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

Zhu, Lian; Ma, Shengjun; Li, Kailin; Xiong, Pei; Qin, Shihan; Cai, Wei

doi:10.3390/molecules27092631

Open AccessArticle

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

by

Lian Zhu

^1,2

,

Shengjun Ma

¹,

Kailin Li

²,

Pei Xiong

²,

Shihan Qin

² and

Wei Cai

^2,*

¹

College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, China

²

Sino-Pakistan Center on Traditional Chinese Medicine, School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua 418000, China

^*

Author to whom correspondence should be addressed.

Molecules 2022, 27(9), 2631; https://doi.org/10.3390/molecules27092631

Submission received: 19 March 2022 / Revised: 18 April 2022 / Accepted: 18 April 2022 / Published: 19 April 2022

(This article belongs to the Special Issue Analytical Methods for Characterization and Isolation of Natural Products)

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Abstract

:

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.

Keywords:

Arnebiae Radix; identification; chemical constituents; UHPLC-Q-Exactive Orbitrap MS

1. 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.

2. Results and Discussion

2.1. 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”.

2.2. 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. In shikonin, the deprotonated molecular ion [M − H]⁻ produced m/z 287.0925 (C₁₆H₁₅O₅) m/z 218.0213 and 190.0261 as the predominant fragment ions by the loss of C₅H₉ and CO, respectively. For acetylshikoninor β,β′-dimethylacrylalkannin, the deprotonated molecular ion [M − H]⁻ at m/z 329.1031 (C₁₈H₁₇O₆) and 369.1344 (C₂₁H₂₁O₆) produced m/z 269.0819, 251.0709 and 241.0869 as the predominant fragment ions.

The quasi-molecular ion of the reference standards salvianolic acid B is m/z 717.14610 in negative mode. The parent ion yielded the fragment ions m/z 339.0507 (C₁₈H₁₁O₇) and 321.0401 (C₁₈H₉O₆) by the loss of C₁₈H₁₈O₉ and C₁₈H₂₀O₁₀, respectively. With the breaking of the ester bond between the carbonyl group and the oxygen atom, 197.0446 (C₉H₉O₅) was obtained. In addition, m/z 197.0446 (C₉H₉O₅) produced the fragment ions m/z 179.0448 (C₉H₇O₄) and 135.0439 (C₈H₇O₂) by the loss of H₂O and COOH. All of the above ions can be used as DFIs of phenolic acid.

Quercetin yielded a deprotonated molecular ion [M − H]⁻ at m/z 301.0359 (C₁₅H₉O₇), which initially produced 151.0025 (C₇H₆O₂) and 178.9977 (C₈H₄O₅) by Retro Diels–Alder (RDA) rearrangement. The diagnostic ion 178.9977 was shown to eliminate a molecule of CO₂ to yield relative fragment ions of 121.0283 (C₇H₄O₄).

2.3. 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, MS² 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.

2.3.1. 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].

Type II Shikonins

Compounds 150 and 174 found at 21.89 and 30.19 min, yielded deprotonated ions [M − H]⁻ m/z 329.10306 (C₁₈H₁₇O_6, −0.19 ppm) and 369.13436 (C₂₁H₂₁O_6, −0.39 ppm), respectively. Through a comparison with the reference standards, they were identified as acetylshikonin and β,β′-dimethylacrylshikonin. Compounds 102, 143, 179, and 183, which were found at 15.74, 20.16, 30.98, and 33.05 min, showed a common precursor ion at [M − H]⁻ m/z 371.1500 (C₂₁H₂₄O₆). They mainly yielded fragment ions at m/z 269.0819, 241.0869, and 251.0709 and were tentatively characterized as α,α-dimethylpropionylshikonin, α-methylbutyrylshikonin, isovalerylshikonin, and isovalerylshikonin isomer through a comparison with the literature and MS² fragmentation [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 III Shikonins

Due to the presence of a double bond between C-12 and C-13, the α-cleavage mechanism of the ester carbonyl is the main fragmentation pathway of the type III shikonins producing product ion at m/z 285.0766. This ion formed the diagnostic generation product ions 257.0807 and 267.0674 by successive neutral loss of H₂O and CO, respectively [27]. Compounds 146 and 162 had the same molecular formula C₂₁H₂₄O₇. In addition, diagnostic products at m/z 285.0766, 267.0663, and 257.0815 were observed through MS² fragmentation ion. Based on a literature search of all shikonins reported [29], compounds 146 and 162 were tentatively identified as lithospermidin A and lithospermidin B, respectively. The ESI-MS of compounds 119, 148, 152, and 158 gave deprotonated molecular ions at m/z 345.09797, 445.15040, 373.12927, and 385.12927, which matched the molecular formulas C₁₈H₁₈O₇, C₂₃H₂₆O₉, C₂₀H₂₂O₇_, and C₂₁H₂₂O₇, respectively. Consequently, these compounds were tentatively identified as lithospermidin C, lithospermidin D, lithospermidin E, and lithospermidin F [27]. Compound 126 was eluted at 18.65 min and generated the same MS and MS² fragmentation results as compound 119. Thus, this compound was tentatively identified as the lithospermidin C isomer. Compounds 153, 156, and 164 appeared at retention times (tR) of 23.03 min, 24.23 min, and 26.38 min, respectively. They had the same MS and MS² fragmentation ions as compound 148 and were tentatively identified as lithospermidin D isomers.

Type IV Shikonins

Diagnostic ions were identified at m/z 299.0921, 284.0687, and 271.0973 for type IV shikonins, and fragmentation ions were obtained at m/z 284.0687 and at m/z 271.0973 via the neutral loss of CH₃ and CO from 299.0921, respectively [27]. As an example, compound 138 with a retention time at 19.74 min showed a deprotonated molecular ion at m/z 359.11362 (C₁₉H₁₉O₇, 0.82 ppm) by ESI-MS. Diagnostic product ions were located at 299.0921, 284.0697, and 271.0973 in MS² and the compound was tentatively identified as 1/4-methoxylithospermidin C [27,28]. According to the literature [29,30] and the different retention times, isomers in each group were eluted based on a predefined order. Compounds 147, 151, 154, 157, 159, 161, 163, 172, and 175 were predicted to be 1/4-methoxylithospermidin H, 1/4-methoxylithospermidin I, 1/4-methoxylithospermidin E, 1/4-methoxylithospermidin J, 1/4-methoxylithospermidin F, 1/4-methoxylithospermidin A, 1/4-methoxylithospermidin D, 1/4-methoxylithospermidin L, and 1/4-methoxylithospermidin B from the experimental chromatographic peaks [27,28]. By comparing MS and MS² fragmentation in the literature, compound 145 was tentatively assigned as 1-methoxy-β-hydroxyisovalerylshikonin [28].

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 H₂O from the deprotonated molecular ion [27]. Compounds 149, 165, 167, 173, 178, and 184 were eluted at 21.76, 26.38, 28.04, 30.11, 30.89, and 34.64 min and showed a deprotonated molecular ion [M − H]⁻ at m/z 271.098 (C₁₆H₁₅O₄). According to a previously published paper [27,28] and the MS² fragmentation results, these compounds were tentatively assigned as deoxyshikonin or its isomers based on the different base peak ions in the MS² 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 H₂O and CO, respectively. The compounds 111, 127, 136, 141, and 142 were eluted at 16.76, 18.83, 19.52, 20.06, and 20.15 min and showed deprotonated molecular ions [M − H]⁻ at m/z 315.12379 (−0.06 ppm, C₁₈H₁₉O₅), 343.15509 (−0.03 ppm, C₂₀H₂₃O₅), 355.15509 (−1.49 ppm, C₂₁H₂₃O₅), 357.17074 (−0.61 ppm, C₃₇H₃₅O₁₀), and 357.17074 (−0.95 ppm, C₃₇H₃₅O₁₀), suggesting that they are shikonofurans. Therefore, 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 MS² 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 H₂O from DFI 273.1133 [27]. Compounds 104, 107, 108, 110, 121, 122, 123, 124, 129, 132, 133, 135, 137 and 140 eluted at 15.96, 16.13, 16.31, 16.46, 18.15, 18.17, 18.45, 18.47, 18.92, 19.16, 19.33, 19.49, 19.67, and 19.80 were predicted to be hydroxyshikonofurans due to the fragment ions yielded at m/z 273.1133 and m/z 255.1024. Therefore, they were tentatively identified as hydroxyshikonofuran J, hydroxyshikonofuran A, hydroxyshikonofuran K, hydroxyshikonofuran A isomer, hydroxyshikonofuran F, hydroxyshikonofuran G, hydroxyshikonofuran H, hydroxyshikonofuran D, hydroxyshikonofuran I, hydroxyshikonofuran E, hydroxyshikonofuran B, hydroxyshikonofuran L, hydroxyshikonofuran C, and hydroxyshikonofuran M [27,28]. Compounds 128 and 134 were found at 18.89 and 19.33 min and showed a common precursor ion at [M − H]⁻ m/z 343.11871. They were tentatively characterized as 1-methoxyacetylshikonin and its isomer, respectively [28].

