Systematic Screening of the Chemical Constituents of Lanqin Oral Liquid by Ultra-High-Performance Liquid Chromatography Combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

A rapid and sensitive method that combined ultra-high-performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) was used to identify the chemical constituents in Lanqin oral liquid. On the basis of UHPLC-FT-ICR-MS analysis, systematic characterization of the chemical profile of Lanqin oral liquid was carried out, and a total of 441 compounds were identified or tentatively characterized including alkaloids, flavonoids, terpenoids, organic acids, phenylpropanoids, and other types. The results provide a reference for improving quality control, contribute to establishing higher quality standards, provide a scientific basis for further research on the pharmacodynamic material basis, and help illustrate the relationship between the complicated constituents and therapeutic effects of Lanqin oral liquid.

Traditional Chinese medicine (TCM) prescriptions collectively exert therapeutic effects and display a synergistic action via the multicomponents-to-multitargets mechanism guided by the compatibility rules of TCM theory.Given the highly complicated and diverse components in these systems, it is inevitably difficult to identify compounds because these ingredients have rich structural types, wide polarity ranges, and significant differences in content; this always makes the separation and identification challenging and, accordingly, creates difficulties in quality control and the elucidation of pharmacodynamic substances, which hinders modernization.Hence, to ensure the safety and effectiveness of TCM, it is unquestionable that systematic and comprehensive chemical characterization using analytical instruments with high resolution, sensitivity, and accuracy are needed.Recently, the Molecules 2023, 28, 7053 2 of 42 chemical compositions in TCM are increasingly being studied systematically through the use of high-performance liquid chromatography (UHPLC) combined with high-resolution mass spectrometry (HRMS) [7,8].The overall separation of complex compounds can be efficiently carried out by UPLC; then, these compounds can be accurately characterized and identified by HRMS.A total of 170 and 175 compounds in Lanqin oral liquid were identified or putatively characterized in the previous literature [9,10].Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has been widely used in the identification and characterization of chemical constituents in TCM for high mass accuracy, good sensitivity, high resolution, and rich data [11,12].A combination of UPLC separation with an FT-ICR-MS system (UPLC-FT-ICR-MS) is suitable for screening and identifying chemical components in complex matrices in terms of the advantages of rapid analysis, high mass accuracy, high resolution, and high detection sensitivity [13,14].
In this study, a rapid, sensitive, and systematic chemical identification and characterization method with UHPLC-FT-ICR-MS was applied to identify and characterize the chemical components of Lanqin oral liquid.A total of 441 compounds including alkaloids, flavonoids, terpenoids, organic acids, phenylpropanoids, and other types were detected, and their structures were tentatively identified based on the retention time, accurate molecular weight measurement, and characteristic mass fragment information with correlative reference standards or reference data.Results of this investigation lay a foundation for the quality control of Lanqin oral liquid; provide a reference and promising experimental data support for further study of the relationship between the effective substances and their pharmacology; illustrate the relationship between the complicated constituents and the therapeutic effects; and, thus, promote the development of Lanqin oral liquid.

