brewing technique on the concentrations of CAFF and ChGA given the growing interest in their nutritional benefits and bioactive roles. Therefore, in this research, we employed commonly used cookware materials in Egyptian markets, such as stainless-steel, along with direct heat brewing, to determine their effect on both CAFF and ChGA content in Coffea arabica samples. The ChGA and CAFF contents of dark coffee (DC) were compared to those of green coffee (GC) under the same circumstances.

Although many methods have been described for the quantification study of bioactive substances such as ChGA and CAFF in foods and beverages, − high-performance liquid chromatography (HPLC) is still among the most frequently utilized techniques that yields more accurate results. − Using various chromatographic conditions of elution systems and different stationary phases, the literature has revealed that HPLC has been employed for the individual and simultaneous determination of CAFF and ChGA in various matrices. However, conventional HPLC methods employed for determining these bioactive compounds in coffee have presented separation times ranging from 35 to 95 min. − This time-consuming aspect restricts their practicality for industrial applications. Other studies have reported liquid chromatography mass spectrometry (LC–MS) techniques for measuring CAFF and ChGA. − Nevertheless, not all laboratories conducting quantitative analysis have access to MS technologies. Additionally, LC–MS approaches are susceptible to matrix effects and require skilled analysts. Despite the significant advancements in LC–MS, HPLC-DAD remains essential as a straightforward, cost-effective, and readily accessible analytical tool for accurately measuring the CAFF and ChGA concentrations in coffee. Thus, in the current investigation, we have explored the potential of establishing a rapid and simple segmented gradient-based elution HPLC method with a diode array detector (SGE-HPLC-DAD) to accurately quantify ChGA and CAFF in DC and GC samples. This approach also seeks to study the impact of the traditional coffee preparation method used in the popular district cafes in Egypt on the studied bioactive compounds and compare the results with those prepared by glass cookware.

2. Experimental Section

2.1. Chemicals and Reagents

Trifluoracetic acid (TFA) and acetonitrile were obtained from Merck (Merck KGaA, Darmstadt, Germany) and CAFF and ChGA standards were acquired from Nawah Scientific (Cairo, Egypt). All chemicals and solvents are HPLC grades. The water utilized in all the analyses was deionized and distilled.

2.2. Instrumentation and Chromatographic Conditions

Analysis of CAFF and ChGA was conducted by employing a Waters Alliance 2690 HPLC chromatographic system (Waters, Milford, MA) equipped with a Waters 996 photodiode array detector. The sample was separated on a Luna-Cyano column (5 μm, 25 × 0.46 cm) (Phenomenex, Torrance, CA, USA). The mobile phases for the gradient elution mode were solvents A (1% TFA in water) and B (acetonitrile). The gradient program consisted of two linear transitions: the first from A-B (95:5, v/v) to A-B (92:8, v/v) over 0 to 4 min and the second from A-B (92:8, v/v) to A-B (0:100, v/v) between 4 and 5 min. This was followed by an isocratic elution with 100% solvent B from 5 to 7 min. A third linear transition was performed from A-B (0:100, v/v) to A-B (95:5, v/v) from 7 to 8 min, and the process concluded with an isocratic elution of A and B (95:5, v/v) from 8 to 11 min. The flow rate was set at 1.5 mL/min, and the eluted compounds were monitored at 254 nm. The Waters Empower Software and Chromatography Data System assisted in all phases of the workflow. The column temperature was maintained at ambient (22 ± 1 °C) and held constant based on preliminary studies, which showed nonsignificant influence on resolution under our specific gradient profile. All gradient solvents, A and B, were employed freshly, filtered through 0.22 μm membrane filters (Millipore, Bedford, MA, USA), and degassed using ultrasonication.

2.3. Calibration Standards

The ChGA and CAFF stock solutions, each at a concentration of 2.00 mg/mL, were prepared separately. Subsequently, various consecutive dilutions of ChGA and CAFF mixtures, as calibration standards, with concentrations varying from 0.60 to 1000 and 0.40 to 1000 μg/mL were prepared, respectively. Each of the calibration standards underwent filtration by employing a 0.22 μm disc filter (Millipore). Following filtration, 10 μL was precisely injected and completed as stated in the developed SGE-HPLC-DAD method. Calibration curves of peak areas versus concentrations of CAFF and ChGA were plotted to obtain the regression equation of the best line of fit. The CAFF and ChGA contents (μg/mL) of DC and GC samples brewed under different cookware materials were determined by the resulted least-squares regression equations.

