• Users Online: 157
  • Print this page
  • Email this page

ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry and database-driven automatic peak annotation for the rapid profiling and characterization of the multicomponents from Stephaniae Tetrandrae radix (Fang-Ji)

1 State Key Laboratory of Component-based Chinese Medicine; Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin, China
2 State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China

Click here for correspondence address and email

Date of Submission16-Jul-2020
Date of Acceptance10-Aug-2020
Date of Web Publication15-Oct-2020


Quality control of traditional Chinese medicine (TCM) begins with elucidation of its chemical basis. The root of Stephania tetrandra, Stephaniae Tetrandrae radix (STR; Fang-Ji), has long been utilized as an antirheumatic, analgesic, and diuretic TCM. Powerful analytical strategies that would enable a multicomponent characterization of STR are still lacking. In the present study, we established a rapid, reliable, and enhanced profiling approach, using ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (MS) and automatic peak annotation facilitated by computational matching of an in-house library. This approach was used to characterize the multicomponents of STR. Good chromatographic separation was achieved within 17 min on a reversed-phase BEH C18 column eluted with acetonitrile/0.1% ammonium hydroxide in water, whereas data-independent high-definition MSE in the positive mode was applied to acquire the MS2 data using a Vion™ IM-QTOF instrument, which, in theory, could cover all the profiled precursor ions. To overcome the interference of three predominant peaks, a knockout strategy was utilized by automated valve switching. An in-house library of 163 compounds was established and incorporated into the UNIFI™ platform. By applying this method, we could identify or tentatively characterize 76 alkaloids from the methanolic extract of STR, including 14 aporphine-type, four morphine-type, 48 bisbenzylisoquinoline-type, seven tetrahydroprotoberberine-type, one protopine-type, one benzylisoquinoline-type, and one other. For each component, four-dimensional information, such as retention time, collision cross-section, high-accuracy MS1, and high-accuracy MS2 data, was utilized to achieve the systematic multicomponent characterization of STR.

Keywords: Ion mobility/quadrupole time-of-flight mass spectrometry, multicomponent characterization, Stephania tetrandra, ultra-high performance liquid chromatography

How to cite this URL:
Qian YX, Xie HM, Zuo TT, Li X, Hu Y, Wang HD, Gao XM, Yang WZ. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry and database-driven automatic peak annotation for the rapid profiling and characterization of the multicomponents from Stephaniae Tetrandrae radix (Fang-Ji). World J Tradit Chin Med [Epub ahead of print] [cited 2022 Aug 8]. Available from: https://www.wjtcm.net/preprintarticle.asp?id=298248

  Introduction Top

Traditional Chinese medicine (TCM) is increasingly recognized worldwide and has been shown to be particularly effective in treating some chronic and prevalent diseases, such as COVID-19.[1] However, the complexity and diversity of the chemical compositions in TCM, in particular, the co-existence of the primary and secondary metabolites along with a wide range of acid–base properties, molecular mass, polarity, and content, make it difficult to clarify the multicomponents.[2],[3] Quality control is a practical solution to ensure the efficacy and safety of TCM for clinical use, and the first step is the comprehensive elucidation of the chemical components.[4]

Hyphenated chromatography/mass spectrometry (MS), especially reversed-phase liquid chromatography coupled with high-resolution MS, has become the preferred analytical tool in comprehensive multicomponent characterization for TCM.[5] To deconvolute the chemical complexity of TCM, the data-dependent acquisition (DDA) method in an untargeted mode has been the first choice, as no preknowledge regarding the analytes is needed in this strategy.[6],[7],[8] However, in case of a complex matrix, targeted ingredients are subject to severe interference, resulting in very limited coverage. To overcome this shortcoming, an inclusion list containing targeted masses is set in DDA, by which the fragmentation information of the minor-targeted components can be acquired.[9],[10],[11] Moreover, the combination of DDA and subsequent exclusion list-driven DDA has been shown to be a powerful tool in profiling and characterizing the multi-type components in TCM.[12] On the other hand, with data-independent acquisition (DIA), we can record the fragmentation information of all precursor ions within a defined m/z range; however, spectral deconvolution is required to match the precursors and the product ions before data interpretation. Using DIA approaches, the precursor ions covering the whole scan range (such as MSE and all ion fragmentation)[13],[14] or a split, fixed-mass range (sequential window acquisition of all theoretical mass spectra)[15] can be automatedly delivered to the collision cell for MS/MS fragmentation. Recently, development of the fit-for-purpose DIA approaches has become a hot topic in the life sciences with a focus on data processing. A notable achievement in the field of TCM component analysis is the introduction of ion mobility MS (IM-MS), such as the SYNAPT High-Definition MS (HDMS) Series and Vion IMS/QTOF from Waters[16],[17] and 6560 IM/QTOF from Agilent.[18] IM-MS can provide one additional dimension of ion separation, which is orthogonal to MS, thus offering more reliable MS information in support of metabolite characterization. With respect to gas-phase ions, the collision cross-section (CCS) derived from IM separation is a stable parameter among different instruments and different laboratories, which allows for more reliable characterization and differentiation of metabolite isomers.[19],[20]

