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Article

Qualitative and Quantitative Analysis of Banhasasim-Tang Using UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS

by
Seol Jang
and
Youn-Hwan Hwang
*
KM Convergence Research Division, Korea Institute of Oriental Medicine, Yuseong-daero 1672, Yuseong-gu, Daejeon 34054, Republic of Korea
*
Author to whom correspondence should be addressed.
Processes 2024, 12(8), 1563; https://doi.org/10.3390/pr12081563
Submission received: 24 June 2024 / Revised: 15 July 2024 / Accepted: 22 July 2024 / Published: 26 July 2024
(This article belongs to the Section Pharmaceutical Processes)
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Abstract

:
In this study, we analyzed the components of Banhasasim-tang (BHSST), a traditional prescription used to treat gastrointestinal disorders. Qualitative analysis was performed using ultra-high-performance liquid chromatography/quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS), and a total of 42 compounds were accurately and unambiguously identified by comparison with the corresponding reference standards and mass spectrometry (MS) results. A simultaneous quantitative method for analysis of the 22 identified compounds was established using UHPLC coupled with a triple-quadrupole tandem mass spectrometer (UHPLC-TQ-MS/MS) in multiple reaction monitoring mode. The established method was validated in terms of linearity (R2, 0.9990–0.9996), recovery (RSD, 0.37–3.60%), and intraday/interday precision (RSD, 0.35–8.90%). This method was applied to three batches of BHSST samples and revealed that two flavonoids from S. baicalensis, baicalin and wagonoside, were the most abundant components. This study presents the first comprehensive method for simultaneously identifying and quantifying the components of BHSST. Our method offers a valuable tool for the quality control of BHSST and serves as a foundational reference for further research on similar preparations.

1. Introduction

Traditional oriental medicine has long been used for the prevention and treatment of various diseases, exhibiting promising anti-inflammatory, antioxidant, and anti-cancer effects [1,2,3]. Banhasasim-tang (BHSST, the Banxia Xiexin decoction in traditional Chinese medicine) is a typical prescription that originated from the Treatise on Febrile Diseases, commonly used to treat gastrointestinal disorders [4]. BHSST consists of seven herbal extracts (Pinellia ternata Breitenbach, Zizyphus jujuba Miller var. inermis Rehder, Scutellaria baicalensis Georgi, Zingiber officinale Roscoe, Panax ginseng C. A. Meyer, Glycyrrhiza uralensis Fischer, and Coptis chinensis Franchet) and has antioxidant and anti-inflammatory properties [5]. BHSST is effective in the treatment of ulcerative colitis and has been shown to reduce the symptoms of chronic gastritis [1,6]. Additionally, BHSST is potentially effective against gastric cancer, as it may inhibit the invasive behavior and metastasis of gastric cancer cells [7]. Some studies have examined the efficacy and mechanism of action of BHSST in treating depression [8], while clinical trials have revealed its efficacy against ulcerative colitis and irritable bowel syndrome without exhibiting side effects [9,10]. A study on gastric ulcers and functional dyspepsia revealed that such symptoms were reduced via BHSST treatment, with BHSST being effective in clinical trials involving patients with hepatocellular carcinoma [11,12,13].
Previous studies have shown that BHSST contains several active components, including flavonoids, alkaloids, and saponins [14]. According to the composition of each herbal extract, it contains flavonoids such as baicalin, baicalein, and wogonin from S. baicalensis and liquiritin and isoliquiritin from G. uralensis [15,16]. In addition, alkaloids such as berberine and coptisine, which are the main components of C. japonica, and ginsenosides Rg1 and Rb1, which are saponin compounds of P. ginseng, are included in BHSST [17,18].
Prescriptions such as BHSST which combine extracts from several herbs exhibit complex chemical properties that can affect each other; therefore, each component must be comprehensively identified and quantified [10,19]. Previously, representative components of BHSST have been analyzed via high-performance liquid chromatography (HPLC) analysis, with ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) methods being developed for the simultaneous analysis of multiple components for the quality control of BHSST [20,21,22]. However, such studies have only included the representative components of BHSST, and few studies have reported the analysis of the chemical profile of BHSST and the simultaneous quantification of the identified components. Therefore, an efficient method must be developed to analyze the components of BHSST qualitatively and quantitatively.
UHPLC-MS/MS is a useful method offering efficient component separation and accurate analysis of their structural characteristics for the identification of unknown components [23]. Analytical techniques using Orbitrap mass spectrometry are employed for screening unknown components and for comprehensively characterizing the components [19]. Additionally, UHPLC coupled with a triple-quadrupole tandem mass spectrometer (UHPLC-TQ-MS/MS) is a fast and sensitive quantitative method widely used to provide precise and accurate detection and analysis of complex compounds [24]. In particular, multiple reaction monitoring (MRM) mode serves as a highly sensitive tool to selectively detect and quantify compounds based on ion transition [25].
In this study, we established an analytical method to characterize and identify the components of Banhasasim-tang (BHSST) using ultra-high-performance liquid chromatography/quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS). A total of 42 components were identified via comparison with retention times and MS results of reference standards. Subsequently, a simultaneous quantitative analytical method using UHPLC-TQ-MS/MS and MRM mode was established to quantify the contents of these identified compounds in BHSST. This established method was then applied to investigate the content distribution of 22 components in actual BHSST extracts.

