Research Article | Volume: 16, Issue: 9, September, 2026

Box–Behnken Assisted Development and Validation of an RP-HPLC Method for Determining Caffeic Acid in Robusta Coffee Brews

Titus Marcel Kusraynaldi,  Theresia Niken Larasati,  Angel Yemima Srininta Br Sembiring,  Florentinus Dika Octa Riswanto,  Enade Perdana Istyastono   

Titus Marcel Kusraynaldi1, Theresia Niken Larasati1, Angel Yemima Srininta Br Sembiring1, Florentinus Dika Octa Riswanto1, Enade Perdana Istyastono2,3

1Research Group of Computer-Aided Drug Design and Discovery of Bioactive Natural Products, Faculty of Pharmacy, Sanata Dharma University, Yogyakarta, Indonesia.

2Research Center for Cheminformatics and Molecular Modeling, Atma Jaya Catholic University of Indonesia, North Jakarta, Indonesia.

3Department of Pharmacy, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, North Jakarta, Indonesia.

Open Access   

Published:  Aug 06, 2026

DOI: 10.1177/22313354261466842
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Abstract

Indonesia remains one of the world’s top coffee producers, with Robusta coffee (Coffea canephora) being the variety favored by most consumers. Owing to its prominence, numerous studies have highlighted caffeic acid, a polyphenolic compound in coffee extract that has been shown to demonstrate antidiabetic properties. The primary objective of this research is to measure the caffeic acid levels in Robusta coffee brews. To achieve the optimal conditions, a Box–Behnken Design was used to determine the most suitable setting for the independent variables, including acetonitrile, formic acid, and flow rate. The analytical method validation parameters assessed were linearity and range, selectivity, accuracy, and precision, limit of detection (LOD), and limit of quantification (LOQ). The optimized reversed-phase high-performance liquid chromatography system was methanol:acetonitrile:aqueous formic acid pH 2.9 (10:15:75 v/v/v), flow rate of 1.0 mL/min, column temperature of 27 °C, 10 µL injection volume, and wavelength detection at 326 nm. The method showed good specificity and selectivity (Rs ≥ 1.5), with linearity from 5.39 to 32.36 µg/mL (R2 = 0.9987). LOD and LOQ were found to be 0.08032 and 0.2434 µg/mL, respectively. The recovery percentage for the intra-day assay was 92.26%–102.94%, while the inter-day percentage yield achieved 94.89% to 102.96% (90%–107%). All of the intra-day and inter-day assays met the required relative standard deviation (RSD) ≤ 5.3%. The average caffeic acid in Robusta coffee brews was 0.772 ± 0.008 mg/g (RSD = 1.066%, n = 6).


Keyword:     Caffeic acid BBD response surface methodology validation determination of content

Citation:

Kusraynaldi TM, Larasati TN, Sembiring AYSB, Riswanto FDO, Istyastono EP. Box–Behnken Assisted Development and Validation of an RP-HPLC Method for Determining Caffeic Acid in Robusta Coffee Brews. J Appl Pharm Sci. 2026;16(9):931-940. https://doi.org/10.1177/22313354261466842

Copyright: © The Author(s). This open-access article is distributed under the Creative Commons Attribution-NonCommercial 4.0 International License, which permits non-commercial use, sharing, adaptation, distribution, and reproduction in any medium or format, provided appropriate credit is given, a link to the license is included, and any changes are indicated.
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