Dimeric Shikonin

Compounds 176, 181, 186, 187, and 188 were eluted at 30.64, 31.78, 35.84, 38.59, and 38.89 min and showed deprotonated molecular ions [M − H]⁻ at m/z 599.19227 (C₃₄H₃₁O₁₀), 599.19227 (C₃₄H₃₁O₁₀), 627.22357 (C₃₆H₃₅O₁₀), 639.22357 (C₃₇H₃₅O₁₀), and 639.22357 (C₃₇H₃₅O₁₀). According to a previously published paper [28], they were tentatively assigned as 7-(11′-deoxyalkannin)-acetylshikonin,7-(11′-deoxyalkannin)-acetylalkannin, 7-(11′-deoxyalkannin)-isobutyrylshikonin, 7-(11′-deoxyalkannin)-β,β-dimethylacrylshikonin, and 7-(11′-deoxyalkannin)-β,β-dimethylacrylalkannin based on the different base peak ions in the MS² spectrum listed in Table 1.

2.3.2. Identification of Phenolic Acids

Four compounds—rosmarinicacid (77), lithospermic acid (80), salvianolic acid B (83), and salvianolic acid C (89)—were unambiguously identified by comparing their MS and MS² fragmentation ion and retention times with the reference standards in negative ionization mode.

Compound 69 produced the same MS and MS² fragmentation results as compound 83 (salvianolic acid B). Thus, this compound was tentatively identified as salvianolic acid B isomer. Compounds 51, 55, 56, and 70 were found at 6.54, 6.71, 6.91, and 8.02 min and showed a common precursor ion at [M − H]⁻ at m/z 537.1039 (C₂₇H₂₁O₁₂). They yielded m/z 197.0446 by the breaking of the ester bond between the carbonyl group and the oxygen atom, m/z 179.0448 (C₉H₇O₄) due to the loss of H₂O units, and m/z 135.0439 (C₈H₇O₂) by the loss of COOH. According to a previously published paper [31], they were tentatively assigned as salvianolic acid U, salvianolic acid T, salvianolic acid J, and salvianolic acid isomer based on the different base peak ions in the MS² spectrum. Compounds 25, 26, 43, 65, 71, 84, 87, and 130 were eluted at 2.28, 2.28, 5.22, 7.60, 8.19, 10.33, 11.16, and 18.99 min with deprotonated ions [M − H]⁻ at m/z 197.04554 (C₉H₉O₅), 417.08271 (C₂₀H₁₇O₁₀), 179.03498 (C₉H₇O₄), 359.07724 (C₁₈H₁₅O₈), 357.06159 (C₁₈H₁₃O₈), 357.06159 (C₁₈H₁₃O₈), 373.09289 (C₁₉H₁₇O₈), and 357.20713 (C₂₂H₂₉O₄). They were tentatively identified as danshensu, salvianolic acid D, caffeic acid, didehydiosalvianolic acid B, methyl rosmarinate, and cannabidiolic acid [31,32,33,34,35]. Likewise, compounds 53 and 57; compounds 58, 81, and 82; compounds 68, 75, and 90; and compounds 78, 79, 85, 91, and 93 produced similar diagnostic ions and MS² fragmentation behaviors. They were provisional identified as salvianolic acid F, salvianolic acid A, monomethyl lithospermate, 9″-methyl salvianolate B, and their isomers, respectively.

Compound 3 was eluted at 0.88 min with deprotonated ions [M − H]⁻ at m/z 387.11441 (−1.44 ppm, C₁₃H₂₃O₁₃), and the presence of fragment ions at m/z 341.1084 and 179.0550 was used to tentatively identify it as 2,3,4,5,6-pentahydroxy-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyheptanoic acid [28]. Compounds 4, 5, and 6 were eluted at the same time (0.88 min) and produced the same fragment ions (89.0230, 101.0230, 113.0231), which were initially identified as α, α-trehalose, mannose, and raffinose. Compound 10 was eluted at 0.91 min with deprotonated ions [M − H]⁻ at m/z 149.04554 and was initially identified as arabinose. By searching in the databases (ChemSpider, mzVault, and mzCloud), compounds 7, 8, 11, 12, 13, 16, 19, and 101 were eluted at 0.89, 0.90, 0.92, 0.93, 0.93, 1.17, 1.31, and 15.35 min with deprotonated ions [M − H]⁻ at m/z 135.02989 (C₄H₇O₅), 195.05102 (C₆H₁₁O₇), 193.03537(C₆H₉O₇), 133.01424 (C₄H₅O₅), 177.04046 (C₆H₉O₆), 147.02989(C₅H₇O₅), 161.04554(C₆H₉O₅), and 187.13396 (C₁₀H₁₉O₃), respectively. They were tentatively identified as threonic acid, gluconic acid, β-D-glucopyranuronic acid, malic acid, δ-gluconic acid δ-lactone, α-hydroxyglutaric acid, 3-hydroxy-3-methylglutaric acid, and 3-hydroxydecanoic acid. According to the literature [36,37], compounds 9, 14, and 15, which were eluted at 0.90, 0.94, and 1.17 min were identified as quinic acid, citric acid isomer, and citric acid. Based on the literature [28,31,32,33,34,35,36,37,38] and a database search (ChemSpider, mzVault and mzCloud), compounds 18, 20, 21, 23, 24, 27, 28, 31, 33, 35, 36, 39, 40, 44, 45, 49, 50, 52, 54, 60, 64, 67, 74, 88, and 113 were tentatively assigned as uric acid, 3-methylglutaric acid, gallic acid, salicylic acid, pantothenic acid, vanillic acid, protocatechuic acid, isovanillic acid, 2-isopropylmalic acid, gentisic acid, kynurenic acid, 3-coumaric acid, p-coumaric acid, 2-hydroxyphenylacetic acid, ferulic acid, 4-methoxysalicylic acid, suberic acid, 4-coumaric acid isomer, indole-3-acetic acid, isoferulic acid, salicylic acid isomer, 4-coumaric acid, azelaic acid, 3-tert-butyladipic acid, and octyl ferulate based on the different base peak ions in the MS² spectrum listed in Table 1.

2.3.3. Identification of Flavonoids

A total of 24 individual flavonoid constituents were putatively identified in Arnebiae Radix extract using UHPLC-Q-Exactive Orbitrap MS. Eight compounds, including rutin (63), isoquercitrin (66), nicotiflorin (73), hesperidin (76), quercetin (92), naringenin (95), kaempferol (96), and baicalein (114) were identified as having a pseudomolecular ion [M − H]⁻ at m/z 609.14610 (−0.79 ppm, C₂₇H₂₉O₁₆), 463.08819 (−0.28 ppm, C₂₁H₁₉O₁₂), 593.15119 (−0.43 ppm, C₂₇H₂₉O₁₅), 609.18249 (1.61 ppm, C₂₈H₃₃O₁₅), 301.03537 (−0.22 ppm, C₁₅H₉O₇), 271.06119 (0.31 ppm, C₁₅H₁₁O₅), 285.04046 (1.30 ppm, C₁₅H₉O₆), and 283.06119 (−0.03 ppm, C₁₆H₁₁O₅), respectively. These were unambiguously identified by comparing their accurate mass information and chromatography retention times with reference standards in negative ionization mode. Compounds 37, 41, 47, 59, 61, 62, and 72 yielded deprotonated molecular ions [M − H]⁻ at m/z 465.10384 (−0.20 ppm, C₂₁H₂₁O₁₂), 465.10384 (−0.26 ppm, C₂₁H₂₁O₁₂), 449.10893 (0.17 ppm, C₂₁H₂₁O₁₁), 433.11402 (−0.17 ppm, C₂₁H₂₁O₁₀), 449.10893 (0.17 ppm, C₂₁H₂₁O₁₁), 433.11402 (−0.17 ppm, C₂₁H₂₁O₁₀) and 277.14453 (−0.99 ppm, C₁₆H₂₁O₄), which initially produced 151.0025 (C₇H₆O₂), and 178.9977 (C₈H₄O₅) by Retro Diels–Alder (RDA) rearrangement. According to a previous study [39,40] and the DFIs, they were tentatively characterized as taxifolin-glucoside, taxifolin-glucoside, eriodictyol-glucoside, naringenin-glucoside, eriodictyol-glucoside, naringenin-glucoside and De-O-methyllasiodiplodin. Compounds 99, 100, 109, 118, 125, and 131 were tentatively assigned as hispidulin, bilirubin, glycitein, pectolinarigenin, apigenin, and medicarpin by searching in the databases such as the chemical structure database (ChemSpider) and MS² database (mzVault and mzCloud). Peaks 38, 46, and 117 were eluted at 4.62, 5.94, and 19.11 min with deprotonated ions [M − H]⁻ at m/z 289.07176 (C₁₅H₁₃O₆), 375.13100 (C₁₇H₁₉N₄O₆), and 345.09797 (C₁₈H₁₇O₇). They were tentatively characterized as catechin, riboflavin, and kaempferide [39,40,41,42,43].

