Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry

Arnebiae Radix (dried root of Arnebia euchroma (Royle) Johnst.) has been used in traditional Chinese medicine (TCM) to treat macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, and inflammation. Previous studies have shown that shikonins and shikonofurans are two of their main bioactive ingredients. However, systematic investigations of their constituents have rarely been conducted. It is necessary to establish a rapid and effective method to identify the chemical constituents of Arnebiae Radix. This will help to further improve the effective resource utilization rate of this plant. In this study, a rapid and effective UHPLC-Q-Exactive Orbitrap mass spectrometry method was established to simultaneously analyze chemical ingredients in Arnebiae Radix within a short period of time. Based on the results of a full scan MS, the MS2 database (mzVault and mzCloud), the diagnostic fragment ions, the retention time, and the bibliography, a total of 188 compounds were identified, with 114 of those being reported from Arnebiae Radix for the first time. The results of this study lay the foundation for obtaining a thorough understanding of the active ingredients in Arnebiae Radix and its quality control. This method may be widely used for the chemical characterization of different samples.


Introduction
Arnebiae Radix, commonly called known as "Zicao" in traditional Chinese medicine (TCM), is the root of Arnebia euchroma (Royle) Johnst. It is primarily distributed in Mongolia, Xinjiang, and Northeast China [1,2]. It has been widely used as a folk medicine for clearing heat (Qingre) and for detoxification (Jiedu) by oral administration and for promoting blood circulation in local wounds via external application [3,4]. Arnebiae Radix has been used for many years for the treatment of macular eruptions, measles, sore throat, carbuncles, burns, skin ulcers, inflammation, allergic contact dermatitis(ACD) [5][6][7][8] and recently, for the treatment of cancer [9,10]. Previous studies have acknowledged the richness and complexity of its chemical composition. Its main active ingredients are the naphthoquinone compound, which has exhibited extensive antioxidant, antimicrobial, anti-inflammatory, and antitumor activities [11][12][13]. Many compounds have been reported to exist in Arnebiae Radix, including shikonins and shikonofurans [14,15]. However, the characterization of the constituents of Arnebiae Radix is still insufficient. Therefore, it is necessary to develop a systematic strategy for the rapid detection and identification of the constituents of Arnebiae Radix, as this will be very helpful for understanding its material basis and quality control.
The complexity of chemicals contained in TCM has presented a significant challenge regarding the rapid identification and characterization of components. Liquid chromatography-mass spectrometry (LC-MS), especially ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), has been used extensively for qualitative analysis, quantitative analysis, and quality control of TCM due to its validity, sensitivity, and specificity [16,17]. HRMS provides high mass accuracy measurements for fragment ions with fast scan speeds. These features help with the identification of constituents with excellent accuracy and high reproducibility [18][19][20][21]. Cai et al. [22] used UHPLC-HRMS with parallel reaction monitoring (PRM) mode to unanimously and tentatively identify 149 chlorogenic acid derivatives from D. nervosa, which widely extended the knowledge on the chemical constituents of D. nervosa, facilitating the understanding of effective substances and quality control. Xiong et al. [23] used UHPLC-Q-Exactive Orbitrap mass spectrometry to systematically identify 106 constituent tannins in Paeoniae Radix Alba in negative ion mode. A systematic strategy was proposed for the rapid detection and identification of the chemical constituents of Arnebiae Radix using UHPLC-Q-Exactive Orbitrap mass spectrometry based on the expected compound and diagnosis fragmentation ion techniques.
The aim of the present investigation was to detect and identify the chemical constituents of Arnebiae Radix by UHPLC-Q-Exactive Orbitrap MS. In total, 188 compounds were identified in Arnebiae Radix, 114 of which are reported for the first time here. This result will improve the in-depth understanding of the pharmacological actions of Arnebiae Radix and lay a foundation for quality control of the drug for future clinical use.

Optimization of the UHPLC-Q-Exactive Orbitrap MS Condition
In order to acquire a better chromatographic peak shape and separation resolution, various factors were set to carry out a detection and identification process, including the column (Thermo Scientific Hypersil GOLD TM aQ 100 mm × 2.1 mm, 1.9 µm and Waters ACQUITY BEH C18 column, 100 mm × 2.1 mm, 1.7 µm), column temperature (30,35,40 • C), and mobile phase gradient. The chemical constituents showed a high resolution and high sensitivity level based on the LC-MS conditions of "Section 3.3".