2.4. Sample Preparation and Application

Two coffee samples, GC and DC, were acquired from local Egyptian supermarkets and stored at ambient temperature (22 ± 1 °C) prior to their preparation, using two different cookware materials and direct heat as follows.

  • 1.

    DC brewed in stainless-steel cookware (DCS)

  • 2.

    DC brewed in glass cookware (DCG)

  • 3.

    GC brewed in stainless-steel cookware (GCS)

  • 4.

    GC brewed in glass cookware (GCG)

All coffee samples were placed in the corresponding cookware and poured with 100 mL of cold water. Cookware was put on direct flame and heated until the foam on the surface occurred. Each sample was prepared in triplicate. An aliquot of 1 mL of brewed GC or DC underwent a 2-fold dilution in water before being filtered using a 0.22 μm disc filter (Millipore). A portion of 10 μL was then injected and analyzed by the SGE-HPLC-DAD method. The study is illustrated through a schematic diagram in Figure S2. All values were normalized by dividing them by the weight of the obtained residue and the equivalent amount of coffee added (in grams), after which the percentages were calculated.

3. Results and Discussion

3.1. Chromatographic Optimization

3.1.1. Segmented Gradient Elution Preset Program and Mobile Phase Constituents

The gradient shape and choice of organic modifier are the two main variables of the new SGE-HPLC-DAD methodology that should be optimized. The results indicate that careful selection of a binary solvent mixture is a crucial factor in attaining an appropriate separation of CAFF and ChGA from the DC and GC matrices. It has been demonstrated that the selection of organic modifier for solvent B and the proportion in which it is combined with the solventless solvent A determine the resolution selectivity of CAFF and ChGA from the endogenous matrix’s components of coffee. The commonly used organic modifiers in HPLCacetonitrile and methanolwere evaluated for their effectiveness in separating ChGA and CAFF from DC and GC matrices. Acetonitrile exhibits lower polarity and viscosity compared to methanol. These features facilitate improved peak shape and efficient separation of analytes of different polarities while consuming less organic content and shortening analysis time when employing a cyano-based analytical column. Therefore, acetonitrile exhibits improved separation efficiency compared to methanol for separating ChGA and CAFF from coffee matrices. By using a segmented gradient elution mode of acetonitrile in accordance with a preset program, it was shown that the ChGA and CAFF are better separated from one another as well as from the other endogenous elements of the coffee matrix.

The time of analysis can be significantly reduced by segmenting and optimizing various components of the elution system, which, in turn, decreases the spacing between the peaks. This optimization process was oriented to a multiple-step elution mode that includes consecutive segmented gradient and isocratic elution modes with different durations and varying mobile phase compositions. Specifically, two segmented gradients were implemented: the first transitioned linearly from A-B (95:5, v/v) to A-B (92:8, v/v) over 0 to 4 min, while the second moved from A-B (92:8, v/v) to A-B (0:100, v/v) between 4 and 5 min. This was followed by an isocratic phase using 100% solvent B from 5 to 7 min. Subsequently, a third gradient transitioned from A-B (0:100, v/v) to A-B (95:5, v/v) between 7 and 8 min, concluding with an isocratic phase of A and B (95:5, v/v) from 8 to 11 min. Figure S3 shows the chromatogram obtained by the SGE-HPLC-DAD technique used to analyze a mixture of ChGA and CAFF standards under the best optimal conditions. For measuring ChGA and CAFF in brewed coffee samples, initial trials were performed with DC and GC brewing in glass cookware. In the first period, an initial concentration of solvent B (5%) proved effective in achieving a satisfactory separation of the ChGA from the early coeluted endogenous highly polar constituents in both the DC (Figure A) and GC (Figure B) matrices when gradually raised to 8% over a period of 4 min. The analytical column was then eluted with the recommended operating conditions for the second gradient elution period by ramping up the organic content rapidly to 100% solvent B at the end of 5 min. This high increment of solvent B content yielded the optimal conditions for achieving a thorough separation of CAFF from ChGA, along with the early eluted endogenous peaks present in the coffee matrices. It was found that when the analytical column was eluted through the first isocratic mode with (100% solvent B) from 5 to 7 min run time followed by the third gradient elution step from B (100%) to A-B (95:5, v/v) at the time point of 8 min, all late-eluted endogenous components of the DC and GC matrices were effectively cleaned up (Figure A and B). Also, it is essential to use a re-equilibration time that substantially restores the composition of the column effluent to the initial mobile-phase condition to perform the initial column function. A period of 3 min of isocratic elution with A-B (95:5, v/v) is enough time for column re-equilibration to achieve reliable results in the next run. The SGE-HPLC-DAD technique was similarly applied to separate CAFF and ChGA from DC and GC while using stainless-steel cookware and direct heat (Figure C,D).