2.3.4. Identification of Amino Acids

Compounds 1, 2, 22, 29, 32, 42, and 48 yielded a quasi-molecular ions [M − H]⁻ at m/z 131.04621 (C₄H₇N₂O₃), 145.06186 (C₅H₉N₂O₃), 164.07170 (C₉H₁₀NO₂), 158.08226 (C₇H₁₂NO₃), 203.08260 (C₁₁H₁₁N₂O₂), 172.09791 (C₈H₁₄NO₃), and 206.08226 (C₁₁H₁₂NO₃) and were eluted at 0.83, 0.86, 1.92, 2.96, 3.46, 5.17, and 6.01 min, respectively. Comparing the MS² fragment ions with data from the bibliography [37,44,45], compounds 1, 2, 22, 29, 32, 42, and 48 were tentatively identified as asparagine, glutamine, phenylalanine, N-acetylvaline, tryptophan, N-acetyl-L-leucine, and N-acetyl-L-phenylalanine, respectively.

2.3.5. Others

Compounds 7, 30, 34, and 86 were tentatively identified as threonic acid, ethyl 3,4-dihydroxybenzoate, 7-hydroxycoumarin, and dibutylphthalate by searching in databases such as the chemical structure database (ChemSpider) and MS² database (mzVault and mzCloud).

Compound 97, found at 14.56 min, possessed a quasi-molecular ion [M − H]⁻ at m/z 253.08701 and was tentatively identified as Rhizonone [28].

3. Materials and Methods

3.1. 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).

3.2. 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.

3.3. 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.

Each LC-MS analysis was exercised on a Q-Exactive Focus Orbitrap MS connected to a Thermo Scientific Dionex Ultimate 3000 RS through an ESI source. Chromatographic separation was performed at 35 °C using a Thermo Scientific Hypersil GOLD^TM aQ (100 mm × 2.1 mm, 1.9 μm). The mobile phase was composed of 0.1% formic acid (A) and acetonitrile (B), and the flow rate was 0.3 mL/min. The following gradient was used: 0–2 min, 95–90% A; 2–5 min, 90–80% A; 5–10 min, 80–75% A;10–15 min, 75–50% A; 15–25 min, 50–45% A; 25–40 min, 45–20% A; 40–45 min, 20–5% A; 45–45.1 min, 5–95% A; 45–50 min, 95% A.

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. MS² data at a resolution of 17,500 were obtained by data-dependent MS² 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%.

3.4. 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 MS² 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: C [0–60], H [0–120], O [0–60], and N [0–10]. The mass tolerance of MS and MS² was within 10 ppm.

4. 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 MS², 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.

Author Contributions

L.Z. and S.M. contributed equally; L.Z.: writing—original draft, performed experiments and sorted the data. S.M.: conceptualization, analyzed the validation data. K.L.: data curation. P.X. and S.Q.: performed experiments and analyzed the data. W.C.: writing—review and editing, conceptualization, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region (2020D03002) and the Graduate Student Innovation Project of the Xinjiang Uygur Autonomous Region (XJ2021G175).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be provided upon request.

Conflicts of Interest

All the authors declare that they have no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

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Figure 1. Fragmentation routes of the reference standards: shikonin (A); acetylshikonin (B); salvianolic acid B (C); quercetin (D).

Figure 2. The high-resolution extracted ion chromatogram (HREIC) in 10 ppm for multiple compounds in Arnebiae Radix. (A) m/z 313.07176, 131.04621, 145.06186, 503.16175, 191.05611, 149.04554, 177.04046, 147.02989, 243.06225, 731.16175, 493.11402, 357.06159, 201.11323, 253.08701, 315.12379, 299.05611, 583.25620, 175.06119, 153.01933, 188.03531, 172.09791, 151.04006, 193.05063, 375.13100, 163.04006, 206.08226, 173.08193, 174.05605, 449.10893, 137.02441, 715.13045, 551.11949, 277.14453, 167.02106, 161.04554, 145.05063, 164.07170, 218.10339, 158.08226, 181.05063, 203.08260, 187.13396, 371.15001, 269.08193, 333.13436, 345.09797, 357.20713, 375.18131, 387.14492; (B) m/z 491.09837, 609.14610, 731.16175, 135.02989, 195.05102, 193.03537, 191.01972, 197.04554, 167.03498, 161.02441, 179.03498, 163.04006, 493.11402, 463.08819, 593.15119, 187.09758, 277.14453, 373.09289, 305.17583, 313.07176, 363.10854, 283.06119, 347.11362, 349.12927, 299.05611, 361.16566, 269.04554, 355.15509, 425.12419, 357.17074, 459.16605, 373.12927, 445.15040, 285.07684, 399.14492, 385.12927, 401.16057, 599.19227, 465.10384, 433.11402; (C) m/z 179.05611, 287.09249, 555.16605, 405.15549, 137.02441, 329.10306, 369.13436, 627.22357, 537.10384, 429.15549, 445.15040, 271.09758, 343.15509, 433.18679, 285.04046, 343.11871, 373.16566, 357.13436, 357.06159, 391.13984, 169.01424, 417.08271, 465.10384, 289.07176, 433.11402, 403.13984, 391.17622; (D) m/z 363.10854, 343.15509, 433.18679, 285.04046, 343.11871, 373.16566, 357.13436, 357.06159, 391.13984, 169.01424, 417.08271, 465.10384, 289.07176, 433.11402, 403.13984, 391.17622, 283.06119, 347.11362, 349.12927, 405.15549, 299.05611, 361.16566, 269.04554, 355.15509, 359.11362, 425.12419, 357.17074, 387.14492, 459.16605, 373.12927, 445.15040, 285.07684, 399.14492, 385.12927, 401.16057, 369.13436, 599.19227, 627.22357; (E) m/z 131.04621, 465.10384, 433.11402, 449.10893, 463.08819, 493.11402, 551.11949, 731.16175, 271.06119, 583.25620, 283.06119, 357.20713, 537.10384, 133.01424, 429.15549, 341.10893, 387.14492, 359.11362, 387.11441, 639.22357, 329.10306, 717.14610,359.07724; (F) m/z 609.18249, 301.03537, 357.17074, 271.09758, 369.13436, 313.07176, 131.04621, 145.06186, 503.16175, 191.05611, 149.04554, 177.04046, 147.02989, 243.06225, 731.16175, 493.11402, 357.06159, 201.11323, 253.08701, 315.12379, 299.05611 ((A–D,E,F) correspond to the EIC of Samples B, C, A, respectively).

Figure 3. Chemical structures of 7 different types of shikonin compounds found in Arnebiae Radix.

Table 1. Chromatographic and mass data of the components detected in Arnebiae Radix though UHPLC-Q-Exactive Orbitrap MS.