Stephania tetrandra Radix (STR; Fang-Ji) is a well-known TCM first recorded as medicine by Shen Nong Ben Cao Jing during the Han Dynasty.[21] Currently, only the roots of S. tetrandra S. Moore (Menispermaceae) are officially used as the botanical source for STR.[22] In phytochemical studies of S. tetrandra, researchers have documented alkaloids isolated from the roots and another five components (bioflavones and sterols) from the aerial part of the plant.[23] Diverse pharmacological properties, such as antimicrobial, antiparasitic, antibacterial, antifungal, antiviral, anti-inflammatory, anticancer, immunomodulatory, antifibrotic, antidiabetic, and antiplatelet, and effects on the central nervous system and cardiovascular system have been reported, and most of these activities are associated with the alkaloids found in this species. A number of studies have been carried out to rapidly identify and quantify the bioactive alkaloids and to differentiate easily confused species (e.g., Sinomenium acutum, Qing-Feng-Teng; Aristolochia fangchi, Guang-Fang-Ji; Cocculus orbiculatus, Mu-Fang-Ji).[24],[25],[26],[27],[28] For the chemical basis elucidation of STR, one issue is that those alkaloids with low content are difficult to characterize with conventional LC/MS.

To profile and characterize the multicomponents of S. tetrandra, we developed a rapid approach that integrates ultra-high performance liquid chromatography/IM-quadrupole time-of-flight MS (UHPLC/IM-QTOF-MS)-based HDMSE profiling and in-house library-driven automated peak annotation. HDMSE is a new powerful DIA approach that enables IM separation, and the bioinformatics platform UNIFI™ can achieve efficient peak annotation by searching against the commercial or in-house built database.[29],[30] Key parameters of chromatography-MS (e.g., stationary phase, capillary voltage, cone voltage, and ramp collision energy) were optimized to improve the overall performance of separation and detection. We established an in-house database, consisting of 163 components isolated from S. tetrandra, which was incorporated into the UNIFI software to trigger the automated annotation of the acquired positive-mode HDMSE data. In addition, a knockout strategy was applied to enhance the exposure of those minor alkaloids. With the results of this work, we have demonstrated a practical and powerful approach for efficient and comprehensive multicomponent characterization of TCM, which will be beneficial in quality control of TCM.

  Experimental Top

Chemicals and reagents

In the current work, a total of 20 compounds used as reference compounds [Figure 1] and [Table S1], including 14 alkaloids, two glycosides, two triterpenes, and two nitroxylic acids, were purchased either from Shanghai Standard Biotech Co., Ltd. (Shanghai, China) or from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China). Acetonitrile, methanol (Thermo Fisher, Fair Lawn, NJ, USA), and formic acid (Sigma-Aldrich, St. Louis, MO, USA) were of HPLC grade. Ultra-pure water was in-house prepared from a Milli-Q Integral 5 water purification system (Millipore, Bedford, MA, USA). The raw materials of S. tetrandra were collected from Jingdezhen (Jiangxi Province, China) in September 2019 and authenticated based on fingerprint analysis and Flora of China. The voucher specimens were deposited at the authors' laboratory in Tianjin University of TCM (Tianjin, China).
Figure 1: Chemical structures of 20 reference compounds used in the current work

Click here to view

Sample preparation

Pulverized powder of S. tetrandra (500 mg) was dispersed in 10 mL 70% aqueous methanol (v/v),[24],[27] vortexed for 2 min, and extracted in a water bath assisted with ultrasound at 40 kHz for 30 min. After being centrifuged at 14,000 rpm for 10 min, the supernatant was collected as the stock solution, which was subsequently diluted to reach a concentration of 25 mg/mL, with 70% methanol, as the test solution for LC/MS analysis.

Ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry

Chromatographic separation of the multicomponents from S. tetrandra by UHPLC was performed on an ACQUITY® UPLC I-CLASS system (Waters, Milford, MA, USA) equipped with an ACQUITY® UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) maintained at 35°C. A binary mobile phase, consisting of water containing 0.1% ammonia hydroxide (A) and acetonitrile (B), was run at a flow rate of 0.3 mL/min according to a gradient elution program: 0–2 min, 2%–20% B; 2–4 min, 20%–30% B; 4–7 min, 30%–55% B; 7–9 min, 55%–80% B; and 9–15 min, 80–90% B. A “purge–wash–purge” cycle was set on the autosampler; by using 10% CH3 CN in H2O (v/v) as the purge solvent and 50% CH3 CN in H2O as the wash solvent, we aimed to minimize the carry-over effect between injections.[29],[30]

High-resolution HDMSE data were recorded on a Vion™ IM-QTOF mass spectrometer equipped with an electrospray ionization (ESI) source in the positive mode (Waters). The source parameters were set as follows: capillary voltage, 2.5 kV; cone voltage, 60 V; source offset, 80 V; source temperature, 120°C; desolvation gas temperature, 500°C; desolvation gas flow (N2), 800 L/h; and cone gas flow (N2), 50 L/h. The mass analyzer scanned over a mass range of 50–650 Da under a low energy of 6 eV, whereas a high-energy ramp of 30–50 eV was set for HDMSE at 0.3 s per scan. Data calibration was performed using an external reference (LockSpray™) by constantly infusing leucine enkephalin solution (200 pg/μL; Sigma-Aldrich) at a flow rate of 10 μL/min, based on the ion m/z 556.2766 for the positive mode. The parameters were set at default values for the traveling wave IM separation. The calibration of CCS was conducted according to the manufacturer's guidelines using mixed calibrants.[31] Data were acquired using the UNIFI™ software (Waters).

A knockout strategy was utilized to enhance the exposure of minor alkaloids from the methanolic extract of S. tetrandra by using the valve switching function of the Vion IM-QTOF instrument. Based on the base peak chromatography, three retention time periods corresponding to the elution of three major peaks (8.3–8.5 min, 8.85–9.2 min, and 9.4–10.1 min) were switched to the waste. The concentrations of STR samples used for preknockout and knockout experiments were 25 mg/mL (injection volume: 3 μL) and 50 mg/mL (injection volume: 5 μL), respectively.