1.

1

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Chromatograms of the SGE-HPLC-DAD method for separating CAFF and ChGA from DC (A and B) and GC (C and D) influenced by glass cookware (A and C) and stainless-steel cookware (B and D), along with the spectrum index plots of the analyzed compounds.

The spectrum index plot provides a visual representation of the maximum absorbance of the analyzed compounds within the PDA wavelength range of 200–400 nm, aligning with the specific retention times and peak areas of the compounds under investigation. This approach has proven to be advantageous, aiding in the identification of eluted compounds throughout the selected wavelength range. The chromatographic profile for ChGA and CAFF was assessed at a maximum UV absorption wavelength of 254 nm, which facilitated the comparison of retention times and UV absorption spectra index plots. The results revealed that the average retention times were 3.945 min for ChGA and 4.542 min for CAFF. The CAFF and ChGA chromatographic peaks in the DC and GC samples were identified by comparing their spectra index plots with those in the standard chromatograms as well as the spectra index plots for the same samples (Figure ). Notably, the mean retention times of the chromatographic peaks for ChGA and CAFF in the GC and DC samples were closely aligned with those of the standards. The spectra index plots for both the ChGA and CAFF standards (Figure S3, Supporting Information), along with those extracted from the GC and DC samples brewed under different conditions (Figure ), exhibited consistent UV spectrum index plots across the same chromatographic peaks, demonstrating effective separation performance of the developed method for the tested ChGA and CAFF without interference from other coeluting coffee matrix’s constituents. Thus, the spectrum index plot of ChGA and CAFF standards further confirms the detection of the same bioactive compounds in DC and GC samples. One of the key findings besides the effectiveness of the SGE-HPLC-DAD technique is that when DC is brewed with glass or stainless-steel cookware, chromatograms show increased intensities of the late eluting peaks of the endogenous matrix components (Figure A and B) in comparison to GC (Figure C,D). Furthermore, DC has higher levels of CAFF compared to GC, while GC has a significantly higher ChGA concentration than DC.

3.1.2. Analytical Column

During the development phase, our primary goal was to find an appropriate analytical column for the fast and efficient separation of CAFF and ChGA from coffee matrices. In the current study, we conducted experiments using ODS, phenyl, and cyano-analytical columns (4.6 mm × 250 mm, 5 μm). The polarity of the cyano-analytical column makes it a very versatile column that facilitates an easy separation of various polar compounds. This feature is particularly advantageous for lowering retention times and improving peak shapes. Following extensive optimization of the mobile phase, the cyano-analytical column demonstrated its effectiveness in retaining and separating both CAFF and ChGA from GC and DC matrices within a short time. The chromatograms in Figure show well-defined, sharp, and well-resolved peaks for ChGA and CAFF, making the cyano-analytical column the optimal choice for the current SGE-HPLC-DAD method. It showed good performance for the rapid separation of the early eluted highly polar constituents from tested ChGA in coffee matrices. Additionally, the cyano-analytical column demonstrates shorter retention times for ChGA and CAFF, offering further benefits for high-throughput analysis of DC and GC products in quality control applications.

3.1.3. Flow Rate and Injection Volume

Two additional variables were undertaken to improve the chromatographic performance and acquisition parameters. Adjustments were made to the SGE-HPLC-DAD method’s mobile phase flow rate as well as injection volume. The study established that the ideal parameters for obtaining consistent and rapid results in the effective separation and quantification of ChGA and CAFF in both GC and DC were a flow rate of 1.5 mL/min along with a 10 μL sample volume.

3.2. Method Validation

3.2.1. Linearity, Detection, and Quantification Limits

Two external calibration curves were constructed to evaluate the linearity of ChGA and CAFF, covering concentration ranges of 0.6–1000 and 0.4–1000 μg/mL, respectively. The detector responses of the peak areas for ChGA and CAFF were plotted against their corresponding concentrations. The coefficients of determination (R 2) of 0.9991 and 0.9996 were obtained, indicating linear relationships of peak areas and the corresponding concentrations of ChGA and CAFF, respectively (Table ). These results verified the suitability of the suggested SGE-HPLC-DAD method to measure both bioactive compounds in different coffees.

1. Characteristic Regression Data of the SGE-HPLC-DAD Method for the Analysis of Standards CAFF and ChGA.
parameters CAFF ChGA
calibration range (μg/mL) 0.40–1000 0.60–1000
detection limit (μg/mL) 0.10 0.16
quantification limit (μg/mL) 0.35 0.53
regression equation (Y)    
slope (b) 0.0001 0.0001
intercept (a) –1.2487 4.6995
correlation coefficient (r 2) 0.9996 0.9991
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a

Y = a + bx, where Y is the peak area and x is the concentration.

