UHPLC-Q Exactive Orbitrap HRMS-Based Metabolite Profiling and Determination of Total Phenolic and Flavonoid Contents in Piper betle L. Leaf Ethanolic Extract

Agus Kurniawan; Syamsudin  Abdillah; Ernawati  Sinaga; Muhammad  Hanafi; Esti  Mumpuni

doi:10.58344/ihj.v5i3.935

Authors

Agus Kurniawan Universitas Pancasila
Syamsudin Abdillah Universitas Gunadarma
Ernawati Sinaga Universitas Nasional
Muhammad Hanafi Badan Riset dan Inovasi Nasional
Esti Mumpuni Universitas Pancasila

DOI:

https://doi.org/10.58344/ihj.v5i3.935

Keywords:

Piper betle L., UHPLC-Q Exactive Orbitrap HRMS, metabolite profiling, total phenolic content, total flavonoid content, phytochemical characterization

Abstract

Piper betle L. leaves contain diverse phytochemical constituents; however, variations in extract composition highlight the need for complementary analytical characterization. This study aimed to characterize the metabolite profile of Piper betle L. leaf ethanolic extract and determine its total phenolic content (TPC) and total flavonoid content (TFC). Metabolite profiling was performed using ultra-high-performance liquid chromatography coupled with Q Exactive Orbitrap high-resolution mass spectrometry (UHPLC–HRMS) in positive and negative electrospray ionization (ESI) modes. Metabolites were tentatively annotated based on accurate mass measurements, tandem mass spectrometric fragmentation patterns, and database matching. TPC and TFC were determined using colorimetric assays with gallic acid and quercetin as reference standards, respectively, and all measurements were performed in triplicate. Thirteen metabolites were tentatively annotated, including phenylpropanoids, flavonoids, phenolic amides, and terpenoids. Hydrocinnamic acid exhibited the highest reported chromatographic signal intensity, followed by tetramethoxyflavanone, cinnamic acid, and coumaric acid derivatives. The TPC was 64.37 ± 4.01 mg gallic acid equivalents per gram of extract (mg GAE/g extract), whereas the TFC was 213.74 ± 1.93 mg quercetin equivalents per gram of extract (mg QE/g extract). These measurements provided complementary information regarding the phytochemical characteristics of the extract but could not be directly interpreted as the relative proportions of individual constituents. Overall, the integrated analytical approach provided a preliminary phytochemical profile of the investigated Piper betle L. leaf ethanolic extract, establishing a foundation for subsequent metabolite identification, targeted quantitative analysis, and comparative quality assessment. Further biological investigations are warranted to elucidate potential relationships between the identified phytochemical constituents and their biological activities.

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