Chemical Profiling of Lanqin Oral Liquid
As shown in Figure 1, the representative base peak intensity chromatograms (BPCs) in the positive and negative modes showed the whole profiling of chemical constituents in Lanqin oral liquid.Compounds were identified based on the extracted ion chromatograms, retention time, accurate precursor mass measurements, and diagnostic fragments data.The isomers of compounds with the same molecular formula but different chemical structures were distinguished by characteristic MS/MS fragments and retention time compared with the reference standards and the literature data.In total, 441 chemical constituents, including 134 flavonoids, 88 alkaloids, 108 terpenoids, 51 organic acids, 29 phenylpropanoids, and 31 other compounds in Lanqin oral liquid were unambiguously identified when reference standards were available or tentatively characterized when reference standards were not available by analyzing their MS/MS fragmentation patterns and retention behaviors in comparison with reference standards and the literature data [7][8][9][10][14][15][16][17][18][19][20][21][22][23][24][25][26][27].The details of the identified compounds are summarized in Tables 1-5.The precursor ion [M-H] − of compound F12 was at m/z 461.07229 and was observed at 8.22 min.The molecular formula was C21H18O12, as predicted by the precursor ion.The fragment ion at m/z 285.04039 was observed, indicating the elimination of a glucuronosyl unit in its structure.The compound was identified as scutellarin [9].Compound F31 was found at 9.72 min, and the molecular formula was C24H26O13, as established by the [M-H] − peak at m/z 521.12952.The characteristic fragments were m/z 359.07727, m/z 344.05343, and m/z 329.02992, corresponding to an excimer ion [M + H] + successively removing a fragment ion of glucose, one and two methyl groups.It was tentatively characterized as 5,2′,6′-trihydroxy-6,7,8-trimethoxyflavone-2′-O-glucoside [7].Compound F76 with a retention time of 13.97 min generated the precursor ion of [M + H] + at m/z 491.11836 in the negative ion mode, and the molecular formula was predicted as C23H22O12.The characteristic fragment were m/z 315.08567, m/z 300.06218, and m/z 285.03875, corresponding to an excimer ion [M + H] + successively removing a fragment ion of glucose, one and two methyl groups.It was tentatively characterized as 5,7-dihydroxy-6,8-dimethoxyflavone-7-O-glucuronide [7].The extracted ion chromatogram and MS/MS mass spectrum of 5,7-dihydroxy-6,8-dimethoxyflavone-7-O-glucuronide are shown in Figure 3.The cleavage at the glycosidic linkages was the main fragmentation pa ern of O-glycoside.

Characterization and Identification of Alkaloids
Alkaloids are a class of basic organic compounds containing nitrogen that exist in nature.A total of 88 alkaloids were tentatively identified in Lanqin oral liquid, mainly containing tetrahydroperoberberines such as phellodendrine, aporphines such as magnoflorine, benzylisoquinoline such as N-methylhigenamine 7-glucopyranoside, protoberberines such as palmatine, and other types such as indirubin.The chemical structures of the major alkaloid compounds are shown in Figure 5.

Characterization and Identification of Alkaloids
Alkaloids are a class of basic organic compounds containing nitrogen that exist in nature.A total of 88 alkaloids were tentatively identified in Lanqin oral liquid, mainly containing tetrahydroperoberberines such as phellodendrine, aporphines such as magnoflorine, benzylisoquinoline such as N-methylhigenamine 7-glucopyranoside, protoberberines such as palmatine, and other types such as indirubin.The chemical structures of the major alkaloid compounds are shown in Figure 5.The precursor ion of compound A28 at m/z 342.16909 was observed at 6.05 min.We speculated that the molecular formula was C20H24NO4.In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 192.10132, corresponding to fragment ion [M-C9H10O2] + .The compound was tentatively characterized as phellodendrine [9,10].The extracted ion chromatogram and MS/MS mass spectrum of phellodendrine are shown in Figure 6.The precursor ion of compound A28 at m/z 342.16909 was observed at 6.05 min.We speculated that the molecular formula was C 20 H 24 NO 4 .In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 192.10132, corresponding to fragment ion [M-C 9 H 10 O 2 ] + .The compound was tentatively characterized as phellodendrine [9,10].The extracted ion chromatogram and MS/MS mass spectrum of phellodendrine are shown in Figure 6.
The precursor ion of compound A34 at m/z 342.16988 was observed at 6.52 min.We speculated that the molecular formula was  The compound was tentatively characterized as magnoflorine [16].
The precursor ion of compound A29 at m/z 448.19537 was observed at 6.15 min.We speculated that the molecular formula was C23H29NO8.In the MS/MS spectrum, the characteristic fragment ions at m/z 286.14311 and m/z 255.10092 were detected, which corresponded to [M + H-glucose] + and [M + H-glucose-NH2CH3] + .The compound was tentatively characterized as N-methylhigenamine 7-glucopyranoside [10].The extracted ion chromatogram and MS/MS mass spectrum of N-methylhigenamine 7glucopyranoside are shown in Figure 7.According to the above analysis results, chemical identification of the compounds was performed based on an integrated consideration of accurate MS data, characteristic fragment ions, and retention time using a reference substance or the reference literature.A total of 441 compounds in Lanqin oral liquid were identified in this study.In the previous literature, a total of 170 compounds were identified or putatively characterized by ultra-high-performance liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry [9], and 175 compounds were identified or putatively characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in Lanqin oral liquid [10], while 441 compounds were identified or tentatively characterized in Lanqin oral liquid by UHPLC-FT-ICR-MS/MS.It was found that the applied UHPLC-FT-ICR-MS/MS method with high mass accuracy, good sensitivity, and high resolution was more sensitive for detecting and identifying the chemical compounds in Lanqin oral liquid.According to the above analysis results, chemical identification of the compounds was performed based on an integrated consideration of accurate MS data, characteristic fragment ions, and retention time using a reference substance or the reference literature.A total of 441 compounds in Lanqin oral liquid were identified in this study.In the previous literature, a total of 170 compounds were identified or putatively characterized by ultra-high-performance liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry [9], and 175 compounds were identified or putatively characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in Lanqin oral liquid [10], while 441 compounds were identified or tentatively characterized in Lanqin oral liquid by UHPLC-FT-ICR-MS/MS.It was found that the applied UHPLC-FT-ICR-MS/MS method with high mass accuracy, good sensitivity, and high resolution was more sensitive for detecting and identifying the chemical compounds in Lanqin oral liquid.