An in-house library for Stephania tetrandra

To facilitate the rapid and more reliable characterization of the multicomponents from STR, a library was built in-house by summarizing the literature with respect to the phytochemistry studies of S. tetrandra up to 2020.[10],[29],[30] This in-house library of S. tetrandra included the trivial name, molecular formula, and chemical structure of each compound. First, the structure information was input into an Excel file according to a required format. We then drew the structure of each compound using ChemDraw Professional, and the drawing was subsequently saved as an .mol file. The .mol file was named with the trivial name consistent with the Excel file. Finally, the Excel file and all structure files were incorporated into the UNIFI™ software.

Date processing

Automated annotation of the HDMSE data was achieved using the UNIFI™ software by searching the incorporated library. The uncorrected HDMSE data, in continuum format, were initially corrected using the UNIFI™ software (Waters) with reference to m/z 556.2766 in the positive mode. Programmed peak annotation was efficiently accomplished and generated a table of the primary identified components. Key parameters of the UNIFI are depicted as follows. Find 4D peaks: MS ion intensity threshold, 500.0 counts; MS-MS ion intensity threshold, 200.0 counts. Target by mass: target match tolerance, 10.0 ppm; screen on all isotopes in a candidate, generate predicted fragments from structure, and look for in-source fragments were enabled; fragment match tolerance, 10.0 ppm. Adducts: Positive adducts including +H,-e. Lock mass: combine width, three scans; mass window, 0.5 m/z; reference mass, 556.2766; reference charge, +1.

  Results Top

Optimization and development of an ultra-high performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight-high-definition mass spectrometryE approach dedicated to separating and characterizing the multicomponents of Stephania tetrandra

To provide an efficient method for profiling and characterizing more chemical components from the methanolic extract of S. tetrandra using the advanced Vion IM/QTOF high-resolution LC/MS platform, we aimed to develop a UHPLC/IM-QTOF-HDMSE approach by optimizing the key chromatography and MS parameters.

The stationary phase is a crucial factor that affects chromatography performance.[5] Targeting the total extract of S. tetrandra, six chromatographic columns with different bond technologies were compared: BEH C18, CSH Phenyl-Hexyl, HSS C18 SB (Waters), Zorbax Eclipse Plus C18, Zorbax Extend C18 (Agilent), and Kinetex 1.7 μ EVO C18 (Phenomenex). Three of these, CSH Phenyl-Hexyl, HSS C18 SB, and Zorbax Eclipse Plus C18, were eluted by the mobile phase containing formic acid (FA; 0.1%) as an additive in the aqueous phase, while ammonia hydroxide (AH; 0.05%) was used for the elution of the other three columns. We present, in [Figure 2], the base peak chromatograms (BPCs) of S. tetrandra obtained from these six candidate columns and the number of ions that were resolved. Comparatively, BEH C18, Kinetex EVO C18, and Zorbax Eclipse Plus C18 could better resolve the main components in STR. In terms of the separated ions, BEH C18 was superior to all the other five columns and was thus our final selection. In addition, a minor adjustment was made on the gradient program to achieve the satisfactory performance of chromatographic separation of the total extract of S. tetrandra within 17 min, indicating a high-throughput approach.
Figure 2: Selection of the stationary phase for the reversed-phase ultra-high performance liquid chromatography separation of the multicomponents from Stephania tetrandra. The left and the right columns separately showing the base peak chromatograms obtained on different sub-2 μm particles packed chromatographic columns, eluted using the base and acid additives in the water phase

Click here to view

The conditions of the Vion IM-QTOF instrument, as the final detector, determine the performance of the established approach in the multicomponent characterization of S. tetrandra, and therefore, three parameters, namely capillary voltage, cone voltage, and the collision energy ramp of HDMSE in the positive mode, were optimized. The purpose of capillary voltage optimization (varying from 1.0 to 3.0 kV) was to obtain a high response for S. tetrandra components in full-scan mode, by evaluating the peak area variations of five alkaloids (tetrandrine and neferine: Bisbenzylisoquinoline alkaloid; protopine and tetrahydroberberine: Protoberberine alkaloid; corypalmine: Tetrahydroprotoberberine alkaloid). As shown in [Figure 3], it was clear that, with the increase of capillary voltage, the ion response of neferine, protopine, and tetrahydroberberine positively correlated with the variation of spray voltage in the test range, whereas the best ionization for tetrandrine and corypalmine was observed when 2.5 kV of spray voltage was set, in contrast to a sharp decrease at 3.0 kV. Taken together, these results indicated that 2.5 kV was the best choice for setting the spray voltage. Cone voltage is also an important factor that can affect the ion response and even induce in-source fragmentation. In this section, three different levels of cone voltage (20 V, 40 V, and 60 V) were set to determine their influence on the ionization of five alkaloid compounds, as described above. Different variation trends were observed, and the setting of 60 V was found to be the best selection for protopine and tetrahydroberberine and acceptable for the other three alkaloid components. As a result, we set cone voltage at 60 V for detecting the multicomponents of S. tetrandra.
Figure 3: Optimization of two key source parameters (spray voltage and cone voltage) of the Vion with ion mobility/quadrupole time-of-flight mass spectrometer, by evaluating the peak area variations of five representative alkaloid compounds (n = 3)

