J. Pharm. Pharmacogn. Res., vol. 11, no. 2, pp. 243-254, March-April 2023.
DOI: https://doi.org/10.56499/jppres22.1566_11.2.243
Original Article
System optimization and validation to improve thin-layer chromatography of roselle calyces (Hibiscus sabdariffa L.) required by the Indonesian Herbal Pharmacopoeia Edition II
[Optimización y validación del sistema para mejorar la cromatografía en capa fina de cálices de rosa mosqueta (Hibiscus sabdariffa L.) requerida por la Farmacopea Herbal Indonesia Edición II]
Bhujangga Agung Ayu Sri Kartika Dewi, Kartini Kartini*
Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Surabaya, Surabaya, Indonesia.
*E-mail: kartini@staff.ubaya.ac.id
Abstract
Context: Roselle (Hibiscus sabdariffa L.) is one of the traditional crude drugs monographed in the Indonesian Herbal Pharmacopoeia Edition II (IHP II). As per IHP II requirements, a TLC pattern should be used in its authentication. However, the TLC system described for this purpose does not produce a clear reference chromatogram, often leading to inconclusive results.
Aims: To develop and validate TLC systems of H. sabdariffa calyces for a better-quality chromatogram than those listed in the IHP II, thereby facilitating crude drugs authentication.
Methods: To optimize TLC for H. sabdariffa, sixteen systems, differing in stationary phase, mobile phase composition, and/or visualization reagent were tested. The TLC system was then validated using several parameters such as analyte stability during chromatography, analyte stability in the extract solution and on the TLC plate, stability of the derivatization result, and precision on a plate as well as intermediate precision.
Results: Two systems (I and II) were successfully designed and applied to evaluate H. sabdariffa quality. System I used Si gel 60 F254 for the stationary phase, ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:2) for the mobile phase, H. sabdariffa’s ethanol extract (5%, 20 µL) for the test solution, cyanidin 3-O-glucoside (100 ppm, 4 µL) as a reference, no derivatization, and detection with visible light. System II combined Si gel 60 F254 for the stationary phase, ethyl acetate-formic acid-glacial acetic acid-toluene-water (80:11:11:20:19), H. sabdariffa’s ethanol extract (5%, 20 µL) for the test solution, chlorogenic acid (1000 ppm, 2 µL) and caffeic acid (50 ppm, 2 µL) as references, the visualizing reagent NP/PEG, and investigation under 366 nm UV light.
Conclusions: Both systems are simple but have good stability and precision, thus facilitating H. sabdariffa calyx authentication and paving the way for developing content analysis methods for H. sabdariffa markers.
Keywords: authenticity; herbal pharmacopoeia; quality evaluation; roselle; TLC pattern.
Resumen
Contexto: La rosa mosqueta (Hibiscus sabdariffa L.) es una de las drogas crudas tradicionales monografiadas en la segunda edición de la Farmacopea Herbal Indonesia (PHI II). Según los requisitos de la PHI II, se debe utilizar un patrón de TLC para su autentificación. Sin embargo, el sistema de TLC descrito para este fin no produce un cromatograma de referencia claro, lo que a menudo conduce a resultados no concluyentes.
Objetivos: Desarrollar y validar sistemas de TLC de cálices de H. sabdariffa para obtener un cromatograma de mejor calidad que los listados en el PHI II, facilitando así la autentificación de drogas crudas.
Métodos: Para optimizar la TLC para H. sabdariffa, se probaron dieciséis sistemas, que diferían en la fase estacionaria, la composición de la fase móvil y/o el reactivo de visualización. A continuación, se validó el sistema de TLC utilizando varios parámetros, como la estabilidad del analito durante la cromatografía, la estabilidad del analito en la solución del extracto y en la placa de TLC, la estabilidad del resultado de la derivatización y la precisión en una placa, así como la precisión intermedia.
Resultados: Se diseñaron y aplicaron con éxito dos sistemas (I y II) para evaluar la calidad de H. sabdariffa. El sistema I utilizó Si gel 60 F254 como fase estacionaria, acetato de etilo-ácido fórmico-ácido acético glacial-agua (100:11:11:2) como fase móvil, extracto de etanol de H. sabdariffa (5%, 20 µL) como solución de prueba, cianidina 3-O-glucósido (100 ppm, 4 µL) como referencia, sin derivatización, y detección con luz visible. El sistema II combinó Si gel 60 F254 para la fase estacionaria, acetato de etilo-ácido fórmico-ácido acético glacial-tolueno-agua (80:11:11:20:19), extracto de etanol de H. sabdariffa (5%, 20 µL) para la solución de prueba, ácido clorogénico (1000 ppm, 2 µL) y ácido cafeico (50 ppm, 2 µL) como referencias, el reactivo de visualización NP/PEG, y la investigación bajo luz UV de 366 nm.
Conclusiones: Ambos sistemas son sencillos pero tienen buena estabilidad y precisión, facilitando así la autentificación del cáliz de H. sabdariffa y allanando el camino para desarrollar métodos de análisis de contenido para marcadores de H. sabdariffa.
Palabras Clave: autenticidad; farmacopea herbal; evaluación de la calidad; rosa mosqueta; patrón TLC.
Citation Format: Dewi BAASK, Kartini K (2023) System optimization and validation to improve thin-layer chromatography of roselle calyces (Hibiscus sabdariffa L.) required by the Indonesian Herbal Pharmacopoeia Edition II. J Pharm Pharmacogn Res 11(2): 243–254. https://doi.org/10.56499/jppres22.1566_11.2.243
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