Chemical Constituent Analysis of Ranunculus sceleratus L. Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole-Orbitrap High-Resolution Mass Spectrometry

Ranunculus sceleratus L.(RS) has shown various pharmacological effects in traditional Chinese medicine. In our previous study, the positive therapeutic effect on α-naphthylisothiocyanate induced intrahepatic cholestasis in rats was obtained using TianJiu treatment with fresh RS. However, the chemical profile of RS has not been clearly clarified, which impedes the research progress on the therapeutic effect of RS. Herein, an ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed to rapidly separate and identify multiple constituents in the 80% methanol extract of RS. A total of sixty-nine compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) were successfully characterized. A total of 12 of these compounds were unambiguously identified by standard samples. Their mass spectrometric fragmentation pathways were investigated. It is worth noting that flavonoids and lignans were identified for the first time in RS. In this study, we successfully provide the first comprehensive report on identifying major chemical constituents in RS by UHPLC-Q-Orbitrap HRMS. The obtained results enrich the RS chemical profile, paving the way for further phytochemical study, quality control, and pharmacological investigation of RS.


Introduction
Ranunculus sceleratus L. (RS), an annual herbaceous plant belonging to the Ranunculus L. family, has been listed among the top herbs in the Shennong Traditional Herbal Scriptures, written in the Western Han Dynasty. Compendium of Materia Medica notes that fresh RS can be pasted onto the acupuncture point of Cunkou overnight, (hyperemia and blistering in the skin), to cure jaundice induced by malaria. TianJiu treatment with fresh RS patches exhibited a positive therapeutic effect on α-naphthylisothiocyanate-intrahepatic cholestasis in rats by pasting on the acupuncture points of Dazhui, Ganshu (both sides), and Jizhong in our previous study [1]. The protoanemonin of fresh RS is commonly used as a robust blistering agent. It has been reported that protoanemonin is poisonous, but toxins will be destroyed when fresh RS is heated or dried. Moreover, the anti-inflammatory activity of the extract of RS administered orally at a dose of 100 mg·kg −1 was obtained in Wistar rats by inhibiting the induced hind paw edema [2]. A dosage between 3 and 9 g is recorded

Optimization of UHPLC-Q-Orbitrap HRMS Conditions
The composition of the UHPLC mobile phase should be systemically optimized to achieve the best chromatographic and mass spectrometric properties for separation and analysis. To assess the resulting peak shape and signal strength, different mobile systems that consisted of methanol−0.1% formic acid aqueous solution or acetonitrile−0.1% formic acid aqueous solution were investigated. In the result, acetonitrile−0.1% formic acid aqueous solution was chosen as the UHPLC mobile phase for gradient elution. In terms of sample processing, 60%, 80%, and 100% methanol (v/v) were examined to obtain the optimal RS extract that contains the more chemical constituents of RS. It was found that 80% methanol showed the best extraction efficiency based on the number of peaks and was selected as the extraction solvent for RS. An 80% methanol extract of RS was then analyzed in the positive and negative ion modes. Most chemical constituents showed higher responses in negative ionization mode than in positive ionization mode. In addition, to improve the sensitivity and accuracy, the ion source voltage and capillary temperature were optimized. The optimal conditions are described in Section 3.3.

Identification of Chemical Constituents in RS
Under the optimal UHPLC-Q-Orbitrap HRMS conditions, the base peak chromatograms obtained in positive and negative ionization modes for the 80% methanol extract of RS are shown in Figure 1. As shown in Table 1, a total of 69 compounds (19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) in the 80% methanol extract of RS were either unambiguously identified (12 compounds) or tentatively characterized (57 compounds). Reference standards of isoscopoletin, scopoletin, and scoparone were detected in positive ionization mode ( Figure S1), and reference standards of aesculetin, quercetin, protocatechuic acid, salicylic acid, ferulic acid, luteolin, caffeic acid, emodin, and oleanic acid were detected in negative ionization mode ( Figure S2). Next, the names and chemical structures of the other 57 compounds were preliminarily inferred by comparing MS ion fragmentation information with relevant literature. Altogether, the chemical structures of identified 69 compounds are summarized in Figure S3. responses in negative ionization mode than in positive ionization mode. In addition, to improve the sensitivity and accuracy, the ion source voltage and capillary temperature were optimized. The optimal conditions are described in Section 3.3.