2. Materials and Methods

2.1. Materials and Reagents

BHSST comprises seven herbal extracts, all of which were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Republic of Korea). Each raw herbal extract was deposited in the KM Convergence Research Division of the Korea Institute of Oriental Medicine; the herbal composition and specimen numbers are as follows: P. ternate (TDC-09); Z. jujuba (TDC-11); S. baicalensis (TDC-10); Z. officinale (TDC-03); P. ginseng (TDC-13); G. uralensis (TDC-12); and C. chinensis (TDC-14). Twenty-two analytical standards were purchased from ChemFaces (Wuhan, China), exhibiting a purity of 98% or higher. Warfarin (IS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Analytical grade methanol, acetonitrile, formic acid, and water were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

2.2. BHSST Sample Preparation

Seven herbal medicines, P. ternate (60 g), Z. jujuba (60 g), S. baicalensis (30 g), Z. officinale (30 g), P. ginseng (30 g), G. uralensis (30 g), and C. chinensis (10 g) were mixed, followed by the addition of a 10-fold mass amount of distilled water. The mixture was refluxed at 100 °C for 3 h, followed by filtration and concentration under reduced pressure using a rotary evaporator. The concentrated water extract was freeze-dried, yielding a powder (28.6%). To prepare the sample for analysis, the freeze-dried BHSST powder was dissolved in methanol (20 mg/mL) and then extracted in an ultrasonic bath for 30 min. The extracted solution was centrifuged at 12,500 rpm for 15 min before analysis.

2.3. Preparation of Standard Solutions and Control Samples

Precisely measured reference standards were dissolved in methanol to prepare stock solutions of target compounds. A mixed solution containing the 22 analytes was prepared and progressively diluted with methanol to obtain a mixed standard working solution with an appropriate concentration range. The prepared solution was used to create a calibration curve for each compound. Calibration samples of the 22 analytes of BHSST were prepared at concentrations within the range of the standard curve. Warfarin was added as IS in all samples at a consistent concentration, after dissolution in methanol. Quality control (QC) samples were prepared at three concentration levels (high, medium, and low) for method validation following the same preparation procedure as the calibration samples.

2.4. Analytical Conditions of UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS

A Dionex UltiMate 3000 system equipped with a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used for qualitative analysis. The 22 compounds identified in BHSST were quantitatively analyzed via an UHPLC-TQ-MS/MS method performed on an Agilent 1290 Infinity II system interfaced with an Agilent 6495C triple-quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The detailed operational conditions for the LC analysis methods used in qualitative and quantitative analyses, as well as for each piece of respective equipment, are summarized in Table 1. Additionally, quantitative analysis of the 22 compounds was performed in the dynamic MRM mode using the positive or negative modes. For each analyte, the MS/MS detection parameters, such as MRM transitions and collision energies, were optimized by setting them to the most appropriate values, and the results are shown in Table 2.

