Rapid Characterization and Identification of Chemical Constituents in Gentiana radix before and after Wine-Processed by UHPLC-LTQ-Orbitrap MSn

Gentiana radix is used in traditional Chinese medicine and has functions of clearing heat and drying dampness, as well as purging liver and gallbladder fire. A highly sensitive and effective strategy for rapid screening and identification of target constituents has been developed by using ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap) in crude and wine-processed Gentiana radix. Based on the accurate mass measurement (<5 ppm), retention times, and MS fragmentation ions, 52 constituents were unambiguously or tentatively characterized from Gentiana radix, including 21 iridoids, 11 flavonoids, 19 xanthones, and a triterpenoid. This study demonstrated that the established method could be a rapid, effective analytical tool for screening and characterization of compounds in the complex systems of Gentiana radix. By comparing the structure and peak areas of chemical constituents in crude and wine-processed Gentiana radix, we found that some compounds in crude and wine-processed Gentiana radix were significantly different.


Introduction
Gentiana radix is the dried root and rhizome of Gentiana manshurica Kitag., Gentiana radix Bge., Gentiana triflora Pall. and Gentiana rigescens Franch. [1]. Gentiana radix is mainly distributed in Heilongjiang, Jilin, Liaoning, Nei Monggol, Zhejiang, Jiangsu, Guangdong, Yunnan, Xinjiang, etc. [2]. Gentiana radix is commonly employed to treat various diseases. For instance, Gentiana radix could be used for treating heat jaundice, pruritus and swells of vulvaered, morbid leucorrhea, eczema, tinnitus, epicophosis, hypochondriac pain, invigorating stomach and convulsions. Modern pharmacological studies have demonstrated that Gentiana radix possesses various biological activities, including anti-inflammatory, anti-oxidative and antiviral [3,4]. Gentiana radix has complicated chemical constituents, including iridoid glycoside, flavonoid glycoside, xanthones, triterpenoids, alkaloids, and so on. Among them, iridoid glycoside, flavonoid glycoside and xanthones are considered the main constituents [5]. There are many processing methods involving Gentiana radix recorded in history, such as wine-processing, bile-processing, honey-processing and ginger-processing [6][7][8]. And now crude and wine-processed Gentiana radix is widely used in clinical practice. According to traditional theory, wine-processing could make the function of the drug move upward, and wine-processing alleviated the bitterness and coldness of crude Gentiana radix. Physical and chemical changes have taken place in the medicinal materials after being wine-processed so that it would lead to changes in the content and types of chemical constituents [9].
In recent years, high-resolution mass spectrometry (HRMS) and linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap) have been exhibiting excellent (i.e. fast and sensitive) performance in traditional Chinese medicines (TCMs) extracts [10,11]. The hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap) has high quality resolution and mass accuracy (within 5 ppm), it combines the high trapping capability with MS n and scanning capability of a linear ion trap [12,13]. LTQ-Orbitrap allows the effective detection of a large amount of data about chemical constituents, and it includes exact mass, elemental compositions, fragmentation pathways, etc. [14]. These capabilities play a vital role in effectively identifying and analyzing the complicated constituents of TCMs. In this paper, a method with UHPLC-LTQ-Orbitrap was established to comprehensively analyze the constituents in Gentiana radix, and this method is applied to the comparative study of the constituents in crude and wine-processed Gentiana radix. Fifty-two constituents were identified, the similarities and differences of the constituents in Gentiana before and after wine processing were determined.