No.	Batch	t_R	Theoretical Mass m/z	Experimental Mass m/z	Error (ppm)	Formula	MS/MS Fragment (-)	Identification
1	A, B, C	0.83 ^#	131.0462	131.0450	−9.05	C₄H₈N₂O₃	MS²[131]: 114.0183(100), 113.0343(65), 70.0284(37), 95.0237(25), 131.0449(22)	Asparagine
2	A, B, C	0.86 ^#	145.0619	145.0606	−8.80	C₅H₁₀N₂O₃	MS²[145]: 127.0500(100), 128.0340(73), 145.0606(97), 102.0546(48), 109.0394(40)	Glutamine
3	A, B, C	0.88	387.1144	387.1139	−1.44	C₁₃H₂₄O₁₃	MS²[387]: 89.0229(100), 119.0336(45), 179.0550(42), 341.1084(33), 161.0444(16), 221.0658(5)	2,3,4,5,6-pentahydroxy-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyheptanoic acid
4	A, B, C	0.88 ^#	341.1089	341.1084	−1.66	C₁₂H₂₂O₁₁	MS²[341]: 89.0230(100), 59.0124(48), 71.0124(40), 101.0230(33), 119.0336(29), 113.0231(25)	α,α-Trehalose
5	A, B, C	0.88 ^#	179.0561	179.0550	−6.27	C₆H₁₂O₆	MS²[341]: 59.0124(100), 89.0230(68), 71.0124(63), 75.0073(48), 101.0230(33), 119.0336(29)	Mannose
6	A, B, C	0.88 ^#	503.1618	503.1612	−1.19	C₁₈H₃₂O₁₆	MS²[503]: 89.0230(100), 101.0230(50), 113.0230(38), 179.0551(26)	Raffinose
7	A, B, C	0.89 ^#	135.0299	135.0286	−9.68	C₄H₈O₅	MS²[135]: 75.0073(100), 135.0287(51), 72.9917(13), 89.0230(11), 59.0124(10)	Threonic acid
8	A, B, C	0.90 ^#	195.0510	195.0500	−5.26	C₆H₁₂O₇	MS²[195]: 195.0501(100), 75.0073(82), 129.0180(76), 99.0074(22), 87.0073(21), 59.0124(12)	Gluconic acid
9	A, B, C	0.90 ^#	191.0561	191.0551	−5.25	C₇H₁₂O₆	MS²[191]: 111.0074(100), 87.0073(35), 85.0280(28), 191.0551(12)	Quinic acid
10	A, B, C	0.91 ^#	149.0455	149.0443	−8.17	C₅H₁₀O₅	MS²[149]: 149.0443(100), 89.0230(76), 59.0124(32), 75.0073(32)	Arabinose
11	A, B, C	0.92 ^#	193.0354	193.0344	−5.16	C₆H₁₀O₇	MS²[193]: 103.0023(100), 59.0124(29), 85.0280(16), 193.0708(10)	β-D-Glucopyranuronic acid
12	A, B, C	0.93 ^#	133.0142	133.0130	−9.60	C₄H₆O₅	MS²[133]: 115.0023(100), 71.0124(44), 133.0130(32)	Malic acid
13	A, B, C	0.93 ^#	177.0405	177.0395	−5.60	C₆H₁₀O₆	MS²[177]: 59.0124(100), 129.0181(42), 99.0074(34), 89.0230(32), 177.0397(31)	δ-Gluconic acidδ-lactone
14	A, B, C	0.94 ^#	191.0197	191.0188	−5.01	C₆H₈O₇	MS²[191]: 111.0074(100), 87.0073(35), 85.0280(28), 191.0188(11)	Citric acid isomer
15	A, B, C	1.17 ^#	191.0197	191.0188	−5.01	C₆H₈O₇	MS²[191]: 111.0074(100), 87.0073(34), 85.0281(23), 191.0188(10)	Citric acid
16	A, B, C	1.17 ^#	147.0299	147.0287	−8.35	C₅H₈O₅	MS²[147]: 129.0180(100), 85.0280(28), 103.0386(20), 147.0287(18), 101.0230(17)	α-Hydroxyglutaric acid
17	A, B, C	1.17 ^#	243.0623	243.0617	−2.35	C₉H₁₂N₂O₆	MS²[243]: 110.0234(100), 128.0340(24), 200.0556(21), 152.0342(16), 140.0341(11)	Uridine
18	A, B, C	1.17	167.0211	167.0199	−6.79	C₅H₄N₄O₃	MS²[167]: 167.0200(100), 124.0140(38)	Uric acid
19	A, B, C	1.31 ^#	161.0455	161.0444	−7.37	C₆H₁₀O₅	MS²[161]: 99.0437(100), 57.0332(63), 101.0230(33), 161.0444(29), 59.0124(22)	3-Hydroxy-3-methylglutaric acid
20	A, B, C	1.42 ^#	145.0506	145.0495	−8.02	C₆H₁₀O₄	MS²[145]: 145.0494(100), 101.0594(50)	3-Methylglutaric acid
21	A, B, C	1.66 ^#	169.0142	169.0133	−5.49	C₇H₆O₅	MS²[169]: 125.0231(100)	Gallic acid
22	A, B, C	1.92	164.0717	164.0707	−6.23	C₉H₁₁NO₂	MS²[164]: 147.0440(100), 164.0706(59), 72.0077(28), 96.9587(13)	Phenylalanine
23	A, B, C	1.96 ^#	137.0244	137.0232	−8.74	C₇H₆O₃	MS²[137]: 93.0331(100), 137.0231(37)	Salicylic acid
24	A, B, C	2.14 ^#	218.1034	218.1028	−2.60	C₉H₁₇NO₅	MS²[218]: 88.0389(100), 146.0810(50), 218.1027(13), 71.0124(9)	Pantothenic acid
25	A, B, C	2.28 ^#	197.0455	197.0447	−4.40	C₉H₁₀O₅	MS²[167]: 72.9917(100), 135.0439(67), 179.0340(58), 123.0439(50), 197.0445(11)	Danshensu
26	A, B, C	2.28 ^#	417.0827	417.0800	−6.62	C₂₀H₁₈O₁₀	MS²[417]: 219.0268(100), 197.0445(21), 179.0339(5)	Salvianolic acid D
27	A, B, C	2.35 ^#	167.0350	167.0339	−6.36	C₈H₈O₄	MS²[167]: 167.0339(100), 123.0439(87)	Vanillic acid
28	A, B, C	2.81 ^#	153.0193	153.0183	−3.10	C₇H₆O₄	MS²[153]: 109.0281(100), 153.0182(30), 126.0911(19)	Protocatechuic acid
29	B, C	2.96 ^#	158.0823	158.0811	−7.38	C₇H₁₃NO₃	MS²[158]: 116.0704(100), 158.0811(21)	N-Acetylvaline
30	A, B, C	3.33 ^#	181.0506	181.0497	−5.43	C₉H₁₀O₄	MS²[181]: 163.0389(100), 181.0496(68), 135.0439(54), 137.0231(42), 119.0489(34)	Ethyl 3,4-dihydroxybenzoate
31	A, B, C	3.35 ^#	167.0350	167.0339	−6.24	C₈H₈O₄	MS²[167]: 123.0438(100), 167.0339(10)	Isovanillic acid
32	A, B, C	3.46 ^#	203.0826	203.0818	−3.85	C₁₁H₁₂N₂O₂	MS²[203]: 116.0492(100), 203.0816(70), 74.0233(36), 72.0077(32), 159.0916(31), 142.0650(29)	Tryptophan
33	A, B, C	3.66 ^#	175.0612	175.0601	−6.16	C₇H₁₂O₅	MS²[175]: 146.9600(100), 115.0387(80), 175.0601(69), 113.0594(32), 85.0644(31)	2-Isopropylmalic acid
34	A, B, C	3.77 ^#	161.0244	161.0231	−8.25	C₉H₆O₃	MS²[161]: 161.0233(100), 133.0282(66)	7-Hydroxycoumarin
35	A, B, C	3.80 ^#	153.0193	153.0182	−3.82	C₇H₆O₄	MS²[153]: 109.0282(100), 153.0181(57)	Gentisic acid
36	A, B, C	4.16 ^#	188.0353	188.0345	−4.51	C₁₀H₇NO₃	MS²[188]: 144.0443(100), 188.0343(4)	Kynurenic acid
37	C	4.51 ^#	465.1038	465.1038	−0.20	C₂₁H₂₂O₁₂	MS²[465]: 285.0403(100), 125.0232(85), 275.0563(53), 177.0189(26), 151.0033(21), 303.0518(19)	Taxifolin-glucoside
38	B, C	4.62 *^#	289.0718	289.0721	1.00	C₁₅H₁₄O₆	MS²[289]: 245.0816(100), 289.0721(94), 125.0233(62), 109.0283(58), 179.0340(48), 151.0390(30), 161.0594(20)	Catechin/Catechin hydrate
39	A, B, C	4.78 ^#	163.0401	163.0390	−6.61	C₉H₈O₃	MS²[163]: 163.0389(100), 120.0522(32)	3-Coumaric acid
40	A, B, C	5.02 ^#	163.0401	163.0390	−6.74	C₉H₈O₃	MS²[163]: 119.0489(100), 163.0390(16)	p-Coumaric acid
41	A, C	5.08 ^#	465.1038	465.1037	−0.26	C₂₁H₂₂O₁₂	MS²[465]: 285.0403(100), 125.0232(39), 275.0558(12), 177.0183(14), 151.0033(21), 303.0507(8)	Taxifolin-glucoside isomer
42	B, C	5.17 ^#	172.0979	172.0969	−5.85	C₈H₁₅NO₃	MS²[172]: 130.0861(100), 172.0969(16), 128.1068(2)	N-Acetyl-L-leucine
43	A, B, C	5.22	179.0350	179.0340	−5.49	C₉H₈O₄	MS²[179]: 135.0439(100), 179.0340(29)	Caffeic acid
44	B, C	5.33 ^#	151.0401	151.0390	−7.14	C₈H₈O₃	MS²[151]: 107.0489(100), 151.0387(5)	2-Hydroxyphenylacetic acid
45	A, B, C	5.41 ^#	193.0506	193.0497	−4.68	C₁₀H₁₀O₄	MS²[193]: 134.0361(100), 149.0596(34), 193.0499(13)	Ferulic acid
46	A, B, C	5.94 ^#	375.1310	375.1307	−0.96	C₁₇H₂₀N₄O₆	MS²[375]: 255.0884(100), 212.0821(18), 151.0388(18), 161.0234(14)	Riboflavin
47	C	6.01 ^#	449.1089	449.1090	0.17	C₂₁H₂₂O₁₁	MS²[449]: 259.0608(100), 59.0124(94), 269.0455(78), 125.0233(37), 287.0564(32), 178.9974(18)	Eriodictyol-glucoside
48	A, B, C	6.01 ^#	206.0823	206.0817	−3.00	C₁₁H₁₃NO₃	MS²[206]: 164.0706(100), 147.0440(28), 58.0285(24), 206.0814(21), 70.0285(14)	N-Acetyl-L-phenylalanine
49	A, B, C	6.16 ^#	167.0350	167.0340	−5.88	C₈H₈O₄	MS²[167]: 167.0339(100), 152.0103(19)	4-Methoxysalicylic acid
50	A, B, C	6.49 ^#	173.0819	173.0809	−5.79	C₈H₁₄O₄	MS²[173]: 111.0802(100), 173.0809(55), 129.0908(6), 112.0835(5)	Suberic acid
