Characterization and identification of iridoid glucosides, flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS(n)). The optimized separation condition was obtained using an Agilent ZorBax SB-C18 column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC-ESI-MS(n) method.

The Hedyotis diffusa Willd. (Rubiaceae): A Review on Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics

“…Accompanied with the analysis of the NMR spectra of the pure compounds, the methods of tandem mass spectrometry (MS n ) and time-of-flight mass spectrometry (TOF/MS) have become more popular for the identification of these compounds [11,14,15,25,52]. To date, thirty-two iridoids and their iridoid glucosides (1-32) have been isolated and identified from H. diffusa (Figure 1).…”

“…diffusa is an annual herb, widely distributed in the orient and tropical Asia, such as China, Japan and Indonesia [1,8]. Generally, the plant grows in humid fields and ridges of farmlands, ascending to procumbent, to 50 cm tall; the stem is slightly flattened to terete, glabrescent to glabrous and the papilla was observed in the transverse section of the stem; the leaves are opposite, sessile or subsessile and blade drying membranous, linear, narrowly elliptic, 1-4ˆ0.1-0.4 cm; the flowers with pedicels are [15,25] 52…”

Liquid chromatography with mass spectrometry method based two‐step precursor ion scanning for the structural elucidation of flavonoids

“…Sample extraction procedure was applied based on a reference . Basically, each frozen tobacco milled sample (0.20 g, fresh) was mixed with 3 mL of 75% methanol in water and sonicated for 30 min.…”

“…As the development of chromatography and its connection with UV, MS, and NMR, the integrated separation and identification systems were developed . Of these methods, LC–UV–MS/MS is the most popularly used . With this method, the UV 254, 280, and 330 nm chromatograms are usually used for flavonoids searching, the MS 2 fragment spectra and the chromatographic retention rules are then used for structural elucidation of the flavonoids found by UV.…”

Structural characterization of secoiridoid glycosides by high‐performance liquid chromatography/electrospray ionization mass spectrometry

“…To date, the characterization of iridoid and secoiridoid glycosides (SIGs) from G. triflora is mainly performed by nuclear magnetic resonance (NMR), which is time‐consuming and difficult to obtain minor compounds. With the development of mass spectrometry (MS) techniques, high‐performance liquid chromatography/mass spectrometry (HPLC/MS) has become a powerful technique for the identification of constituents in complex matrices . This method allows the separation and identification of constituents with low levels of sample consumption and high sensitivity, and provides molecular weight (MW) and structural information online .…”