Identification of the Constituents by UHPLC-LTQ-Orbitrap MS n
UHPLC-ESI-LTQ-Orbitrap was employed for comprehensive analysis in positive and negative modes to identify the chemical constituents in Gentiana radix. In order to determine the molecular formula of compounds, it was necessary to compare it with the HRMS molecular formula database built in-home, the high-accuracy protonated precursors with errors less than 5 ppm and related literature. As a result, a total of 52 compounds (Table 1, Figure 1) were screened and identified from Gentiana radix extract, including 21 iridoids, 11 flavonoids, 19 xanthones and a triterpenoid. By comparing the constituents in crude and wine-processed Gentiana radix, the results showed that there were 34 identical chemical constituents in them, 10 constituents characteristic of crude Gentiana radix, and 8 constituents characteristic of wine-processed Gentiana radix. And the peak area of some constituents increased or decreased after Gentiana radix was processed with wine. A typical total ion chromatogram (TIC) of crude and wine-processed Gentiana radix in positive and negative ion mode is showed in Figure 2.
. The proposed spectra of chromatograms of fragmentation MS n of compound 5 is shown in Figure 3, and the proposed fragmentation pathway of compound 5 is shown in Figure 4. So, compound 5 was tentatively determined as loganic acid [42].
. The proposed spectra of chromatograms of fragmentation MS n of compound 5 is shown in Figure 3, and the proposed fragmentation pathway of compound 5 is shown in Figure 4. So, compound 5 was tentatively determined as loganic acid [42].            (Figures 5 and 6). Therefore, compound 8 was tentatively deduced as 6 -O-β-D-glucosyl swertiamarin, compared with the t R values and mass spectra with the reference standard, 14 and 21 were tentatively annotated as swertiamarin and sweroside [43].  (Figures 5 and 6). Therefore, compound 8 was tentatively deduced as 6′-O-β-D-glucosyl swertiamarin, compared with the tR values and mass spectra with the reference standard, 14 and 21 were tentatively annotated as swertiamarin and sweroside [43].                 (Figures 7 and 8). Therefore, combined with bibliography data and fragmentation pathways, these four compounds were tentatively identified as isovitexin-2 -O-B-D-glucosyle, saponarin, isovitexin, isopyrenine-7-O-glucosyle.               (Figures 9 and 10). Compared with the t R values and mass spectra with the reference standard, compound 19 was ascertained as mangiferin [55][56][57].
A LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) was connected to the UHPLC system via an electrospray ionization (ESI) interface. The effluent was introduced into the ESI source in a post-column splitting ratio of 1:4. The analysis was performed in both negative and positive ion mode with a mass range of m/z 100-200.
The analysis was performed in both negative and positive ion mode with a mass range of m/z 100-1500. The optimized ESI parameters in negative ion mode were set as follows: capillary temperature of 350 • C; sheath gas (nitrogen) flow of 30 arb.; auxiliary gas (nitrogen) flow of 10 arb.; source voltage of 3.0 kV; capillary voltage of −35 V; tube lens voltage of −110 V. The capillary voltage was 25 V, source voltage of 4.0 kV and tube lens voltage was 110 V in positive ion mode; and other parameters were same as those of negative ion mode. The resolution of the orbitrap mass analyzer was set at 30,000. The isolation width was 2 amu, and the normalized collision energy (CE) was set to 35%. Collision-induced dissociation (CID) was conducted in LTQ with an activation q of 0.25 and activation time of 30 ms. All instruments were controlled by the Xcalibur data system, and the data acquisition was carried out by analyst software Xcalibur (version 2.1) from Thermo Electron Corp (Waltham, MA, USA).

Conclusions
In this study, an effective and sensitive analytical method by UHPLC-LTQ-Orbitrap-MS n was established for systematically characterizing constituents in crude and wine-processed Gentiana radix. By this method, the structure of constituents could be identified and the peak area of constituents could be determined. The wine-processing would change the structure of chemical constituents of Gentiana scabra, which resulted in partial loss and abscission of unstable chemical groups (hydroxyl, glucosyle, carboxyl, etc.), and carbon-carbon double bond would be displaced or broken in the structure of some constituents.
By comparing the structure and peak area of the constituents, we could see that there are differences between crude and wine-processed Gentiana radix, which showed wine-processing can change the structure and contents of some constituents in Gentiana radix. The result of this paper can be used to explain the processing principles of Gentiana radix to some extent.