Click here to view

To enable adequate fragmentation and generate a sufficient number of product ions (facilitating the structural elucidation of the multicomponents), we optimized the collision energy ramp of HDMSE on the Vion IM-QTOF mass spectrometer. Based on the in-house compiled database, the main components contained in the roots of S. tetrandra are alkaloids, generally with a molecular mass <700 Da. The collision-induced dissociation-MS2 (CID-MS2) behavior of four alkaloid compounds – tetrandrine, fangchinoline, cepharanthine, and corydalmine – was investigated by setting four levels of collision energy ramps: 10–30 eV, 20–40 eV, 30–50 eV, and 40–60 eV [Figure S1]. Evidently, these four reference compounds, upon CID, were able to yield more balanced, diversified fragments in the low mass region of spectra with increasing of the energy ramp. Although the collision energy ramp of 40–60 eV, compared with 30–50 eV, could give more low-mass fragments, the abundance of these product ions decreased significantly. Considering that the minor components may be over fragmented at higher collision energy, we selected the energy ramp of 30–50 eV in setting the HDMSE acquisition method.

Application of a knockout strategy to enhance the exposure of minor components

One issue that can hinder the exposure of minor compounds in TCM is the presence of predominant components. In the case of STR, the BPC in the positive mode only gave three major peaks, corresponding to several major alkaloids [Figure 4]. Direct analysis of the obtained data could only characterize fewer than 30 alkaloids.[24] To improve the performance with respect to alkaloid constituents, we applied additional minor components characterized from S. tetrandra using a knockout strategy (similar to what was described in a previous report[29]) that is based on the automatic valve switching of the Vion IM-QTOF instrument. Initially, 25 mg/mL of the total extract was injected with 3 μL to record the positive HDMSE data. Subsequently, a volume of 5 μL of the total extract at 50 mg/mL was injected onto the column, and the eluates from three time periods were automatedly delivered to the waste, as shown in [Figure 4]. Therefore, the sample amount loaded onto the column in the knockout experiment was 3.33-fold that in the preknockout experiment. An intuitive result, benefited from these efforts, was the remarkable enrichment for those minor components. The primary characterization results, by applying UNIFI to process the HDMSE data obtained in the preknockout experiment and knockout experiment, were compared. As a result, the number of “identified components” and “unknown components” in the preknockout experiment was 228 and 8,906, respectively, and increased to 450 (1.97-fold) and 10,491 (1.18-fold) in addition to those contained in the knockout peaks. These results seem to demonstrate the superiority of the knockout strategy in profiling and characterizing minor TCM components. Moreover, the knockout process was completed in the automated mode; thus, the technique can be easily performed on LC/MS instruments that have a switching valve.
Figure 4: Illustration for the knockout strategy for the enhanced exposure of minor components from Stephania tetrandra

Click here to view

Systematic characterization of the multicomponents in Stephania tetrandra based on an intelligent workflow facilitated by the UNIFI platform

To identify the multicomponents of S. tetrandra, the HDMSE data obtained in both the preknockout experiment and knockout experiment were analyzed following an intelligent workflow facilitated by the UNIFI platform. Efficient peak annotation could be completed by matching the experimental data with the predicted fragments, generating the lists of “identified components” and “unknown components.” Various pieces of information – such as the observed m/z, formula, observed tR, mass error, adducts, and CCS values – can be offered by the software. The final identification list was obtained by re-analyzing and confirming the results listed as the “identified components.” In particular, the identity of each component given by the UNIFI software was carefully analyzed by referring to the in-house library.

In total, 76 alkaloids were identified or tentatively characterized from S. tetrandra, including 14 of the aporphine-type, four morphine-type, 48 bisbenzylisoquinoline-type, seven tetrahydroprotoberberine-type, one protopine-type, one benzylisoquinoline-type, and one other type. The details of these alkaloids are given in [Table S2]. Notably, no nonalkaloid components were characterized from the roots of this medicinal plant, which is consistent with the review literature.[23] On CID in the positive ESI mode, the corresponding characteristic product ions (CPIs) were observed for each of the five subtypes of alkaloids: M/z 297.11/296.10 and 265.09 for the aporphine-type, m/z 181.06 and 153.07 for the morphine-type, m/z 192.20/190.10, 365.15/381.18, and 178.08 for the bisbenzylisoquinoline-/benzylisoquinoline-type, and m/z 206.08, 190.10, 177.07, 151.07, and 149.08 for the tetrahydroprotoberberine-/protoberberine-type. All of these ions can be obtained due to the loss of (CH3)2 NH and CH3 OH, cleavage of the piperidine ring and loss of amine, elimination of the benzene ring for single isoquinoline moiety, and the retro-Diels-Alder (RDA) fragmentation occurring to C-ring and direct B-ring, which are diagnostic in identifying alkaloids and even distinguishing isomers. In addition, for bisbenzylisoquinoline- and benzylisoquinoline-type alkaloids, the characteristic neutral losses of 162 and 132 Da were detected.[24],[32],[33],[34],[35]