51	A, B, C	6.54 ^#	537.1038	537.1036	0.52	C₂₇H₂₂O₁₂	MS²[537]: 339.0504(100), 229.0137(64), 295.0609(56), 197.0446(31), 135.0439(25), 179.0338(14)	Salvianolic acid U
52	A, B, C	6.54 ^#	163.0401	163.0390	−6.61	C₉H₈O₃	MS²[163]: 119.0490(100), 163.0389(14), 120.0522(6)	4-Coumaric acid isomer
53	A, B, C	6.61 ^#	313.0718	313.0715	−0.74	C₁₇H₁₄O₆	MS²[313]: 109.0281(100), 147.0439(38), 159.0440(27), 269.0816(14)	Salvianolic acid F
54	A, B, C	6.67 ^#	174.0561	174.0550	−5.82	C₁₀H₉NO₂	MS²[174]: 146.9600(100), 174.0550(67), 130.0650(35)	Indole-3-acetic acid
55	A, B, C	6.71 ^#	537.1038	537.1036	0.52	C₂₇H₂₂O₁₂	MS²[537]: 197.0446(100), 135.0439(80), 339.0505(68), 229.0137(64), 295.0609(63), 179.0340(42)	Salvianolic acid T
56	A, B, C	6.91 ^#	537.1038	537.1033	−0.97	C₂₇H₂₂O₁₂	MS²[537]: 197.0447(100), 135.0439(71), 339.0506(64), 295.0609(58), 229.0137(47), 179.0340(41)	Salvianolic acid J
57	B, C	6.91 ^#	313.0718	313.0711	−2.09	C₁₇H₁₄O₆	MS²[313]: 269.0818(100), 313.0716(46), 203.0341(44), 159.0443(31), 109.0281(22)	Salvianolic acid F isomer
58	A, B, C	6.91 ^#	493.1140	493.1135	−1.08	C₂₆H₂₂O₁₀	MS²[493]: 197.0446(100), 135.0439(63), 295.0609(34), 179.0339(33), 72.9917(24), 269.0818(21)	Salvianolic acid A isomer
59	C	7.03 ^#	433.1140	433.1140	−0.17	C₂₁H₂₂O₁₀	MS²[433]: 271.0609(100), 151.0030(30), 98.9477(74), 119.0489(11)	Naringenin-glucoside
60	A, B, C	7.13 ^#	193.0506	193.0497	−4.83	C₁₀H₁₀O₄	MS²[193]: 134.0361(100), 193.0496(13), 149.0596(7)	Isoferulic acid
61	C	7.22 ^#	449.1089	449.1087	−0.53	C₂₁H₂₂O₁₁	MS²[449]: 151.0025(100), 287.0558(65), 135.0439(46), 98.9477(41), 96.9587(12)	Eriodictyol hexoside 1
62	C	7.30 ^#	433.1140	433.1134	−1.44	C₂₁H₂₂O₁₀	MS²[433]: 271.0610(100), 151.0025(33), 98.9476(10), 119.0492(6)	Naringenin-glucoside isomer
63	A, B, C	7.50 *^#	609.1461	609.1456	−0.79	C₂₇H₃₀O₁₆	MS²[609]: 300.0273(100), 301.0345(57)	Rutin
64	A, B, C	7.57 ^#	137.0244	137.0231	−9.40	C₇H6O3	MS²[137]: 93.0332(100), 137.0232(52)	Salicylic acid isomer
65	B, C	7.60 ^#	715.1305	715.1303	−0.30	C₃₆H₂₈O₁₆	MS²[715]: 197.0446(100), 151.0390(54), 135.0437(40), 177.0182(39), 179.0339(35)	DidehydioSalvianolic acid B
66	C	7.77 *^#	463.0882	463.0881	−0.28	C₂₁H₂₀O₁₂	MS²[463]: 300.0272(100), 301.0346(56), 151.0025(4), 178.9980(2)	Isoquercitrin
67	A, B, C	7.81 ^#	163.0401	163.0390	−6.74	C₉H₈O₃	MS²[163]: 163.0389(100), 137.0596(91), 119.0489(46), 162.8380(17)	4-Coumaric acid
68	A, B, C	7.88 ^#	551.1195	551.1198	0.46	C₂₈H₂₄O₁₂	MS²[551]: 197.0447(100), 135.0440(74), 59.0124(62), 353.0659(50), 179.0341(45), 309.0770(41)	Monomethyl lithospermate isomer
69	A, B, C	7.92 ^#	717.1461	717.1456	−0.75	C₃₆H₃₀O₁₆	MS²[717]: 339.0505(100), 321.0760(40), 295.0611(20), 197.0449(26), 135.0440(16), 179.03469(6)	Salvianolic acid B isomer
70	A, B, C	8.02 ^#	537.1038	537.1034	−0.86	C₂₇H₂₂O₁₂	MS²[537]: 295.0609(100), 339.0504(44), 109.0282(41), 185.0233(31), 277.0504(13)	Salvianolic acid isomer
71	A, B, C	8.19 ^#	357.0616	357.0611	−1.46	C₁₈H₁₄O₈	MS²[357]: 135.0439(100), 229.0135(31), 197.0448(29), 179.0341(23), 109.0280(17)	Salvianolic acid H
72	B, C	8.31 ^#	277.1445	277.1443	−0.99	C₁₆H₂₂O₄	MS²[277]: 259.1336(100), 247.1335(88), 174.0675(82), 121.0282(76), 241.1230(47)	De-O-Methyllasiodiplodin
73	A, B, C	8.37 *^#	593.1512	593.1509	−0.43	C₂₇H₃₀O₁₅	MS²[593]: 285.0401(100), 284.0324(49)	Nicotiflorin
74	A, B, C	8.40 ^#	187.0976	187.0966	−5.25	C₉H₁₆O₄	MS²[187]: 125.0959(100), 187.0966(50), 97.0645(4), 169.0859(3)	Azelaic acid
75	A, B, C	8.50 ^#	551.1195	551.1201	1.13	C₂₈H₂₄O₁₂	MS²[551]: 327.0827(100), 135.0439(37), 197.0446(33), 217.0499(24), 229.0137(22), 353.0664(22)	Monomethyl lithospermate
76	A, B	8.65 *^#	609.1825	609.1829	1.61	C₂₈H₃₄O₁₅	MS²[609]: 301.0714(100), 302.0744(8)	Hesperidin
77	A, B, C	8.84 *	359.0772	359.0769	−0.95	C₁₈H₁₆O₈	MS²[359]: 161.0233(100), 197.0446(37), 179.0339(16), 72.9917(11), 135.0440(6)	Rosmarinic acid
78	A, B, C	8.88 ^#	731.1618	731.1613	−0.60	C₃₇H₃₂O₁₆	MS²[731]: 109.0282(100), 335.0921(89), 353.0670(70), 197.0446(61), 489.1185(45)	9″-Methyl salvianolate B isomer
79	A, B, C	9.00 ^#	731.1618	731.1620	0.32	C₃₇H₃₂O₁₆	MS²[731]: 367.0821(100), 353.0667(97), 109.0282(74), 197.0446(58), 335.0924(53), 489.1198(35)	9″-Methyl salvianolate B
80	A, B, C	9.22 *	537.1038	537.1036	−0.51	C₂₇H₂₂O₁₂	MS²[537]: 339.0511(100), 197.0447(82), 135.0440(74), 295.0613(66), 179.0338(25)	Lithospermic acid
81	A, B, C	9.22 ^#	493.1140	493.1138	−0.45	C₂₆H₂₂O₁₀	MS²[493]: 185.0235(100), 109.0281(94), 295.0608(77), 203.0343(21), 159.0440(20), 135.0439(19), 197.0448(17), 179.0343(12)	Salvianolic acid A
82	C	9.68 ^#	493.1140	493.1140	−0.09	C₂₆H₂₂O₁₀	MS²[493]: 197.0446(100), 135.0439(82), 295.0608(55), 179.0338(38), 185.0234(33), 109.0281(33), 269.0817(30)	Salvianolic acid A isomer
83	C	10.19 *	717.1461	717.1460	−0.15	C₃₆H₃₀O₁₆	MS²[717]: 321.0401(100), 339.0507(29), 295.0609(16), 185.0237(10), 197.0444(3), 135.0438(2), 179.0339(2)	Salvianolic acid B
84	A, B, C	10.33 ^#	357.0616	357.0616	−0.12	C₁₈H₁₄O₈	MS²[357]: 135.0439(100), 337.0353(56), 72.9917(27), 179.0339(26), 197.0446(25), 321.0403(18)	Salvianolic acid I
85	A, B, C	10.66 ^#	731.1618	731.1625	1.07	C₃₇H₃₂O₁₆	MS²[731]: 229.0136(100), 339.0506(76), 313.0716(59), 203.0340(47), 267.0659(32)	9″-Methyl salvianolate B isomer
86	A, B, C	11.04 ^#	277.1445	277.1441	−1.56	C₁₆H₂₂O₄	MS²[277]: 277.1443(100), 233.1541(74), 203.1433(11)	Dibutylphthalate
87	B, C	11.16 ^#	373.0929	373.0926	−0.73	C₁₉H₁₈O₈	MS²[373]: 135.0439(100), 175.0391(65), 197.0447(57), 179.0340(27), 72.9917(23), 161.0235(15)	Methyl rosmarinate
88	A, B, C	11.20 ^#	201.1132	201.1125	−3.79	C₁₀H₁₈O₄	MS²[201]: 139.1116(100), 201.1123(92), 183.1017(47)	3-tert-Butyladipic acid
89	A, B, C	11.65 *	491.0984	491.0980	−0.84	C₂₆H₂₀O₁₀	MS²[491]: 311.0559(100), 135.0439(35), 197.0447(4), 179.0341(2)	Salvianolic acid C
90	C	11.87 ^#	551.1195	551.1193	−0.44	C₂₈H₂₄O₁₂	MS²[551]: 321.0400(100), 231.0292(22), 109.0281(21), 293.0455(17), 135.0440(11), 197.0447(8), 179.0337(4)	Monomethyl lithospermate isomer
91	B, C	11.92 ^#	731.1618	731.1627	1.32	C₃₇H₃₂O₁₆	MS²[731]: 335.0560(100), 353.0666(60), 309.0762(38), 135.0439(33), 197.0446(20)	9″-Methyl salvianolate B isomer
92	A	12.49 *^#	301.0354	301.0353	−0.22	C₁₅H₁₀O₇	MS²[301]: 151.0026(100), 301.0359(89), 178.9977(53), 121.0283(19)	Quercetin
93	C	13.01 ^#	731.1618	731.1630	1.73	C₃₇H₃₂O₁₆	MS²[731]: 339.0507(100), 229.0137(42), 295.0607(30), 359.0772(19)	9″-Methyl salvianolate B isomer
94	A, B, C	13.13 ^#	363.1085	363.1086	0.03	C₁₈H₂₀O₈	MS²[363]: 218.0214(100), 190.0262(79), 303.0872(74), 227.0343(70), 219.0251(5)	Shikonin derivative
95	C	13.25 *^#	271.0612	271.0613	0.31	C₁₅H₁₂O₅	MS²[271]: 151.0025(100), 271.0612(62), 119.0490(42), 227.1071(23), 107.0125(14), 93.0332(14), 177.0185(11)	Naringenin