Identification of Chemical Constituents in RS
Under the optimal UHPLC-Q-Orbitrap HRMS conditions, the base peak chromatograms obtained in positive and negative ionization modes for the 80% methanol extract of RS are shown in Figure 1. As shown in Table 1, a total of 69 compounds (19 flavonoids,  22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones) in the 80% methanol extract of RS were either unambiguously identified (12 compounds) or tentatively characterized (57 compounds). Reference standards of isoscopoletin, scopoletin, and scoparone were detected in positive ionization mode ( Figure S1), and reference standards of aesculetin, quercetin, protocatechuic acid, salicylic acid, ferulic acid, luteolin, caffeic acid, emodin, and oleanic acid were detected in negative ionization mode ( Figure S2). Next, the names and chemical structures of the other 57 compounds were preliminarily inferred by comparing MS ion fragmentation information with relevant literature. Altogether, the chemical structures of identified 69 compounds are summarized in Figure S3.     Flavonoids refer to a class of naturally occurring bioactive compounds in herbal medicine. The principal mass spectrometric fragmentation mechanisms for flavonoids are loss of neutral fragments, such as H 2 O, CH 3 , CO, CO 2 , and the cleavage of retro-Diels-Alder (RDA) at glycosyl bonds. The product ion spectrum of compound 39 shown in Figure 2A  were produced by successive loss of glucuronide [28]. Glucuronide residues at m/z 175.0233 and m/z 113.0229 could always be observed in glycosides. The mass spectra and proposed major fragmentations with structures are shown in Figure 2B. Except for luteolin (55) and quercetin (56)

Organic Acids
Organic acids widely occur in natural plants, especially in herbs. In negative ionization mode, organic acids exhibited deprotonated molecular peaks and easily lost CO2 and CO to generate corresponding fragment ion peaks. Moreover, the losses of small molecules (like H2O) or radicals (like CH3) sometimes occurred.

Organic Acids
Organic acids widely occur in natural plants, especially in herbs. In negative ionization mode, organic acids exhibited deprotonated molecular peaks and easily lost CO 2 and CO to generate corresponding fragment ion peaks. Moreover, the losses of small molecules (like H 2 O) or radicals (like CH 3 Figure 3A. Compound 21 was unambiguously identified as protocatechuic acid by comparing its MS/MS fragmentation pattern and retention time of reference standard. Likewise, compounds 32, 46, 53, and 69 were identified as caffeic acid, ferulic acid, salicylic acid, and oleanic acid by reference standards. The deprotonated molecule [M − H] − of compound 25 peak was at m/z 299.0777, and its chemical structure and fragmentation pathway are shown in Figure 3B. Compound 25 was identified as salicylic acid-O-glucopyranoside according to the literature [19]. The other compounds were identified according to their molecular mass, formulas, MS/MS fragments, and related literature studies, including gluconic acid (5), malic acid (8), aconitic acid (12), furoic acid (13), citric acid (14), succinic acid (16) Figure 3A. Compound 21 was unambiguously identified as protocatechuic acid by comparing its MS/MS fragmentation pattern and retention time of reference standard. Likewise, compounds 32, 46, 53, and 69 were identified as caffeic acid, ferulic acid, salicylic acid, and oleanic acid by reference standards. The deprotonated molecule [M − H] -of compound 25 peak was at m/z 299.0777, and its chemical structure and fragmentation pathway are shown in Figure 3B. Compound 25 was identified as salicylic acid-Oglucopyranoside according to the literature [19]. The other compounds were identified according to their molecular mass, formulas, MS/MS fragments, and related literature studies, including gluconic acid (5), malic acid (8), aconitic acid (12), furoic acid (13), citric acid (14), succinic acid (16)

Nitrogenous Compounds
Amino acids, nucleobases, and other nitrogenous compounds respond strongly in positive ionization mode, and most of the second mass spectra are broken in the center of N + . In the present study, 3 nucleobases, 7 amino acids, and other nitrogenous compounds in RS were characterized. The usual fragmentation pathways, including the losses of NH3, H2O, and HCOOH, were observed in these compounds. Compound 6 was identified as proline based on short retention time and specific fragments. It has a pseudomolecular ion of m/z 116.0704, indicative of the molecular formula C5H9NO2. The fragment ion at m/z 70.0656 for [M + H − HCOOH] + agrees with the literature studies due to losing one carboxyl group [11]. The chemical structures and fragmentation pathways are shown in Figure 5. In this way, other amino acids can be successfully characterized according to related literature studies, including asparagine (1), glutamic acid (2), threonine (3), pyroglutamic acid (4), phenylalanine (17), and tryptophan (23). In addition, nucleobases (compounds 7, 9, and 15) and other nitrogenous compounds (compounds 10, 11, 34, and 67) were also detected and tentatively identified based on databases and the literature.  [19]. The possible fragmentation mechanism of compound 38 is depicted in Figure 4B. Compounds 49, 52, and 57 were tentatively identified as matairesinoside, 1-hydroxypinoresinol, and matairesinol according to their MS/MS fragments and related literature studies [19].