Characterization of aporphine-type alkaloids

Due to the structure of conjugated rings in alkaloids of this type, it is difficult to obtain the product ions by cleavage of the rings.[24] The fragment ions [M+H–45Da]+, [M+H–31Da]+, and [M+H–17Da]+ were always observed, depending on the number of N-methyl groups. Further fragmentation pathways involved the loss of NH2 CH3, NHC2H6, CH2O, OCH3, CH3 OH, and CO groups.[36] The MS/MS spectrum of #3 [tR2.34 min; C20H23 NO4; [Table S2] produced CPIs, such as m/z 297.11 [M–(CH3)2 NH]+, 282.08 [M–(CH3)2 NH–CH3]+, 265.08 [M–(CH3)2 NH–CH3 OH]+, and 237.06 [M–(CH3)2 NH–CH3 OH–CO]+, as a result of the loss of A-ring amine and the subsequent loss of the side chains attached at C-1, C-2, C-10, and C-11 positions. These fragmentation and retention times were consistent with that of reference magnoflorine; thus, compound #3 was identified as magnoflorine. Compound #9 (tR3.17 min; C20H19 NO5), #10 (tR3.25 min; C20H21 NO4), #11 (tR3.33 min; C19H18 NO4+), #12 (tR3.46 min; C20H24 NO4+), and #19 (tR4.71 min; C18H19 NO4), were identified as N-formylnornantenine, nantenine, dehydrophanostenin/dehydrostesakine, and N-methyllaurotetanine, respectively. Under the current conditions, except the rich protonated precursor ions, on the basis of aporphine structure, the loss of N-methyl groups (NH(CH3)2, NH2 CH3, and NH3 of A-ring usually occurred easily, engendering the fragments of m/z 308.1291 ([M+H–NH2 CHO–H]+), 308.1290 ([M+H–NH2 CH3]+), 309.1004 ([M+H–NH2 CH3]+), 312.1236 ([M+H–NH2 CH3]+), and 282.1130 ([M+H–CH3 OH]+), respectively. Based on the product ions caused by the dissociation of different groups, we were able to infer the substitution of CHO, CH3, CH3 and CH3 of the amino groups for compounds #9, #10, #11, and #12, respectively. By comparison with compound #3, the structures of #11, #12, and #19 were all deduced to contain OH adjacent to OCH3. The above-mentioned fragments for compounds #9, #10, #11, #12, and #19 were consistent with those reported in the literature, which helped characterize their structures.[34],[35],[36],[37]

Characterization of bisbenzylisoquinoline alkaloids

The bisbenzylisoquinoline-type alkaloids occupy a large proportion – up to 61.84% (47 in total) – of the STR components characterized in the current work. As an obvious feature, they have larger molecular mass (>500 Da) than the other subtypes,[37] and their structures can be regarded as the dimerization of two benzyltetrahydroisoquinoline units via two ether bonds. One of the main CID-MS2 features of bisbenzylisoquinoline alkaloids is the generation of (M+H–242Da)+ ion caused by the preferred cleavage of the β carbon–carbon bonds to the nitrogen and the two aromatic systems, followed by the O-demethylation of an aromatic methoxyl group.[31] The elemental compositions and accurate mass of CPIs for all bisbenzylisoquinoline-type alkaloids observed in this work are summarized in [Table S2]. By comparing with the reference compounds, we identified compounds #56 (tR8.90 min; C37H40N2O6), #62 (tR9.57 min; C38H42N2O6), and #63 (tR9.72 min; C37H38N2O6) as fangchinoline, tetrandrine, and cepharanthine, respectively. For compound #56, as exhibited in [Figure 5], the main CPI at m/z 367.1657 [M+H–C15H15O2–CH3]+ was produced by dibenzylic cleavage of the alkaloid skeleton and loss of CH3 and m/z 192.1022 [M+H–C26H27 NO5]+ was produced by fragmentation of the dimeric isoquinoline fragment. The characteristic fragment ions m/z 580.2699 ([M+H–CH3 CH2N]+), 381.1816 ([M+H–C15H15O2–CH3]+), and 174.0920 ([M+H–C15H15O2–CH3–C11H14 NO2–H2O]+) for compound #62 and m/z 564.2397 ([M+H–NH2 CH3]+), 365.1506 ([M+H–C15H15O2]+), and 227.1078 ([M+H–C22H25N2O4]+) for compound #63 were diagnostic in their identification.[35],[36],[38] The abundant [M + H]+ ions of compounds #55 (an unknown compound with tR8.69 min; C36H36N2O6) at m/z 593.2540 can be observed, indicating a mass of 14 Da higher than compound #63. We tentatively identified compound #55 as 2-norcepharanthine or its isomer,[38] depending on the CPIs at m/z 351.1348 and 192.1025 [Figure 5]. The MS/MS spectrum of compound #43 (tR7.98 min; C35H34N2O5) displayed CPIs at m/z 213.0914, 178.0789, 353.1503, and 564.2385. By comparing its MS/MS spectrum to that of fangchinoline (compound #56), we tentatively inferred that the OH group on the E ring was superseded by OCH3 − and amine substitution on the A ring. Thus, compound #43 was tentatively identified as trilobamine, previously reported in Cenarrhenes trilobus.[26]
Figure 5: Annotation of the CID-MS2 spectra and proposed fragmentation pathways of representative bisbenzylisoquinoline (#56 and #55) alkaloids characterized from Stephania tetrandra