2.5. Validation of the Quantitative Method

A mixed-standard solution containing 22 analytes and IS was serially diluted to different concentrations for linearity evaluation: 0.02–6.25 ng/mL (rutin, scutellarin, jatrorrhizine, isoliquiritin, wogonin, oroxylin A, and 6-shogaol), 0.05–12.50 ng/mL (acteoside, coptisine, isoliquiritin apioside, ginsenoside Re, baicalein, and 6-gingerol), 0.10–25.00 ng/mL (liquiritin, ginsenoside Rg1, palmatine, and ginsenoside Rb1), 0.20–50.00 ng/mL (liquiritin apioside and berberine), 0.39–100.00 ng/mL (glycyrrhizic acid), and 1.56–400.00 ng/mL (baicalin and wogonoside). Calibration curves were constructed for each analyte using the ratio of the peak area of the analyte to the IS versus the analyte concentration. The lower limit of quantification, which represents the sensitivity of the analytical method, was determined as the lowest measured concentration at which accuracy and precision were exhibited. To recover the analyte, various concentrations (high, middle, and low levels) of each standard were added to a sample solution. The recovery was evaluated by comparing the difference between the known added content and the detected concentration for each analyte. The precision of the analytical method was assessed by analyzing intra- and interday variations: Intraday precision was measured by preparing six QC samples mixed with standard solutions of analytes in one day. Interday precision was measured by preparing one sample and analyzing it for three consecutive days following the same method. Both analyses were performed using QC samples at three concentrations, and the precision of all analytes was assessed using the relative standard deviation of the repeatedly measured values.

3. Results

3.1. Characterization of Chemical Compounds in BHSST via UHPLC-Q-Orbitrap-MS

The BHSST extract was analyzed using UHPLC-Q-Orbitrap-MS based on a qualitative analysis method in which 42 chemical compounds constituting BHSST were identified and characterized. The base peak chromatograms of BHSST were obtained in both positive and negative ion modes, as shown in Figure 1. Among the 42 detected compounds, 39 compounds were unambiguously identified by comparing the retention times and MS results of the corresponding reference standards. The other three compounds were tentatively characterized by comparing their respective MS data with previous reports, and the retention times and MS results of the identified compounds are summarized in Table 3 and Figure S1.

3.2. Quantitation of Chemical Compounds in BHSST via UHPLC-TQ-MS/MS

Based on the results of the compounds identified through qualitative analysis of BHSST, quantitative analysis was performed on the 22 major components of each herbal medicine. These chemical compounds are distributed in the constituent herbal medicines of BHSST as follows: 6-shogaol from P. ternate; rutin from Z. jujuba; scutellarin, acteoside, baicalin, wogonoside, baicalein, wogonin, and oroxylin A from S. baicalensis; 6-gingerol from Z. officinale; ginsenoside Re, ginsenoside Rg1, and ginsenoside Rb1 from P. ginseng; liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid from G. uralensis; and jatrorrhizine, coptisine, palmatine, and berberine from C. chinensis. The 22 compounds detected in BHSST were quantified via UHPLC-TQ-MS/MS analysis, with all compounds being detected simultaneously within 20 min. All compounds were analyzed individually in both positive and negative ion modes to establish the optimal MS/MS conditions. Consequently, scutellarin, jatrorrhizine, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, wogonin, oroxylin A, 6-gingerol, and 6-shogaol exhibited higher sensitivity in the positive ion mode. Conversely, the remaining compounds exhibited higher sensitivity in the negative ion mode.
In this study, the dynamic MRM mode was applied to perform sensitive and accurate quantitative analysis. The best MRM transition for quantitative analysis was determined by identifying the appropriate precursor ion for each analyte and selecting the most specific product ion for MRM analysis. In addition, to optimize the quantitative analysis method, the fragmentor was set to 166 V, and the collision energy was determined to be the optimal value for each compound in the range from 10 to 40 V. The optimized analytical conditions of the MRM parameters for all analytes, including the internal standard (IS), are summarized in Table 2. The obtained chromatogram is presented in Figure 2.