The limit of detection (LOD) refers to the minimum concentration of CAFF and ChGA that can be reliably distinguished from the surrounding matrix background, whereas the limit of quantification (LOQ) represents the lowest concentration at which these compounds can be quantified with acceptable accuracy and precision. , Chromatograms of the injected samples of CAFF and ChGA at the lowest concentration levels were examined to determine LOD and LOQ based on signal-to-noise (S/N) ratios of 3 and 10, respectively. The LODs were determined to be 0.10 μg/mL and 0.16 μg/mL for CAFF and ChGA, while LOQs were 0.35 μg/mL and 0.53 μg/mL, respectively (Table ).

3.2.2. Intra- and Interday Accuracy and Precision

The recovery percentages for CAFF and ChGA were assessed at three different concentration levels. For intraday validation, the average recoveries were found to be 100.97% for CAFF and 101.33 for ChGA. In the interday validation, the average recoveries for CAFF and ChGA were calculated at 100.21 and 100.10%, respectively. The experimental recoveries demonstrate the high accuracy of the suggested SGE-HPLC-DAD method (Table ). The intraday and interday precision assessments of the SGE-HPLC-DAD methodology revealed relative standard deviation (RSD) percentages below 1% for CAFF and ChGA, which confirms the method’s effectiveness for its intended purpose.

2. Intra-and Interday Accuracy and Precision for the Validated SGE-HPLC-DAD Method.
number concentration (μg/mL) retention time (min) recovery % concentration (μg/mL) retention time (min) recovery %
    CAFF     ChGA  
intraday repeatability
1 300 4.540 100.25 1000 3.958 101.26
2 300 4.545 100.53 1000 3.936 100.90
3 300 4.540 102.12 1000 3.942 101.84
average 4.542 100.97   3.945 101.33  
SD 0.003 1.01   0.011 0.47  
RSD % 0.066 1.00   0.279 0.46  
interday repeatability
1 350 4.416 99.52 780 4.013 99.87
2 350 4.495 100.01 780 3.985 100.29
3 350 4.472 101.11 780 3.958 100.13
average 4.461 100.21   3.985 100.10  
SD 0.041 0.81   0.028 0.21  
RSD % 0.917 0.81   0.702 0.21  
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3.2.3. Selectivity

The degree of selectivity refers to the capability of the SGE-HPLC-DAD method to accurately quantify CAFF and ChGA in the presence of other closely related water-soluble components within the GC and DC sample matrices. Figure clearly demonstrates the successful separation of CAFF and ChGA from the early and late eluted peaks and the various endogenous components found in the DC and GC matrices.

3.2.4. Robustness

The developed method’s robustness was tested by making slight adjustments (±2%) to the flow rate and composition of the elution system, which consists of solvents A and B. The results illustrated that the levels of the bioactive compounds tested were not significantly impacted by the varying conditions, demonstrating the reliability of the SGE-HPLC-DAD method for conducting quality control evaluations of DC and GC products.

3.3. Features of the SGE-HPLC-DAD Method in Terms of Its Speed and Sensitivity

This section focused on assessing the enhanced SGE-HPLC-DAD method in comparison with previously reported chromatographic techniques for measuring CAFF and ChGA. Both the SGE-HPLC-DAD and the alternative HPLC methods provided strong validation approaches, particularly for the analysis of CAFF and ChGA in coffee samples. The SGE-HPLC-DAD methodology has demonstrated superiority over the other reported HPLC methods that utilized UV or PDA detection systems, achieving remarkable sensitivity along with high accuracy and precision in a short analytical time frame. Table lists the LODs, LOQs, and run times for various published HPLC techniques used for CAFF and ChGA analysis. While one HPLC-UV method demonstrated a shorter retention time compared to the SGE-HPLC-DAD technique, it exhibited lower sensitivity. Furthermore, the rapid and simple purification method for CAFF and ChGA from coffee matrices was efficient in the reduction of matrix effects and run times upon utilizing the SGE-HPLC-DAD technique. Consequently, it was essential to employ the newly developed, simple, and rapid SGE-HPLC-DAD method for the analysis of CAFF and ChGA in DC and GC samples.