96	A, B, C	14.54 ^#	285.0405	285.0408	1.30	C₁₅H₁₀O₆	MS²[285]: 285.0406(100), 227.0711(16), 241.0508(11), 215.0701(11)	Kaempferol
97	A, B, C	14.56	253.0870	253.0869	−0.67	C₁₆H₁₄O₃	MS²[253]: 237.0551(100), 238.0615(31), 270.0533(20), 253.0505(14)	Rhizonone
98	A, B, C	14.57	315.1238	315.1239	0.43	C₁₈H₂₀O₅	MS²[315]: 241.0865(100), 256.1102(65), 300.1002(27), 271.0973(22), 285.0762(11)	Ethylshikonin
99	A, B, C	14.61 ^#	299.0561	299.0561	0.03	C₁₆H₁₂O₆	MS²[301]: 284.0323(100), 255.0293(16), 299.0560(12), 285.0359(10)	Hispidulin
100	C	15.35 ^#	583.2562	583.2564	0.24	C₃₃H₃₆N₄O₆	MS²[583]: 285.1242(100), 297.1239(15), 241.1341(9), 213.1030(3)	Bilirubin
101	A, B, C	15.35 ^#	187.1340	187.1331	−4.53	C₁₀H₂₀O₃	MS²[187]: 59.0124(100), 125.0960(21), 141.8672(14), 187.1324(14)	3-Hydroxydecanoic acid
102	A, B, C	15.74 ^#	371.1500	371.1497	−0.87	C₂₁H₂₄O₆	MS²[371]: 271.0970(100), 253.0863(52), 99.0439(49), 241.0868(40), 225.0916(38)	Valerylshikonin isomer
103	A, B, C	15.85 ^#	403.1398	403.1398	−0.03	C₂₁H₂₄O₈	MS²[403]: 303.0875(100), 218.0215(96), 227.0345(65), 190.0258(75), 99.0435(83)	Shikonin derivative
104	B, C	15.96	391.1762	391.1762	−0.20	C₂₁H₂₈O₇	MS²[391]: 255.1024(100), 273.1133(92), 190.0262(18), 117.0544(17), 227.0340(14)	Hydroxyshikonofuran J
105	A, B, C	15.99 ^#	391.1398	391.1399	0.21	C₂₀H₂₄O₈	MS²[363]: 218.0215(100), 303.0872(98), 227.0343(87), 190.0262(82), 87.0437(71)	Shikonin derivative
106	A, B, C	16.05	269.0819	269.0819	−0.09	C₁₆H₁₄O₄	MS²[269]: 136.0153(100), 251.0712(58), 223.0753(44), 269.0817(41), 241.0870(31)	Dehydratedshikonin
107	A, B, C	16.13	333.1344	333.1341	−0.82	C₁₈H₂₂O₆	MS²[333]: 255.1023(100), 273.1129(66), 219.0292(15), 254.0937(2), 237.0917(1)	Hydroxyshikonofuran A
108	B, C	16.31	391.1762	391.1763	0.27	C₂₁H₂₈O₇	MS²[391]: 255.1023(100), 273.1133(91), 117.0545(25)	Hydroxyshikonofuran K
109	C	16.36 ^#	283.0612	283.0611	−0.45	C₁₆H₁₂O₅	MS²[283]: 283.0608(100), 240.0423(27), 257.0451(19), 239.0373(3)	Glycitein
110	A, B, C	16.46 ^#	333.1344	333.1343	−0.07	C₁₈H₂₂O₆	MS²[333]: 255.1023(100), 273.1130(65), 315.1576(30), 254.0945(2), 237.0918(2)	Hydroxyshikonofuran A isomer
111	A, B, C	16.76	315.1238	315.1238	−0.06	C₁₈H₂₀O₅	MS²[315]: 255.1023(100), 59.0125(29), 227.1077(13), 237.0922(11), 121.0281(7), 187.0390(4)	Shikonofuran A
112	A, B, C	16.76	347.1136	347.1134	−0.57	C₁₈H₂₀O₇	MS²[347]: 241.0867(100), 287.0922(73), 269.0824(69), 59.0124(68), 227.1079(23)	Shikonin acetate
113	A, B, C	16.79 ^#	305.1758	305.1758	1.63	C₁₈H₂₆O₄	MS²[305]: 135.0803(100), 249.1492(62), 174.9551(46), 235.0195(26), 146.9600(26)	Octyl ferulate
114	C	16.87 *^#	283.0612	283.0612	−0.03	C₁₆H₁₂O₅	MS²[283]: 283.0608(100), 268.0377(67), 265.1805(10)	Baicalein
115	A, B, C	17.67 ^#	349.1293	349.1292	−0.37	C₁₈H₂₂O₇	MS²[349]: 255.1023(100), 227.1071(43), 237.0921(13)	Shikonofurans derivative
116	A, B, C	17.00	405.1555	405.1558	0.74	C₂₁H₂₆O₈	MS²[405]: 303.0870(100), 218.0214(59), 190.0262(58), 227.0343(56), 245.0451(54), 101.0594(53)	Dihydrohydroxyshikonin tiglate
117	A, B, C	17.11 *	299.0561	299.0560	−0.38	C₁₆H₁₂O₆	MS²[299]: 299.0558(100), 254.0581(27), 237.0556(25), 281.0452(22), 284.0333(13)	Kaempferide
118	A, B, C	17.14 ^#	313.0718	313.0716	−0.46	C₁₇H₁₄O₆	MS²[313]: 298.0482(100), 202.1159(82), 283.0243(53), 312.2260(47), 255.0294(24)	Pectolinarigenin
119	B, C	17.19	345.0980	345.0975	−1.47	C₁₈H₁₈O₇	MS²[345]: 285.0766(100), 267.0674(32), 257.0807(25), 59.0123(20)	Lithospermidin C
120	A, B, C	18.04 *	287.0925	287.0923	−0.62	C₁₆H₁₆O₅	MS²[287]: 218.0214(100), 219.0255(9), 190.0261(2)	Shikonin
121	B, C	18.15	433.1868	433.1871	0.65	C₂₃H₃₀O₈	MS²[433]: 255.1023(100), 273.1131(84), 273.9557(16), 59.0126(5)	Hydroxyshikonofuran F
122	A, B, C	18.17	361.1657	361.1655	−0.54	C₂₀H₂₆O₆	MS²[361]: 255.1022(100), 273.1129(73), 259.0607(15), 87.0439(4)	Hydroxyshikonofuran G
123	B, C	18.45	433.1868	433.1866	−0.56	C₂₃H₃₀O₈	MS²[433]: 255.1023(100), 273.1131(81), 59.0123(6), 237.0913(3)	Hydroxyshikonofuran H
124	A, B, C	18.47	361.1657	361.1658	0.39	C₂₀H₂₆O₆	MS²[361]: 255.1022(100), 273.1128(72), 218.0214(10), 87.0438(3)	Hydroxyshikonofuran D
125	A, B, C	18.57 ^#	269.0455	269.0454	3.57	C₁₅H₁₀O₅	MS²[301]: 269.0454(100), 149.0229(1)	Apigenin
126	A, B, C	18.65 ^#	345.0980	345.0975	−1.47	C₁₈H₁₈O₇	MS²[345]: 285.0766(100), 267.0659(14), 257.0817(8), 59.0124(4), 239.0709(1)	Lithospermidin C isomer
127	A, B, C	18.83	343.1551	343.1551	−0.03	C₂₀H₂₄O₅	MS²[343]: 283.0974(100), 266.0824(14), 255.1023(35), 87.0438(16), 227.1078(3)	Shikonofuran D
128	A, B, C	18.89	343.1187	343.1184	−0.80	C₁₉H₂₀O₆	MS²[343]: 283.0974(100), 255.1023(35), 87.0438(16), 266.0824(14)	1-Methoxyacetylshikonin
129	A, B, C	18.92	373.1657	373.1658	0.37	C₂₁H₂₆O₆	MS²[373]: 255.1022(100), 273.1131(61), 174.9551(35)	Hydroxyshikonofuran I
130	C	18.99 ^#	357.2071	357.2071	−0.04	C₂₂H₃₀O₄	MS²[357]: 357.2070(100), 339.1964(69), 295.2061(31), 327.1965(30), 269.0819(13)	Cannabidiolic acid
131	A, B, C	19.04 ^#	269.0819	269.0812	−0.20	C₁₆H₁₄O₄	MS²[269]: 269.0816(100), 254.0582(80), 149.0231(42), 133.0646(37), 210.0677(35)	Medicarpin
132	A, B, C	19.16	373.1657	373.1655	−0.36	C₂₁H₂₆O₆	MS²[373]: 255.1025(100), 273.1130(67), 174.9550(24)	Hydroxyshikonofuran E
133	B, C	19.33	375.1813	375.1812	1.03	C₂₁H₂₈O₆	MS²[375]: 255.1025(100), 273.1132(71)	Hydroxyshikonofuran B
134	A, B, C	19.33	343.1187	343.1185	−0.53	C₁₉H₂₀O₆	MS²[343]: 283.0973(100), 255.1021(13)	1-methoxyacetylshikonin isomer
135	A, B, C	19.49	375.1813	375.1810	0.72	C₂₁H₂₈O₆	MS²[375]: 255.1023(100), 273.1132(74)	Hydroxyshikonofuran L
136	A, B, C	19.52	355.1551	355.1546	−1.49	C₂₁H₂₄O₅	MS²[355]: 255.1025(100), 355.3217(70), 99.0438(58), 218.0218(30), 227.1070(15), 237.0919(4)	Shikonofuran E
137	B, C	19.67	375.1813	375.1784	−7.74	C₂₁H₂₈O₆	MS²[375]: 255.1024(100), 273.1131(69), 101.0594(3)	Hydroxyshikonofuran C
138	A, B, C	19.74	359.1136	359.1139	0.82	C₁₉H₂₀O₇	MS²[359]: 299.0921(100), 284.0687(57), 359.1137(11), 161.0234(9), 271.0973(7)	1/4-methoxylithospermidin C
139	A, B, C	19.80	425.1242	425.1245	1.90	C₂₃H₂₂O₈	MS²[425]: 321.1494(100), 178.9977(59), 227.1074(58), 271.0969(27), 363.1226(21), 245.1178(16), 345.1130(12)	Unknown
140	A, B, C	19.80	375.1813	375.1811	−0.52	C₂₁H₂₈O₆	MS²[375]: 255.1023(100), 273.1131(72), 101.0594(3)	Hydroxyshikonofuran M
141	A, B	20.06	357.1707	357.1705	−0.61	C₂₁H₂₆O₅	MS²[357]: 255.1023(100), 101.0594(47), 297.1130(18), 227.1069(9), 121.0281(7), 237.0916(6)	Shikonofuran B
142	A, B, C	20.15	357.1707	357.1704	−0.95	C₂₁H₂₆O₅	MS²[357]: 255.1023(100), 101.0594(45), 227.1068(10), 237.0921(8), 121.0283(8), 172.0517(2), 143.0497(2)	Shikonofuran C
143	A, B, C	20.16	371.1500	371.1497	−0.79	C₂₁H₂₄O₆	MS²[371]: 269.0817(100), 241.0866(16), 251.0706(7)	α,α-dimethylpropionylshikonin