Sample Preparation
1 mL of Lanqin oral liquid was accurately measured and diluted with 1 mL of water.The mixture was vortexed for 3 min, and then the solution was prepared by diluting 5-fold with ultrapure water.The mixture was vortexed for 3 min, then centrifuged at 12,000 rpm for 10 min.The supernatant was collected and filtered through a 0.22 µm filter membrane before UHPLC-FT-ICR-MS/analysis.

Mass Spectrometry
Mass spectrometric detection was carried out on a Solarix 7.0T FT-ICR mass spectrometer (Bruker, Bremen, Germany) coupled with an electrospray ionization (ESI) interface.The full-scan MS data acquired in both positive and negative modes scanned from 100 to 1200 Da.The capillary voltage was 4500 V, the endplate offset was 500 V, dry gas flow was 8 L/min, dry gas temperature was 200 • C, and nebulizer gas pressure was 4 bar.The collision gas and nebulizing gas were high-purity argon (Ar) and high-purity nitrogen (N 2 ), respectively.Full-scan MS data were acquired over an m/z range of 100-1200 Da.And the collision energy were set from 10 to 40 eV for fragmentation information.Bruker Compass Hystar (version 4.1, Bruker Daltonics, Bremen, Germany) and Fourier Transform Mass Spectrometer Control (version 2.1, Bruker Daltonics, Bremen, Germany) were used for instrument control and data acquisition.Data Analysis Software (version 4.4, Bruker Daltonics, Bremen, Germany) was used for data analysis.

Conclusions
In our study, a rapid, sensitive, and specific analytical method using UHPLC-FT-ICR-MS/MS was successfully applied to separate and identify chemical constituents of Lanqin oral liquid.A total of 441 compounds were identified or tentatively characterized in Lanqin oral liquid, revealing that flavonoids, alkaloids, terpenoids, organic acids, and phenylpropanoids were the main components.The applied UHPLC-FT-ICR-MS method with the advantages of good separation, high sensitivity, high resolution, and high accuracy for the detection of chemical constituents in Lanqin oral liquid provided comprehensive chemical information on Lanqin oral liquid and could aid in quality control and an understanding of the pharmacodynamic material basis and thus promote the development of Lanqin oral liquid.
Author Contributions: Conceptualization, investigation, formal analysis, visualization, writingoriginal draft preparation, writing-review and editing, supervision, project administration, funding acquisition, T.L.; writing-review and editing, S.L.All authors have read and agreed to the published version of the manuscript.

2. 1 . 1 . 39 2. 1 . 1 .
Characterization and Identification of Flavonoids Flavonoid compounds are natural compounds containing the basic skeleton C6-C3-C6.A total of 134 flavonoids were tentatively identified in Lanqin oral liquid.The chemical structures of major flavonoid compounds are shown in Figure 2. Molecules 2023, 28, x FOR PEER REVIEW 23 of Characterization and Identification of Flavonoids Flavonoid compounds are natural compounds containing the basic skeleton C6-C3-C6.A total of 134 flavonoids were tentatively identified in Lanqin oral liquid.The chemical structures of major flavonoid compounds are shown in Figure 2.

