Putative Identification of 47 Compounds from Jieyu Anshen Granule and Proposal of Pharmacopeia Quality-Assessment Strategy Using TCM-Specific Library with UHPLC-Q-Exactive-Orbitrap-MS

Abstract

Jieyu Anshen Granule is a traditional Chinese medicine prescription used for depression and primarily comprises five herbal medicines: Zhizi, Chaihu, Zhigancao, Danggui, and Yuanzhi. This study established a traditional Chinese herbal medicine-specific library using emerging ultra-high-performance liquid chromatography-quadrupole-orbitrap-tandem mass spectrometry analysis. Through library comparison, the study has fulfilled isomers distinction. As a result, 47 compounds were simultaneously and putatively identified from Jieyu Anshen Granule, including 12 unexpected compounds and 35 expected compounds. The unexpected compounds comprised cyclocommunol, 5-hydroxyflavone, tangeretin, 3,5,6,7,8,3',4'-heptemethoxyflavone, calycosin-7-O-β-D-glucoside, 7,4'-dihydroxyflavone, naringenin-7-O-β-D-glucoside, matrine, betaine, jervine, alantolactone, and hypericin. Among the 35 expected compounds, saikosaponin A, saikosaponin D, glycyrrhizic acid, geniposide, ligustilide, and polygalaxanthone III were further investigated using a quantum chemistry approach. Based on these, an effective quality assessment strategy is proposed for the Pharmacopeia, involving the simultaneous analysis of glycyrrhizic acid, geniposide, ligustilide, polygalaxanthone III, saikosaponins A and D through ultra-high-performance liquid chromatography-quadrupole-orbitrap-tandem mass spectrometry analysis. This strategy enables the detection of adulteration in relation to Zhizi, Chaihu, Zhigancao, Danggui, and Yuanzhi in Jieyu Anshen Granule. The findings of unexpected compounds will deepen the understanding of chemistry in Jieyu Anshen Granule.

Keywords: Adulterate; Chinese medicine; Glycyrrhizic acid; Saikosaponin A; Saikosaponin D; UPLC−Q- Orbitrap-MS/MS.

Figure 1

Jieyu Anshen Granule appearance (the insert upper right is an enlarged view).

Figure 2

The flowchart of sample solution preparation of lyophilized aqueous extract from Jieyu Anshen Granule.

Figure 3

The total ion current (TIC) chromatogram of Jieyu Anshen Granule in the UHPLC−Q‐Exactive‐Orbitrap‐MS/MS analysis. (A) The negative ion mode; (B) the positive ion mode. The positive ion mode was supplement of negative ion mode.

Figure 4

The structures and configurations of 47 identified compounds from Jieyu Anshen Granule. The red indicates the old Q‐marker, while the purple indicates new Q‐markers. The wave line in 35 indicated the uncertain stereo‐configuration. The symbol “a” means positive ion model. Considering the layout space, the TIC diagram under positive ion model was not shown in the main text. Knot line with “iso” connected two isomers.

Figure 5

The putative identification of glycyrrhizic acid (38). (A) MS/MS spectra and the relevant elucidation of standard glycyrrhizic acid; (B) MS/MS spectra of the peak (R.T. 13.11). The m/z values in purple indicated the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 4.87×10⁻⁷–3.40×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 6

The putative identification of saikosaponin A (42). (A) MS/MS spectra and the relevant elucidation of standard saikosaponin A; (B) MS/MS spectra of the peak (R.T. 13.59). The m/z values in purple indicated the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 0–6.93×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 7

The putative identification of saikosaponin D (43). (A) MS/MS spectra and the relevant elucidation of standard saikosaponin D; (B) MS/MS spectra of the peak (R.T. 13.89). The m/z values in purple indicated the calculated ones. The m/z values in blue are the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 1.28×10⁻⁷–2.28×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 8

The MS fragmenting elucidation of cyclocommunol (40).

Figure 9

The optimized conformation of geniposide (10), polygalaxanthone III (19), ligustilide (35), glycyrrhizic acid (38), saikosaponin A (42), and saikosaponin D (43). The calculation was conducted at (U)B3LYP−D3(BJ)/6‐31+G(d,p) basis set with SMD model using the Gaussian 16 software and Gaussian View 6.1.1.