144	A, B, C	20.50	387.1449	387.1448	−0.30	C₂₁H₂₄O₇	MS²[387]: 117.0544(100), 269.0813(31), 251.0711(31), 59.0123(21), 241.0867(13)	β-hydroxyisovalerylshikonin
145	A, B, C	20.94	401.1606	401.1605	1.16	C₂₂H₂₆O₇	MS²[401]: 299.0923(100), 255.1027(57), 121.0284(15), 313.0705(10), 237.0908(10)	1-Methoxy-β-hydroxyisovalerylshikonin
146	A, B, C	21.33	387.1449	387.1448	−0.23	C₂₁H₂₄O₇	MS²[387]: 101.0594(100), 189.0184(76), 285.0765(60), 217.0135(49), 257.0814(28), 267.0659(19)	Lithospermidin A
147	B, C	21.69	459.1661	459.1664	0.71	C₂₄H₂₈O₉	MS²[459]: 299.0916(100), 59.0124(58), 271.0966(36), 281.0820(21)	1/4-methoxylithospermidin H
148	B, C	21.70	445.1504	445.1503	−0.18	C₂₃H₂₆O₉	MS²[445]: 285.0766(100), 257.0819(9), 59.0124(8), 267.0657(7)	Lithospermidin D
149	A, B	21.76 ^#	271.0976	271.0974	−0.53	C₁₆H₁₆O₄	MS²[271]: 253.0867(100), 271.0968(73), 203.0342(58), 256.0739(44), 238.0630(39)	Deoxyshikonin isomer
150	A, B, C	21.89 *	329.1031	329.1030	−0.19	C₁₈H₁₈O₆	MS²[329]: 269.0818(100), 251.0710(81), 241.0868(48), 59.0124(47)	Acetylshikonin
151	A, B, C	22.80	387.1449	387.1449	0.01	C₂₁H₂₄O₇	MS²[387]: 299.0923(100), 270.0893(94), 285.0756(68), 271.0954(66), 87.0438(63), 253.0864(24)	1/4-methoxylithospermidin I
152	A, B, C	22.84	373.1293	373.1293	0.15	C₂₀H₂₂O₇	MS²[373]: 285.0768(100), 267.0663(10), 257.0817(9), 174.9552(8), 87.0438(6)	Lithospermidin E
153	B, C	23.03 ^#	445.1504	445.1497	−1.61	C₂₃H₂₆O₉	MS²[445]: 285.0768(100), 257.0817(49), 59.0123(18), 267.0655(7)	Lithospermidin D isomer
154	A, B, C	23.13	387.1449	387.1447	−0.56	C₂₁H₂₄O₇	MS²[387]: 299.0903(100), 285.0767(78), 270.0897(74), 87.0438(58), 271.0974(50)	1/4-methoxylithospermidin E
155	A, B, C	24.03	285.0768	285.0767	−0.66	C₁₆H₁₄O₅	MS²[285]: 285.0766(100), 267.0659(55), 227.0344(50), 73.0281(8), 257.0817(6), 239.0708(2)	Sakuranetin
156	B, C	24.23 ^#	445.1504	445.1504	−0.11	C₂₃H₂₆O₉	MS²[445]: 285.0771(100), 257.0809(93), 59.0124(35)	Lithospermidin D isomer
157	A, B, C	24.57	399.1449	399.1445	−1.07	C₂₂H₂₄O₇	MS²[399]: 270.0891(100), 299.0921(98), 271.0978(68), 99.0437(56), 281.0811(21)	1/4-methoxylithospermidin J
158	A, B, C	24.63	385.1293	385.1292	−0.33	C₂₁H₂₂O₇	MS²[385]: 285.0767(100), 257.0817(11), 99.0438(9), 267.0651(8)	Lithospermidin F
159	A, B, C	24.81	399.1449	399.1445	−1.07	C₂₂H₂₄O₇	MS²[399]: 299.0921(100), 271.0957(63), 99.0438(53), 281.0811(25)	1/4-methoxylithospermidin F
160	A, B, C	24.99	343.1187	343.1193	1.69	C₁₉H₂₀O₆	MS²[343]: 57.0332(100), 343.2271(79), 269.0806(55), 251.0713(51), 285.1859(50), 73.0281(43), 241.0872(33)	Propionylshikonin
161	A, B, C	25.48	401.1606	401.1603	−0.67	C₂₂H₂₆O₇	MS²[401]: 299.0919(100), 270.0900(92), 101.0594(61), 271.0976(53), 281.0826(21), 253.0871(17)	1/4-methoxylithospermidin A
162	A, B, C	25.60	387.1449	387.1448	−0.30	C₂₁H₂₄O₇	MS²[387]: 285.0766(100), 257.0816(9), 101.0594(9), 267.0658(8)	Lithospermidin B
163	B, C	26.29	459.1661	459.1654	−1.41	C₂₄H₂₈O₉	MS²[459]: 299.0922(100), 271.0971(10), 59.0124(9)	1/4-methoxylithospermidin D
164	B	26.38 ^#	445.1504	445.1500	−0.94	C₂₃H₂₆O₉	MS²[445]: 285.0768(100), 257.0815(54), 59.0125(21), 267.0662(6)	Lithospermidin D isomer
165	B	26.38 ^#	271.0976	271.0974	−0.86	C₁₆H₁₆O₄	MS²[271]: 253.0867(100), 271.0966(71), 203.0341(57), 256.0736(48), 238.0629(39)	Deoxyshikonin isomer
166	A, B, C	26.60	429.1555	429.1552	−0.59	C₂₃H₂₆O₈	MS²[429]: 269.0822(100), 251.0710(65), 59.0125(65), 241.0866(35)	5-acetoxy-valerylshikonin
167	A, B	28.04 ^#	271.0976	271.0974	−0.53	C₁₆H₁₆O₄	MS²[271]: 253.0866(100), 271.0967(66), 203.0341(54), 256.0738(45), 238.0629(42)	Deoxyshikonin isomer
168	B, C	28.07	429.1555	429.1551	−0.87	C₂₃H₂₆O₈	MS²[429]: 269.0822(100), 251.0706(63), 59.0124(61), 241.0871(37)	β-acetoxyisovalerylshikonin
169	A, B, C	28.08 ^#	425.1242	425.1227	−2.13	C₂₃H₂₂O₈	MS²[425]: 269.0818(100), 251.0710(27), 87.0437(17), 241.0966(10)	Shikonin derivative
170	A, B, C	28.16	357.1344	357.1342	−0.40	C₂₀H₂₂O₆	MS²[357]: 269.0819(100), 251.0713(70), 87.0438(63), 241.0863(50), 223.0820(4)	Butyrylshikonin
171	A, B, C	29.19	357.1344	357.1342	−0.40	C₂₀H₂₂O₆	MS²[357]: 269.0819(100), 251.0707(66), 87.0437(72), 241.0866(43)	Isobutyrylshikonin
172	A, B, C	29.53	399.1449	339.1999	9.71	C₂₂H₂₄O₇	MS²[399]: 299.0923(100), 99.0439(11), 281.0812(10), 271.0970(9)	1/4-Methoxylithospermidin L
173	A, B, C	30.11 ^#	271.0976	271.0974	−0.64	C₁₆H₁₆O₄	MS²[271]: 253.0867(100), 271.0967(74), 203.0341(54), 256.0736(45), 238.0631(41)	Deoxyshikonin isomer
174	A, B, C	30.19 *	369.1344	369.1342	−0.39	C₂₁H₂₂O₆	MS²[369]: 269.0818(100), 251.0710(71), 99.0438(68), 270.0888(57), 241.0867(49)	β,β-dimethylacrylshikonin
175	A, B, C	30.44	401.1606	401.1603	−0.59	C₂₂H₂₆O₇	MS²[401]: 299.0924(100), 121.0283(15), 271.0959(12), 101.0592(11), 281.0879(10)	1/4-methoxylithospermidin B
176	A, B, C	30.64	599.1923	599.1926	1.52	C₃₄H₃₂O₁₀	MS²[599]: 426.1099(100), 412.0935(42), 102.9554(26), 132.4304(21), 116.5537(21), 59.0124(21)	7-(11′-Deoxyalkannin)-Acetylshikonin
177	A	30.72	369.1344	369.1340	−1.07	C₂₁H₂₂O₆	MS²[369]: 269.0817(100), 251.0709(68), 99.0438(61), 241.0864(52)	α-methylene-butenoylshikonin
178	A, B, C	30.89 ^#	271.0976	271.0974	−0.53	C₁₆H₁₆O₄	MS²[271]: 253.0868(100), 271.0968(77), 203.0343(64), 256.0738(47), 238.0630(40)	Deoxyshikonin isomer
179	A, B, C	30.98	371.1500	371.1497	−0.95	C₂₁H₂₄O₆	MS²[371]: 269.0818(100), 251.0710(66), 101.0594(65), 241.0865(43)	α-methylbutyrylshikonin
180	A, B, C	31.62	555.1661	555.1657	−0.63	C₃₂H₂₈O₉	MS²[555]: 486.0952(100), 555.1646(11)	Shikometabolin B
181	A, B, C	31.78	599.1923	599.1936	3.14	C₃₄H₃₂O₁₀	MS²[599]: 412.0944(100), 426.1105(77)	7-(11′-Deoxyalkannin)-Acetylalkannin
182	A	32.05	369.1344	369.1343	−0.14	C₂₁H₂₂O₆	MS²[369]: 269.0816(100), 251.0705(48), 99.0439(43), 241.0871(37)	Tigloylshikonin
183	A, B, C	33.05	371.1500	371.1497	−0.95	C₂₁H₂₄O₆	MS²[371]: 269.0819(100), 101.0594(71), 251.0710(64), 241.0866(47)	Isovalerylshikonin
184	A, B, C	34.64 ^#	271.0976	271.0973	−1.08	C₁₆H₁₆O₄	MS²[271]: 253.0866(100), 271.0974(66), 203.0341(58), 256.0735(45), 238.0630(37)	Deoxyshikonin isomer
185	A	35.15	369.1344	369.1342	−0.47	C₂₁H₂₂O₆	MS²[369]: 269.0829(100), 251.0721(46), 99.0443(54), 241.0867(40)	Angeloylshikonin
186	A, B, C	35.84	627.2236	627.2235	0.69	C₃₆H₃₆O₁₀	MS²[627]: 426.1100(100), 412.0944(80), 87.0437(45), 495.1801(24), 349.1006(12), 290.5349(12)	7-(11′-Deoxyalkannin)-Isobutyrylshikonin
187	A, B, C	38.59	639.2236	639.2231	0.19	C₃₇H₃₆O₁₀	MS²[639]: 537.1556(100), 639.2231(5), 509.1626(4), 519.1473(2), 101.0590(2)	7-(11′-Deoxyalkannin)-β,β-dimethylacrylshikonin
188	A, B, C	38.89	639.2236	639.2232	0.30	C₃₇H₃₆O₁₀	MS²[639]: 537.1553(100), 639.2230(4), 519.1443(2), 101.0589(1)	7-(11′-Deoxyalkannin)-β,β-dimethylacrylalkannin

