Box–Behenken-Supported Development and Validation of UPLC Method for the Estimation of Eugenol in Syzygium aromaticum, Cinnamomum tamala, and Myristica fragrance

Abstract

Eugenol (EUG) is one of the most important components available in several spices, including clove, bay leaves, and nutmeg. These spices are used as flavouring agents in foods and beverages. The aim of the present study is to develop and validate a rapid, simple, sensitive, and robust ultra-performance liquid chromatography (UPLC) technique for the quantitative estimation of EUG in the ultrasound-assisted methanolic extracts of three spices, namely Syzygium aromaticum (L.) Merr. & L.M.Perry (SA), Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm (CT), and Myristica fragrance Houtt. (MF). EUG was isocratically separated on a UPLC C18 column. The acetonitrile:methanol:water (50:40:10, v/v/v) solvent in different proportions was optimized as the mobile phase for the determination of EUG in ultrasound-assisted methanolic extracts of three different spices. The quantitative estimation of EUG was performed at a 281 nm detection wavelength. The column oven temperature was maintained at 35 ± 5 °C, and the flow rate of the mobile phase was 0.2 mL/min using an injection volume of 1 µL. The UPLC technique was validated according to the ICH guidelines and showed an excellent linearity range of 10–100 ng/mL. The robustness of the method was validated using Box–Behenken response surface design (BBD) software, and a 0.2 mL/min flow rate of the mobile phase, a column oven temperature of 308 K, and a 281 nm detection wavelength were found to be the best optimal conditions for obtaining the highest amount and separation of EUG. The content of EUG in ultrasound-assisted methanolic extracts of SA, CT, and MF using the UPLC technique showed 313.67 ± 0.87 mg g−1, 44.95 ± 0.56 mg g−1, and 59.66 ± 0.41 mg g−1, respectively. The antioxidant potentials of EUG, SA, CT, and MF were analysed using the DPPH (2,2-diphenyl-1-pcrylhydrazil radical) method, which revealed the antioxidant potential of EUG (IC50 = 3.12 µg/mL), standard ascorbic acid (IC50 = 7.06 µg/mL), SA ultrasound-assisted methanolic extract (IC50 = 5.97 µg/mL), CT ultrasound-assisted methanolic extract (IC50 = 49.48 µg/mL), and MF ultrasound-assisted methanolic extract (IC50 = 65.16 µg/mL). The proposed UPLC technique can be used to quantitatively assess various spices, plants, pharmaceutical products, and polyherbal formulations containing EUG as an active constituent.