Figure 2 .
Figure 2. Structures of major flavonoid compounds identified in Lanqin oral liquid.Glu A: glucuronic acid; Glc: glucose; Rha: rhamnose.The precursor ion [M-H] − of compound F12 was at m/z 461.07229 and was observed at 8.22 min.The molecular formula was C 21 H 18 O 12 , as predicted by the precursor ion.The

Figure 5 .
Figure 5. Structures of major alkaloid compounds identified in Lanqin oral liquid.

Figure 5 .
Figure 5. Structures of major alkaloid compounds identified in Lanqin oral liquid.

Figure 6 .
Figure 6.The extracted ion chromatogram (A) and MS/MS mass spectrum (B) of phellodendrine in positive mode.The precursor ion of compound A74 at m/z 352.15457 was observed at 11.97 min.We speculated that the molecular formula was C 21 H 22 NO 4 .In the MS/MS spectrum, the characteristic fragment ions at m/z 336.12260, m/z 320.12862, and m/z 308.12759 were detected, which corresponded to [M-CH 4 ] + , [M-CH 2 O] + , and [M-CH 4 -CO] + .The compound was chemically defined as palmatine [9,10].The precursor ion [M + H] + of compound A86 was at m/z 263.08145 and was observed at 23.49 min.The molecular formula was C 16 H 11 N 2 O 2 , as predicted by the precursor ion.In the MS/MS spectrum, the characteristic fragment ions at m/z 245.07024, m/z 235.08585, and m/z 219.09110 corresponded to [M + H-H 2 O] + , [M+H-CO] + , and [M + H-CO 2 ] + .The compound was chemically defined as indirubin.The precursor ion [M + H] + of compound A88 was at m/z 263.08146 and was observed at 21.69 min.The MS spectra of the two compounds were highly similar to each other.The compound was chemically defined as indigo [17].

Figure 7 .
Figure 7.The extracted ion chromatogram (A) and MS/MS mass spectrum (B) of Nmethylhigenamine 7-glucopyranoside in positive mode.The precursor ion of compound A74 at m/z 352.15457 was observed at 11.97 min.We speculated that the molecular formula was C21H22NO4.In the MS/MS spectrum, the characteristic fragment ions at m/z 336.12260, m/z 320.12862, and m/z 308.12759 were detected, which corresponded to [M-CH4] + , [M-CH2O] + , and [M-CH4-CO] + .The compound was chemically defined as palmatine[9,10].The precursor ion [M + H] + of compound A86 was at m/z 263.08145 and was observed at 23.49 min.The molecular formula was C16H11N2O2, as predicted by the precursor ion.In the MS/MS spectrum, the characteristic fragment ions at m/z 245.07024, m/z 235.08585, and m/z 219.09110 corresponded to [M + H-H2O] + , [M+H-CO] + , and [M + H-CO2] + .The compound was chemically defined as indirubin.The precursor ion [M + H] + of compound A88 was at m/z 263.08146 and was observed at 21.69 min.The MS spectra of the two compounds were highly similar to each other.The compound was chemically defined as indigo[17].

Figure 8 .
Figure 8. Structures of major terpenoid compounds identified in Lanqin oral liquid.The precursor ion [M-H] -of compound T5 was at m/z 373.11374 and was observed at 2.34 min, indicating the formation of C16H22O10.The characteristic fragments were m/z

Figure 8 .
Figure 8. Structures of major terpenoid compounds identified in Lanqin oral liquid.

Figure 10 .
Figure 10.The extracted ion chromatogram (A) and MS/MS mass spectrum (B) of obacunone in positive mode.