Establishment of Diagnostic Fragment Ions (DFIs)
The DFI filter is a rapid screening method that is used to identify traditional Chinese medicine chemical components based on the accurate ion mass information provided by high-resolution mass spectrometry and the mass spectrometry fragmentation characteristics of similar chemical components. Thus, it is suitable for the identification of structural analogs. Different from a previous method of referring to the literature to establish a chemical database, the diagnostic fragment ions overcome the limitation of finding potential new compounds and deduce the overall structure of the compounds from the fragmentation information of the mass spectrum of the compound [24][25][26]. Generally, it is well-known that chemical constituents in the same category possess identical carbon skeletons and homologous biosynthetic pathways. It is easily understood that shikonin derivatives, phenolic acids, and flavonoids with the same carbon skeletons will generate similar fragmentation patterns, and these can be defined as DFIs for screening and characterization.
In this study, the fragmentation patterns of 14 reference standards were investigated by UHPLC-Q-Exactive Orbitrap MS in negative mode to establish the DFIs, and the selected fragmentation patterns of the components are shown in Figure 1

Characterization of the Chemical Constituents in Arnebiae Radix
The table lists all the chemical constituents detected in the extracted Arnebiae Radix sample by UHPLC-Q Exactive Orbitrap mass spectrometry based on the diagnostic fragment ions, retention time, MS 2 database (mzVault and mzCloud), and bibliographical identification (Table 1). A total of 188 chemical constituents (114 first report) were accurately or tentatively identified. The extracted ion chromatogram (EIC) in negative ion mode was obtained, as shown in Figure 2. Large differences in chemical constituents were seen in the different batches of Arnebiae Radix samples (A, B, and C), indicating that the chemical constituents of Arnebiae Radix differ significantly in the current Chinese medicine market, possibly due to the different growth environments, growth periods, and medicinal material storage times used [27]. Generally, the samples from batch C from southern Xinjiang showed obvious advantages in terms of their peak intensity and number of peaks.

Identification of Shikonins
It is well-known that the common structural features of most shikonins are a skeleton of naphthoquinone combined with an isohexenyl side chain. Based on a comparison with fragment ions detailed in the literature and reference standards, this study identified seven different types of shikonin compounds in the crude extract, and the chemical structures of these are shown in Figure 3.

Type I Shikonins
Shikonins, composed of a naphthoquinone moiety and an isohexenyl side chain, were identified based on the DFIs at m/z 218.0213 and 190.0261. In total, five type I shikonins (94, 103, 105, 116, and 120) were unambiguously or tentatively identified. For instance, shikonin (120, R.T. = 18.04 min) was unambiguously identified by comparing the listed diagnostic ions and retention times with reference standards. Compounds 94, 103, and 105 were found at 13.13, 15.85, and 15.99 min, and these generated similar fragment ions as the shikonins. Thus, they were tentatively characterized as shikonin derivatives. Compound 116, which was observed at 17.00 min yielded a deprotonated ion [M − H] − m/z 405.15549 and was tentatively identified as dihydrohydroxyshikonin tiglate [28].  [27,28]. Compound 144, found at 20.50 min, showed a precursor ion at [M − H] − m/z 387.14492. Therefore, it was tentatively characterized as β-hydroxyisovalerylshikonin [27,28]. Compounds 166 and 168 appeared at retention times (tR) of 26.60 min and 28.07 min, respectively. The parent ion m/z 429.15549 generated the characteristic fragments m/z 269.0822, 251.0709, and 241.0869, which were tentatively identified as 5-acetoxy-valerylshikonin and β-acetoxyisovalerylshikonin [27]. Similarly, compounds 170 and 171 were tentatively characterized as butyrylshikonin and isobutyrylshikonin. Overall, compounds 177, 182, and 185 demonstrated highly similar product ion spectra and MS/MS fragmentation behaviors but had different retention times, suggesting that they are isobaric compounds. Therefore, they were tentatively identified as α-methylene-butenoylshikonin, tigloylshikonin, and angeloylshikonin, as previously reported [27].

Type V Shikonins
Deoxyshikonin, a precursor for the biosynthesis of other shikonins, was not classified as any type of shikonin or shikonofuran due to the absence of a carboxylic group. The deprotonated molecular ion at m/z 271.0968 for deoxyshikonin gave a product ion base peak at m/z 203.0342. In addition, a product ion at m/z 253.0867 was derived from the neutral loss of H 2 O from the deprotonated molecular ion [27]. Compounds 149, 165 [27,28] and the MS 2 fragmentation results, these compounds were tentatively assigned as deoxyshikonin or its isomers based on the different base peak ions in the MS 2 spectrum.