Nitrogenous Compounds
Amino acids, nucleobases, and other nitrogenous compounds respond strongly in positive ionization mode, and most of the second mass spectra are broken in the center of N + . In the present study, 3 nucleobases, 7 amino acids, and other nitrogenous compounds in RS were characterized. The usual fragmentation pathways, including the losses of NH 3 , H 2 O, and HCOOH, were observed in these compounds. Compound 6 was identified as proline based on short retention time and specific fragments. It has a pseudomolecular ion of m/z 116.0704, indicative of the molecular formula C 5 H 9 NO 2 . The fragment ion at m/z 70.0656 for [M + H − HCOOH] + agrees with the literature studies due to losing one carboxyl group [11]. The chemical structures and fragmentation pathways are shown in Figure 5. In this way, other amino acids can be successfully characterized according to related literature studies, including asparagine (1), glutamic acid (2), threonine (3), pyroglutamic acid (4), phenylalanine (17), and tryptophan (23). In addition, nucleobases (compounds 7, 9, and 15) and other nitrogenous compounds (compounds 10, 11, 34, and 67) were also detected and tentatively identified based on databases and the literature.  Figure 6. In addition, compounds 60, 63, and 66 were tentatively identified as 7-hydroxy-emodin, 1-O-methyl-emodin, and physcion according to their MS/MS fragments and related literature studies [34].

Chemicals and Reagents
High-performance liquid chromatography (HPLC)-grade acetonitrile and formic acid were purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA). Analytical-grade methanol was purchased from Chinasun Specialty Products Co., Ltd. (Jiangsu, China

Chemicals and Reagents
High-performance liquid chromatography (HPLC)-grade acetonitrile and formic acid were purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA

Preparation of Sample and Standard Solutions
An aliquot of 0.1 g fine powder (<65 mesh) of RS samples was accurately weighed and placed in a 50 mL of a conical flask, and then 10 mL of 80% methanol−water (v/v) was added into the conical flask. After sonication for 30 min, the sample solution was cooled to room temperature. All the standards of aesculetin, isoscopoletin, scopoletin, quercetin, scoparone, protocatechuic acid, salicylic acid, ferulic acid, luteolin, caffeic acid, emodin and oleanic acid were dissolved in 80% methanol−water (v/v) at a concentration of 10 µg·mL −1 to prepare standard solutions. All the solutions were filtered through a 0.22 µm filter membrane (Bandao Corp., Shanghai, China) before analysis.

Data Processing and Analysis
Tune 2.9 (Thermo Fisher Scientific, San Jose, CA, USA) was used to control the mass spectrometer, and Xcalibur 4.1 software (Thermo Fisher Scientific, San Jose, CA, USA) was used to control the instrument for data acquisition and analysis. The mass tolerance of MS and MS 2 was within 5 ppm. The chemical formulas for all parent and fragment ions were calculated according to the exact mass, and the parameters are set as follows: C (0-60), H (0-120), O (0-60), and N (0-10).

Conclusions
The inherent variety of natural products in TCM has presented a big challenge in separation and detection techniques for the rapid characterization of its chemical profiling. In the present study, the chemical constituents of RS extract were determined by UHPLC-Q-Orbitrap HRMS. A total of 69 compounds, including 19 flavonoids, 22 organic acids, 6 coumarins, 4 lignans, 14 nitrogenous compounds, and 4 anthraquinones, were identified based on the comparison of their accurate masses, fragment ions, literature studies, and standard samples. Isoscopoletin, scopoletin, scoparone, aesculetin, quercetin, protocatechuic, salicylic acid, ferulic acid, luteolin, caffeic acid, emodin and oleanic acid were identified by standard samples. It is worth noting that flavonoids and lignans were identified for the first time in RS. This work can provide an essential chemical basis for quality control and further studies on the pharmacological and clinical application of RS.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.