Figure 10 .
Figure 10.The extracted ion chromatogram (A) and MS/MS mass spectrum (B) of obacunone in positive mode.2.1.4.Characterization and Identification of Organic Acids Organic acids refer to some organic compounds with acidity, most commonly containing a carboxyl group (-COOH, 44 Da).A total of 51 organic acids were tentatively identified in Lanqin oral liquid.The chemical structures of major organic acid compounds are shown in Figure 11.Compound O1 with a retention time of 0.94 min generated the precursor ion of [M-H] − at m/z 191.05582 in negative ion mode, and the molecular formula was predicted as C 7 H 12 O 6 .In the MS/MS spectrum, the characteristic fragment ions produced were at m/z 173.04533 and m/z 127.03983, corresponding to fragment ions [M-H-H 2 O] − and [M-H-H 2 O-H 2 CO 2 ] − .It was tentatively characterized as quinic acid [8].Compound O17 with a retention time of 6.17 min generated the precursor ion of [M-H] − at m/z 179.03487 in negative ion mode, and the molecular formula was predicted as C 9 H 8 O 4 .In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 135.04512, corresponding to fragment ion [M-H-CO 2 ] − .It was identified as caffeic acid [8,18].The precursor ion [M-H] − of compound O33 was at m/z 559.14509 and was observed at 9.99 min, indicating the formation of C 27 H 28 O 13 .Daughter ions at m/z 379.10338 [M-H-C 9 H 8 O 4 ] − , m/z 364.07967 [M-H-C 10 H 11 O 4 ] − , m/z 353.08759 [M-H-C 11 H 10 O 4 ] − , m/z 335.07676 [M-H-C 11 H 12 O 5 ] − , m/z 223.06100 [M-H-C 16 H 16 O 8 ] − , m/z 205.05048 [M-H-C 16 H 18 O 9 ] − , m/z 191.05598 [M-

Figure 11 .
Figure 11.Structures of major organic acid compounds identified in Lanqin oral liquid.

Figure 11 .
Figure 11.Structures of major organic acid compounds identified in Lanqin oral liquid.

Figure 11 .
Figure 11.Structures of major organic acid compounds identified in Lanqin oral liquid.

Figure 12 .
Figure 12.The extracted ion chromatogram (A) and MS/MS mass spectrum (B) of 3-O-caffeoyl-4-Osinapoylquinic acid in negative mode.2.1.5.Characterization and Identification of Phenylpropanoids Phenylpropanoid compounds are natural compounds with one or more C6-C3 as the basic skeleton.A total of 29 phenylpropanoids were tentatively identified in Lanqin oral liquid.The chemical structures of major phenylpropanoid compounds are shown in Figure 13.The precursor ion [M + H] + of compound P1 was at m/z 341.08642 and was observed at 4.40 min.The molecular formula was predicted as C 15 H 16 O 9 .In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 179.03357 [M + H-glucose] + .The compound was tentatively characterized as esculin [23].The precursor ion [M + COOH] − of compound P2 was at m/z 387.12932 and was observed at 5.03 min, indicating the formation of C 16 H 22 O 8 .In the MS/MS spectrum, the characteristic ions at m/z 341.12388 [M-H] − and m/z 179.07122 [M-H-glucose] − were observed.The compound was tentatively characterized as coniferin [24].The precursor ion [M-H] − of compound P10 was at m/z 523.21827 and was observed at 7.40 min.The molecular formula was predicted as C 26 H 36 O 11 .In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 361.16525, which corresponded to [M-H-C 6 H 10 O 5 ] − .The compound was tentatively characterized as (−)-secoisolariciresinol 4-O-β-D-glucopyranoside [8].The extracted ion chromatogram and MS/MS mass spectrum of (−)-secoisolariciresinol 4-O-β-D-glucopyranoside are shown in Figure 14.

Figure 13 .
Figure 13.Structures of major phenylpropanoid compounds identified in Lanqin oral liquid.Figure 13.Structures of major phenylpropanoid compounds identified in Lanqin oral liquid.

Figure 13 .
Figure 13.Structures of major phenylpropanoid compounds identified in Lanqin oral liquid.Figure 13.Structures of major phenylpropanoid compounds identified in Lanqin oral liquid.

Funding:
This research was supported by the National Natural Science Foundation of China (grant number 82003935), Basic Scientific Research Project of Education Department of Liaoning Province (grant number LJKQZ20222409), Natural Science Foundation of Liaoning Province (grant number 2023-MS-326), and Startup Foundation for Doctors of Shenyang Medical College (grant number 20195077).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 4 .
Organic acids identified in Lanqin oral liquid by UHPLC-FT-ICR MS.

Table 5 .
Phenylpropanoids and other compounds identified in Lanqin oral liquid by UHPLC-FT-ICR MS.