Click here to view

Characterization of tetrahydroprotoberberine alkaloids

Tetrahydroprotoberberine-type alkaloids in the positive ion mode can easily undergo RDA cleavage on the C ring and direct elimination of the B ring.[35],[36],[37] Furthermore, because of the existence of N-methyl and the vicinal hydrogen atom, the isoquinoline structure can form a closed oxygen ring after the loss of CH3 OH. Compounds #4 (tR2.36 min; C20H23 NO4) and #41 (tR7.92 min; C21H25 NO4) were identified as corydalmine and rotundine with the aid of reference compounds comparison.[35],[38] By comparison with compounds #4 and #41, the unknown compounds #13 (tR3.46 min; C20H23 NO4) and #18 (tR4.62 min; C21H25 NO4) were assumed to be the isomers corydalmine and rotundine, respectively, owing to their shared CPIs. The loss of CH3 and H (M–16Da)+ and CH3 OH (M–32Da)+ can be regarded as the valid information to determine the number and position of substituents. Compounds #6 (tR2.69 min; C19H21 NO4) and #16 (tR4.18 min; C19H21 NO4), with the (M+H)+ ion observed at m/z 328 lower than #4 by 14 Da, were primarily characterized with OCH3 replaced by OH. Based on the CPIs at m/z 178.0825 (a RDA fragmentation product ion), 151.0554 (RDA and B ring cleavage), and 163.0641 (a RDA fragment), we could deduce that compounds #6 and #16 possess neighboring hydroxyl and methoxyl groups on rings A and D [Figure 6]. These fragments matched well with those reported for stepholidine,[25] and thereby, compounds #6 and #16 could be identified as stepholidine or its isomer [Table S2].
Figure 6: Annotation of the CID-MS2 spectra and proposed fragmentation pathways of representative tetrahydroprotoberberine (#4 and #6) alkaloids characterized from Stephania tetrandra

Click here to view

Characterization of morphine alkaloids

Morphine alkaloids are another subclass of major bioactive ingredients in STR,[34] for which the common fragmentation pathways involve cleavage of the piperidine ring and loss of an amine, such as the CPIs of (M+H–CH2 CHNHCH3)+, (M+H–CH2 NCH3)+, and (M+H–NH2 CH3)+.[38] By analyzing the positive CID-MS2 data, four compounds of this subclass (#5, #7, #20, and #28) were tentatively characterized from STR. Based on the CPI at m/z 272.1307 ([M+H–CH2 CHNHCH3]+), compound #7 (tR2.93 min; C19H24 NO4+) was identified as a morphine alkaloid with a methyl on the nitrogen, whereas the fragment of m/z 223.0768 ([M+H–CH2 CHNHCH3–H2O–CH3 OH]+) could indicate CH3 OH substitution on the A ring, which were generally consistent with sinomenine as reported earlier.[24],[35] Compound #5 (tR2.46 min; C18H23 NO6) yielded a protonated precursor ion at m/z 350.1608, suggesting an additional 20 Da compared with compound #7. Considering the predominant product ions at m/z 309.0989 ([M+H–CH2 NCH3]+) and 279.0941 ([M+H–CH3 OH]+), we tentatively characterized this compound as tazopsine by comparing with the data reported in the literature.[39],[40]

  Discussion Top

UHPLC, by applying sub-2 μm particles packed column, can greatly elevate the analytical efficiency with less organic solvents consumed;[5] however, it has rarely been applied in the chemical analysis of STR. In the current work, by comparison among six different chromatographic columns, a rapid UHPLC approach capable of good resolution of S. tetrandra components was established on a BEH C18 column, enabling injection analysis within 17 min, which was much faster than the HPLC approaches.[24],[32] On the other hand, DIA is a powerful strategy that can maximally cover the precursor ions, and HDMSE is a new DIA approach with IM separation enabled.[29],[30] UNIFI is a powerful bioinformatic platform that can facilitate the automated annotation of high-resolution MS data obtained on Waters QTOF instruments (such as MSE, HDMSE, and DDA), by searching against commercial or in-house built database. Therefore, the integral strategy we used in this work renders a potent approach to efficient and comprehensive multicomponent characterization of TCM and has the potential for interlab popularization.

  Conclusions Top

We established a UHPLC/IM-QTOF-MS-based profiling approach, which efficiently separated the multicomponents of STR (<17 min). The alkaloids contained received good response under the optimal MS condition, and the use of a collision energy ramp 30–50 eV in the positive-mode HDMSE yielded rich fragments beneficial to the structural elucidation. Application of a knockout strategy could enhance the exposure of those minor components that are difficult to be characterized by conventional injection pattern. By the automated peak annotation using UNIFI, a total of 76 alkaloids were identified or tentatively characterized from STR. Overall, this work presents a practical and powerful approach to comprehensive profiling and characterization of the multicomponents from TCM.


This study was supported by the National Key Research and Development Program of China (Grant No. 2018YFC1704500) and the National Natural Science Foundation of China (Grant No. 81872996).

Financial support and sponsorship

This study was supported by the National Key Research and Development Program of China (Grant No. 2018YFC1704500) and the National Natural Science Foundation of China (Grant No. 81872996).

Conflicts of interest

There are no conflicts of interest.