* Identified by comparison with standards. ^# First reported in Arnebiae Radix.

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Zhu, L.; Ma, S.; Li, K.; Xiong, P.; Qin, S.; Cai, W. Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry. Molecules 2022, 27, 2631. https://doi.org/10.3390/molecules27092631

AMA Style

Zhu L, Ma S, Li K, Xiong P, Qin S, Cai W. Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry. Molecules. 2022; 27(9):2631. https://doi.org/10.3390/molecules27092631

Chicago/Turabian Style

Zhu, Lian, Shengjun Ma, Kailin Li, Pei Xiong, Shihan Qin, and Wei Cai. 2022. "Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry" Molecules 27, no. 9: 2631. https://doi.org/10.3390/molecules27092631

APA Style

Zhu, L., Ma, S., Li, K., Xiong, P., Qin, S., & Cai, W. (2022). Systematic Screening of Chemical Constituents in the Traditional Chinese Medicine Arnebiae Radix by UHPLC-Q-Exactive Orbitrap Mass Spectrometry. Molecules, 27(9), 2631. https://doi.org/10.3390/molecules27092631

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

Abstract

1. Introduction

2. Results and Discussion

2.1. Optimization of the UHPLC-Q-Exactive Orbitrap MS Condition

2.2. Establishment of Diagnostic Fragment Ions (DFIs)

2.3. Characterization of the Chemical Constituents in Arnebiae Radix

2.3.1. Identification of Shikonins

Type I Shikonins

Type II Shikonins

Type III Shikonins

Type IV Shikonins

Type V Shikonins

Shikonofurans

Dimeric Shikonin

2.3.2. Identification of Phenolic Acids

2.3.3. Identification of Flavonoids

2.3.4. Identification of Amino Acids

2.3.5. Others

3. Materials and Methods

3.1. Chemicals and Reagents

3.2. Sample and Standard Preparation

3.3. Instruments and Conditions

3.4. Data Processing and Analysis

4. Conclusions

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Conflicts of Interest

Sample Availability

References

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