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PM · Lens · Analysis of Free Amino Acids in Different Extracts of Orthosiphon stamineus Leaves by High-Performance Liquid Chromatography Combined with Solid-Phase Extraction

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Article · Volume 13, Issue 51S · pp. S385–91

Analysis of Free Amino Acids in Different Extracts of Orthosiphon stamineus Leaves by High-Performance Liquid Chromatography Combined with Solid-Phase Extraction

Armaghan Shafaei1, Nor Hidayah Ab Halim2, Norhidayah Zakaria2, Zhari Ismail1

  1. 1Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, Penang, Malaysia.
  2. 2Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Tembila Campus, Besut Terengganu, Malaysia.

Abstract

Background: Orthosiphon stamineus (OS) Benth is a medicinal plant and native in Southeast Asia. Previous studies have shown that OS leaves possess antioxidant, cytotoxic, diuretic, antihypertensive, and uricosuric effects. These beneficial effects have been attributed to the presence of primary and secondary metabolites such as polyphenols, amino acids, and flavonoids.Objective: To develop and validate an high-performance liquid chromatography (HPLC)-diode array detector (DAD) method combined with solid-phase extraction that involves precolumn derivatization with O-phthaladehyde for simultaneous analysis of free amino acids in OS leaves extracts.Materials and Methods: OS leaves were extracted with water (OS-W), ethanol (OS-E), methanol (OS-M), 50% ethanol (OS-EW), and 50% methanol (OS-MW). The extracts were treated by C18 cartridge before derivatization, resulting in great improvement of separation by Zorbox Eclipse XDB-C18 column.Results: The HPLC–DAD method was successfully developed and validated for analyzing the contents of free amino acids in OS extracts. The results showed that l-aspartic acid with 0.93 ± 0.01 nmol/mg was the major free amino acid in OS-W extract. However, in OS-E, OS-M, OS-EW, and OS-MW, l-glutamic acid with 3.53 ± 0.16, 2.17 ± 0.10, 4.01 ± 0.12, and 2.49 ± 0.12 nmol/mg, respectively, was the major free amino acid. Subsequently, l-serine, which was detected in OS-W, OS-E, and OS-M, was the minor free amino acid with 0.33 ± 0.02, 0.12 ± 0.01, and 0.06 ± 0.01 nmol/mg, respectively. However, l-threonine with 0.26 ± 0.02 and 0.19 ± 0.08 nmol/mL in OS-EW and OS-MW, respectively, had the lowest concentration compared with other amino acid components.Conclusion: All validation parameters of the developed method indicate that the method is reliable and efficient to simultaneously determine the free amino acids content for routine analysis of OS extracts.

Keywords: Derivatization; free amino acids; HPLC-DAD; Orthosiphon stamineus; solid-phase extraction

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