  References Top

Luo L, Jiang J, Wang C, Fitzgerald M, Hu W, Zhou Y, et al. Analysis on herbal medicines utilized for treatment of COVID-19. Acta Pharm Sin B 2020;10:1192-204.  Back to cited text no. 1
Jia-Xi L, Chun-Xia Z, Ying H, Meng-Han Z, Ya-Nan W, Yue-Xin Q, et al. Application of multiple chemical and biological approaches for quality assessment of Carthamus tinctorius L. (safflower) by determining both the primary and secondary metabolites. Phytomedicine 2019;58:152826.  Back to cited text no. 2
Yan Y, Song Q, Chen X, Li J, Li P, Wang Y, et al. Simultaneous determination of components with wide polarity and content ranges in Cistanche tubulosa using serially coupled reverse phase-hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 2017;1501:39-50.  Back to cited text no. 3
Wang M, Franz G. The role of the European Pharmacopoeia (Ph Eur) in quality control of traditional Chinese herbal medicine in European member states. World J Tradit Chin Med 2015;1:5-15.  Back to cited text no. 4
  [Full text]  
Jin H, Liu Y, Guo Z, Wang J, Zhang X, Wang C, et al. Recent development in liquid chromatography stationary phases for separation of Traditional Chinese Medicine components. J Pharm Biomed Anal 2016;130:336-46.  Back to cited text no. 5
Liu X, Fan X, Wang X, Liu R, Meng C, Wang C. Structural characterization and screening of chemical markers of flavonoids in Lysimachiae herba and Desmodii styracifolii herba by ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry based metabolomics approach. J Pharm Biomed Anal 2019;171:52-64.  Back to cited text no. 6
Cao JL, Ma LJ, Wang SP, Deng Y, Wang YT, Li P, et al. Comprehensively qualitative and quantitative analysis of ginsenosides in Panax notoginseng leaves by online two-dimensional liquid chromatography coupled to hybrid linear ion trap Orbitrap mass spectrometry with deeply optimized dilution and modulation system. Anal Chim Acta 2019;1079:237-51.  Back to cited text no. 7
Jia L, Fu L, Wang X, Yang W, Wang H, Zuo T, et al. Systematic profiling of the multicomponents and authentication of Erzhi Pill by UHPLC/Q-Orbitrap-MS oriented rapid polarity-switching data-dependent acquisition and selective monitoring of the chemical markers deduced from fingerprint analysis. Molecules 2018;23:3143.  Back to cited text no. 8
Wang H, Zhang C, Zuo T, Li W, Jia L, Wang X, et al. In-depth profiling, characterization, and comparison of the ginsenosides among three different parts (the root, stem leaf, and flower bud) of Panax quinquefolius L. by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Anal Bioanal Chem 2019;411:7817-29.  Back to cited text no. 9
Fu LL, Ding H, Han LF, Jia L, Yang WZ, Zhang CX, et al. Simultaneously targeted and untargeted multicomponent characterization of Erzhi Pill by offline two-dimensional liquid chromatography/quadrupole-Orbitrap mass spectrometry. J Chromatogr A 2019;1584:87-96.  Back to cited text no. 10
Li M, Wang X, Han L, Jia L, Liu E, Li Z, et al. Integration of multicomponent characterization, untargeted metabolomics and mass spectrometry imaging to unveil the holistic chemical transformations and key markers associated with wine steaming of Ligustri Lucidi Fructus. J Chromatogr A 2020;1624:461228.  Back to cited text no. 11
Pan H, Zhou H, Miao S, Cao J, Liu J, Lan L, et al. An integrated approach for global profiling of multi-type constituents: Comprehensive chemical characterization of Lonicerae japonicae Flos as a case study. J Chromatogr A 2020;1613:460674.  Back to cited text no. 12
Qiu S, Yang WZ, Yao CL, Qiu ZD, Shi XJ, Zhang JX, et al. Nontargeted metabolomic analysis and “commercial-homophyletic” comparison-induced biomarkers verification for the systematic chemical differentiation of five different parts of Panax ginseng. J Chromatogr A 2016;1453:78-87.  Back to cited text no. 13
Naz S, Gallart-Ayala H, Reinke SN, Mathon C, Blankley R, Chaleckis R, et al. Development of a liquid chromatography-high resolution mass spectrometry metabolomics method with high specificity for metabolite identification using all ion fragmentation acquisition. Anal Chem 2017;89:7933-42.  Back to cited text no. 14
Xia YG, Gong FQ, Guo XD, Song Y, Li CX, Liang J, et al. Rapid screening and characterization of triterpene saponins in Acanthopanax senticosus leaves via untargeted MSAll and SWATH techniques on a quadrupole time of flight mass spectrometry. J Pharm Biomed Anal 2019;170:68-82.  Back to cited text no. 15
Zhang H, Jiang JM, Zheng D, Yuan M, Wang ZY, Zhang HM, et al. A multidimensional analytical approach based on time-decoupled online comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of ginsenosides from white and red ginsengs. J Pharm Biomed Anal 2019;163:24-33.  Back to cited text no. 16
Zhang CX, Wang XY, Lin ZZ, Wang HD, Qian YX, Li WW, et al. Highly selective monitoring of in-source fragmentation sapogenin product ions in positive mode enabling group-target ginsenosides profiling and simultaneous identification of seven Panax herbal medicines. J Chromatogr A 2020;1618:460850.  Back to cited text no. 17
Li MN, Wang HY, Wang R, Li CR, Shen BQ, Gao W, et al. A modified data filtering strategy for targeted characterization of polymers in complex matrixes using drift tube ion mobility-mass spectrometry: Application to analysis of procyanidins in the grape seed extracts. Food Chem 2020;321:126693.  Back to cited text no. 18