3.4. Application

3.4.1. Effects of Cookware on the Concentration Levels of CAFF

Understanding the phytochemical composition of coffee is essential to accurately interpret its physiological and pharmacological effects. Also, CAFF and ChGA should receive special attention since the concentrations of these bioactive substances can be greatly affected by the techniques used in processing. Thus, it was necessary to develop and validate an accurate and sensitive SGE-HPLC-DAD method for measuring CAFF and ChGA in GC and DC samples brewed by the most utilized cookware materials found in Egyptian cafes, such as stainless-steel, in combination with direct heat. The results were then compared to those obtained from the same portions of DC and GC brewed by using glass cookware materials under the same conditions.

Figure A and B illustrate the changes in CAFF levels in DC and GC samples brewed using two different types of cookware. These data show that brewing DC in glass cookware resulted in slightly elevated CAFF levels (>2.0%) compared to that observed using stainless-steel (<2.0%). The CAFF concentration levels in GC samples showed comparable results, with glass cookware having slightly higher levels (>1.5%) than stainless-steel (<1.5%) (Figure B). However, it was noticed that the CAFF content was slightly higher in DC brewing using glass or stainless-steel (>1.5%) over direct heat compared to GC brewing in similar conditions (Figure A).

2.

2

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CAFF and ChGA contents (g %) from DC (A) and GC (B) prepared by glass and stainless-steel cookware materials.

3.4.2. Effects of Cookware on the Concentration Levels of ChGA

In the case of ChGA, its levels in GC and DC were examined by the developed SGE-HPLC-DAD method under the same circumstances mentioned for the CAFF contents. The variation of ChGA levels in differently processed DC and GC samples is depicted in Figure . This figure shows that the highest ChGA levels are measured in GC. The highest percentage of ChGA was found to be more than 4.00% when GC was brewed in glass cookware compared to that observed using stainless-steel (<3.50%) (Figure B). On the contrary, DC was shown to contain the lowest content of ChGA (Figure A). Figure A and B also reveals that GC had a slightly higher level of ChGA when brewed in glass, but the lowest ChGA content was observed when DC was brewed in stainless-steel cookware. The percentages of ChGA presented by DC were below 0.5% across all types of cookware materials.

The results concerning the influence of cookware materials on the concentration levels of CAFF and ChGA in GC and DC imply that flavor is a key factor affecting the selection of coffee type and brewing techniques typically found in Egyptian cafes, irrespective of the health benefits that coffee may offer.

4. Conclusions

This study presents a rapid, sensitive, and cost-effective SGE-HPLC-DAD method for the simultaneous quantification of ChGA and CAFF in DC and GC coffee samples. Compared with traditional isocratic methods, the proposed approach offers shorter run times (11.00 min), improved selectivity, and effective separation without interference from matrix components. The use of the PDA detection system provides a practical alternative to LC–MS, with spectral index plots enhancing peak identification. Notably, the study reveals that both the coffee types and cookware materials significantly influence ChGA and CAFF contents, with glass cookware yielding higher concentrations of bioactive compounds. These findings highlight the analytical and practical relevance of the SGE-HPLC-DAD method for routine quality control and underscore the impact of brewing conditions on the phytochemical profile of coffee. While this research demonstrates that the type of cooking materials can significantly affect the concentration of phytochemicals and, consequently, the biological activity of coffee, it indicates that taste is the primary factor, often overshadowing the possible health benefits of coffee when Egyptians choose how to prepare coffee in traditional Egyptian cafes.

Supplementary Material

ao5c03408_si_001.pdf (350.5KB, pdf)

Acknowledgments

The authors sincerely thank the Central Research Laboratory at Misr International University for their collaboration and support in conducting the chromatographic analysis.

Glossary

Abbreviations

SGE-HPLC-DAD

segmented gradient elution high-performance liquid chromatographic methodology with diode array detector

ChGA

chlorogenic acid

and CAFF

caffeine

DC

dark coffee

GC

green coffee

TFA

trifluoracetic acid

DCS

DC brewed in stainless-steel cookware

GCS

GC brewed in stainless-steel cookware

The majority of the data that support the findings of this study are included in the article. Further information can be requested from the corresponding author.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03408.

  • Structures of ChGA and CAFF, a schematic diagram of the study, and chromatogram of ChGA and CAFF standards as well as the Spectrum Index plot of the same tested analytes (PDF)

R.F.A.: methodology, sample analysis, investigation, results interpretation, validation, and writingoriginal draft and English editing. K.M. and S.E.: conceptualization and supervision. A.I.: methodology, sample analysis, results interpretation, validation, and writingoriginal draft and English editing. All authors have given their approval of the published version of the manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

ao5c03408_si_001.pdf (350.5KB, pdf)

Data Availability Statement

The majority of the data that support the findings of this study are included in the article. Further information can be requested from the corresponding author.

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