Shikonofurans
According to previous studies [27], m/z 255.1024 is the DFIs of shikonofurans, and it generates fragment ions at m/z 237.0917 and m/z 227.0340 through the neutral loss of H 2 O and CO, respectively. The compounds 111, 127, 136, 141, and 142 , compounds 111, 127, 136,  141, and 142 were identified as shikonofuran A, shikonofuran D, shikonofuran E, shikonofuran B, and shikonofuran C, respectively, by comparing their respective MS 2 data and data presented in the literature [27]. According to the DFIs, compound 115 was tentatively identified as a shikonofuran derivative. In addition, DFIs at m/z 273.1133 were used for the classification of another shikonofuran indicating that the hydration was likely on the double bond of the side chain. A peak at m/z 255.1024 was observed due to neutral losses of H 2 O from DFI 273.1133 [27]. Compounds 104, 107, 108, 110, 121, 122, 123, 124, 129, 132,  133, 135, 137 and 140

Chemicals and Reagents
MS grade formic acid and acetonitrile were purchased from Thermo Fisher Scientific Co., Ltd. (Waltham, MA, USA). Ultra-pure water was obtained from Guangzhou Watsons Food & Beverage Co., Ltd. (Guangzhou, China). Other solvents were of analytical grade and were supplied by the Aladdin Industrial Corporation (Shanghai, China). The chemical reference standards of shikonin, acetylshikonin, β,β-dimethylacrylshikonin, lithospermic acid, rosmarinic acid, salvianolic acid B, and salvianolic acid C were purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China), while those of nicotiflorin, rutin, isoquercitrin, quercetin, hesperidin, naringenin, kaempferol, and baicalein were provided by Cheng Du Herbpurify Co., Ltd. (Chengdu, China). The purities of all reference standards were above 98% according to an HPLC-UV analysis. The brand and model of the sonication device was KQ-300DE from Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan China).

Sample and Standard Preparation
A total of three batches of Arnebiae Radix were used in this study. Batch A samples were collected from Bortala Mongol Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China; batch B samples (1703060112) were purchased from the herbal medicine markets; and batch C samples were obtained from Zhaosu County and authenticated by Professor Shengjun Ma. The voucher specimens were deposited in the School of Pharmaceutical Sciences, Hunan University of Medicine.
Arnebiae Radix herbs were ground into powder before sample preparation and sieved through No. 40 mesh. Dried Arnebiae Radix powder (10 g) was sonication-extracted (300 W, 40 KHz) in 200 mL of methanol for 1 h at room temperature (16-24 • C), and then the extracted solution was filtered and dried by rotary evaporation. The extracts of these samples were redissolved and centrifuged at 12,000 rpm for 10 min to obtain the supernatant. A volume of 2 µL of supernatant was injected into UHPLC-Q-Exactive Orbitrap MS for analysis.
Standard solutions were prepared in methanol at a concentration of 1.00 mg/mL. The stock solutions of the reference standards were further diluted to obtain working solutions, and then these solutions were stored at 4 • C before analysis.

Instruments and Conditions
In order to acquire a better chromatographic peak shape and separation resolution, various factors were set in the detection and identification process, including a column (Thermo Scientific Hypersil GOLDTM aQ 100 mm × 2.1 mm, 1.9 µm and Waters ACQUITY BEH C18 column, 100 mm × 2.1 mm, 1.7 µm), column temperature (30, 35, 40 • C), and the mobile phase gradient.
All samples were analyzed in negative mode using the following tuning method. In terms of the mass spectrometry conditions, the spray voltage was 3.2 kV, the sheath gas and auxiliary gas operated at flow rates of 35 arb and 10 arb, respectively the temperature of the capillary was 320 • C and that of the auxiliary gas heater was 350 • C, and the S-lens RF level is 60. In the mass range of m/z 100-1200, a high-resolution mass spectrum was obtained at a resolution of 70,000, which was detected by the Orbitrap analyzer. MS 2 data at a resolution of 17,500 were obtained by data-dependent MS 2 scanning or parallel reaction monitoring (PRM) mode. Nitrogen (purity ≥ 99.999%) served as the collision gas, which generated the fragment ions, and the energy level was set as a normalized collision energy of 30%.

Data Processing and Analysis
Xcalibur software version 4.2 (Thermo Fisher Scientific, San Jose, CA, USA) was used to obtain all high-resolution data including the full-scan MS and MS 2 data. Peaks detected with intensities over 10,000 were selected for identification. The chemical formulas for all parent and fragment ions of the selected peaks were calculated from the accurate mass using a formula predictor by setting the parameters as follows:

Conclusions
In this research, an efficient strategy based on UHPLC Q-Exactive Orbitrap MS in negative ion mode was established to detect chemical components in Arnebiae Radix. A total of 188 constituents were identified, of which 114 were reported in Arnebiae Radix for the first time here, including shikonins, phenolic acids, and flavonoids. These were detected and identified based on their chromatographic retention, MS and MS 2 , and bibliography data. These results are very useful references for understanding the bioactive compounds of Arnebiae Radix and their utilization. Overall, the results lay the foundation for in-depth research on the pharmacodynamic material basis of Arnebiae Radix.