Zhou Z, Shen X, Tu J, Zhu ZJ. Large-scale prediction of collision cross-section values for metabolites in ion mobility-mass spectrometry. Anal Chem 2016;88:11084-91.  Back to cited text no. 19
Tu J, Zhou ZW, Li TZ, Zhu ZJ. The emerging role of ion mobility-mass spectrometry in lipidomics to facilitate lipid separation and identification. Trends Anal Chem 2019;116:332-9.  Back to cited text no. 20
Huang HP, Peng HS, Wang DL, Huang P. Textual research on historical evolution of Tradition Chinese medicine of Fang Ji. J Chin Med Mater 2015;38:1533-5.  Back to cited text no. 21
Chinese Pharmacopoeia Commission. The Pharmacopoeia (2015 edition) of the People's Republic of China (First section). Beijing: The Medicine Science and Technology Press of China; 2015. p. 149.  Back to cited text no. 22
Jiang Y, Liu M, Liu H, Liu S. A critical review: Traditional uses, phytochemistry, pharmacology and toxicology of Stephania tetrandra S. Moore (Fen Fang Ji). Phytochem Rev 2020;19:449-89.  Back to cited text no. 23
Sim HJ, Yoon SH, Kim MS, Kim B, Park HM, Hong J. Identification of alkaloid constituents from Fangchi species using pH control liquid-liquid extraction and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2015;29:837-54.  Back to cited text no. 24
Chen J, Zhao Q, Si D, Nie A, Wang Y, Deng Z, et al. Comprehensive profiling of Stephania tetrandra (Fangji) by stepwise DFI and NL-dependent structure annotation algorithm-based UHPLC-Q-TOF-MS and direct authentication by LMJ-HRMS. J Pharm Biomed Anal 2020;185:113225.  Back to cited text no. 25
Sim HJ, Kim JH, Lee KR, Hong J. Simultaneous determination of structurally diverse compounds in different Fangchi species by UHPLC-DAD and UHPLC-ESI-MS/MS. Molecules 2013;18:5235-50.  Back to cited text no. 26
Chuang CC, Su CH, Huang WY, Sheu S. Classification of Fangchi Radix samples by multivariate analysis. J Food Drug Anal 2008;18:48-56.  Back to cited text no. 27
Tankeu S, Vermaak I, Chen W, Sandasi M, Viljoen A. Differentiation between two “fang ji” herbal medicines, Stephania tetrandra and the nephrotoxic Aristolochia fangchi, using hyperspectral imaging. Phytochemistry 2016;122:213-22.  Back to cited text no. 28
Zuo T, Qian Y, Zhang C, Wei Y, Wang X, Wang H, et al. Data-dependent acquisition and database-driven efficient peak annotation for the comprehensive profiling and characterization of the multicomponents from compound Xueshuantong Capsule by UHPLC/IM-QTOF-MS. Molecules 2019;24:3431.  Back to cited text no. 29
Zhang C, Zuo T, Wang X, Wang H, Hu Y, Li Z, et al. Integration of data-dependent acquisition (DDA) and data-independent high-definition MSE (HDMSE) for the comprehensive profiling and characterization of multicomponents from Panax japonicus by UHPLC/IM-QTOF-MS. Molecules 2019;24:2708.  Back to cited text no. 30
Paglia G, Angel P, Williams JP, Richardson K, Olivos HJ, Thompson JW, et al. Ion mobility-derived collision cross section as an additional measure for lipid fingerprinting and identification. Anal Chem 2015;87:1137-44.  Back to cited text no. 31
Huang WY, Su CH, Sheu SJ. Separation and identification of the constituents in Fangchi Radix of different origins. J Food Drug Anal 2006;14:357-67.  Back to cited text no. 32
Jeong EK, Lee SY, Yu SM, Park NH, Lee HS, Yim YH, et al. Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2012;26:1661-74.  Back to cited text no. 33
Qing ZX, Cheng P, Liu XB, Liu YS, Zeng JG. Systematic identification of alkaloids in Macleaya microcarpa fruits by liquid chromatography tandem mass spectrometry combined with the isoquinoline alkaloids biosynthetic pathway. J Pharm Biomed Anal 2015;103:26-34.  Back to cited text no. 34
Xiao J, Song N, Lu T, Pan Y, Song J, Chen G, et al. Rapid characterization of TCM Qianjinteng by UPLC-QTOF-MS and its application in the evaluation of three species of Stephania. J Pharm Biomed Anal 2018;156:284-96.  Back to cited text no. 35
Wu YC. New Research and development on the formosan annonaceous plants. Stud Nat Prod Chem 2006;33:957-1023.  Back to cited text no. 36
Wang R, Zhou J, Shi G, Liu Y, Yu D. Aporphine and phenanthrene alkaloids with antioxidant activity from the roots of Stephania tetrandra. Fitoterapia 2020;143:104551.  Back to cited text no. 37
Shangguan Y, He J, Kang Y, Wang Y, Yang P, Guo J, et al. Structural characterisation of alkaloids in leaves and roots of Stephania kwangsiensis by LC-QTOF-MS. Phytochem Anal 2018;29:101-11.  Back to cited text no. 38
Li XN, Yan HX, Sha N, Hua HM, Wu LJ, Guo DA. Isolation and identification of alkaloids from the root of Stephania tetrandra. J Shenyang Pharm Univ 2009;26:430-3.  Back to cited text no. 39
Li Z, Fan X, Cai M, Yang Z, Ying J, Qiang G, et al. Pharmacokinetics of fangchinoline and tetrandrine in rats. China J Chin Mater Med 2009;34:3110-3.  Back to cited text no. 40

Correspondence Address:
Wen-Zhi Yang,
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/wjtcm.wjtcm_56_20


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


   Ahead Of Print
 Article in PDF
     Search Pubmed for
    -  Qian YX
    -  Xie HM
    -  Zuo TT
    -  Li X
    -  Hu Y
    -  Wang HD
    -  Gao XM
    -  Yang WZ

Article Figures

 Article Access Statistics
    PDF Downloaded203    
    Cited by others 4    

Recommend this journal