BIOANALYTICAL METHOD VALIDATION FOR DETERMINATION OF ROSMARINIC ACID IN SIMULATED BIOLOGICAL MEDIA USING HPLC

KOTCHAPHAN CHOOLUCK; PIYANUCH ROJSANGA; CHUTIMA PHECHKRAJANG; MONTREE JATURANPINYO

doi:10.22159/ijap.2021v13i2.40366

Authors

KOTCHAPHAN CHOOLUCK Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
PIYANUCH ROJSANGA Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
CHUTIMA PHECHKRAJANG Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
MONTREE JATURANPINYO Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand

DOI:

https://doi.org/10.22159/ijap.2021v13i2.40366

Keywords:

HPLC, Liquid chromatography, Rosmarinic acid, Thunbergia laurifolia, validation

Abstract

Objective: This study aims to develop and validate a simple, rapid and accurate HPLC method for the determination of rosmarinic acid (RA), an active marker of Thunbergia laurifolia (TL) tea, in Hank's Balanced Salt Solution (HBSS) using HPLC.

Methods: The separation was performed using a Xterra^® C₁₈ reversed-phase column (150 3.9 mm, 5 µm). The mobile phase consisted of 0.5% (v/v) glacial acetic acid (A) and acetonitrile (B). The flow rate was 1 ml/min and detection was carried out at 330 nm with UV-visible spectrophotometer. The method was validated according to the US FDA guidance on bioanalytical method validation in 2018.

Results: The method was successfully validated in the range of 50-500 ng/ml of RA. Intra-and inter-day precision ranged from 1.6 to 2.1% and 1.4 to 4.5%, respectively. Intra-and inter-day relative errors (% bias) were less than 7.6 and 3.6%, respectively. In addition, it was found that the stability of RA in HBSS could be dependent on the pH and temperature.

Conclusion: The developed method met the validation requirements and could be further applied to the permeability study of RA using an in vitro Caco-2 cell monolayer model.

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