3.3. Method Validation

The BHSST quantitative analytical method was validated for its linearity, recovery, precision, and accuracy. The detailed data of the linear relationship and the lower limit of quantitation (LLOQ) for the 22 analytes are presented in Table 4. Calibration curves for all compounds showed good linearity with correlation coefficients (R2) greater than 0.9990 within specific concentration ranges, indicating an excellent coefficient of determination. Additionally, the LLOQ obtained from UHPLC-TQ-MS/MS analysis was <1.56 ng/mL. Therefore, this method is sufficiently suitable for the quantitative analysis of the 22 target compounds.
The recovery of the method was determined by adding a mixed standard solution containing three different concentrations of the target compound to a sample of known concentration. The average recovery of the 22 analytes ranged from 91.32% to 110.54%, and the relative standard deviation (RSD) values were within 3.60%. The obtained results are summarized in Table 5.
The precision of the method was evaluated via the intraday and interday relative standard deviations (RSD). Intraday precision was evaluated by analyzing six replicates of samples containing the target compounds at three concentrations. Interday precision was evaluated following the same method; however, analyses were performed on three consecutive days. The results of the analyses are presented in Table 6; the RSD for precision was 0.35–6.45% for intraday and 0.46–8.90% for interday evaluation. Additionally, the accuracy range for these analytes was 90.74–112.25% for intraday and 91.33–107.36% for interday analyses.
The above results demonstrate that the dynamic MRM mode method of UHPLC-TQ-MS/MS established for quantitative analysis is accurate and precise, suggesting that it can be applied effectively and efficiently to quantify the compounds present in BHSST.

3.4. Quantitative Application to BHSST Samples

The content of BHSST was analyzed using the validated UHPLC-TQ-MS/MS MRM analytical method, with all compounds being detected simultaneously. Each sample was spiked with IS and injected six times to obtain the mean and standard deviation values. The contents of the three batches of BHSST samples were not significantly different from each other; the detailed data are presented in Table 7.

4. Discussion

This study performed a qualitative analysis to identify the components of BHSST, a representative prescription used to treat gastrointestinal diseases, and a total of 42 compounds were identified. The detected compounds were reported to be constituents of 6-shogaol from P. ternate [26]; rutin from Z. jujuba [27]; scutellarin, acteoside, baicalin, wogonoside, baicalein, wogonin, and oroxylin A from S. baicalensis [15]; 6-gingerol from Z. officinale [28]; ginsenosides Re, Rg, and Rb1 from P. ginseng [18]; liquiritin, isoliquiritin, and glycyrrhizic acid from G. uralensis [16]; and coptisine, jatrorrhizine, palmatine, and berberine from C. chinensis [17]. Among these identified compounds, 24 components were in agreement with compound information reported in the literature related to BHSST [22,29,30].
Previously reported studies were either qualitative analyses that identified constituents of BHSST or quantitative analyses of representative constituents [22,29]. Accordingly, in this study, in order to efficiently analyze the components of BHSST, we established not only a chemical profile analysis but also a quantitative analysis method for some of the identified components. A total of 22 compounds were simultaneously quantitatively analyzed through MRM analysis using UPLC-TQ-MS/MS, and the optimal precursor and product ion for quantitative analysis were set for each component. Rutin exhibited a precursor ion at m/z 609.1, whereas its product ion was detected at m/z 300.1, owing to the loss of the rhamnosyl-glucoside moiety [31]. The two isomeric compounds, liquiritin and isoliquiritin, lost their glucose residue (162 Da) from the precursor ion to form a product ion at m/z 255. The two isomeric compounds, liquiritin apioside and isoliquiritin apioside, also exhibited a product ion of [M – H – xyl – glc]− at m/z 255 [32,33]. Regarding glycyrrhizic acid, its product ion was formed at m/z 351.0 in the negative ion mode, which resulted from the loss of the triterpenic aglycone [34]. Baicalin, wogonoside, and scutellarin generated product ions at m/z 271.0, 285.1, and 287.0, respectively, by neutral loss of the glucuronic acid moiety from the precursor ion [M + H]+ [35,36]. In the case of baicalein, the trihydroxyphenyl moiety at m/z 123.0 was the product ion formed from the precursor ion [M + H]+. Regarding wogonin and oroxylin A, the same product ion was observed at m/z 270.0 via the loss of CH3 [37]. The product ion of acteoside detected at m/z 161.0 was generated via the successive loss of H2O from the CA moiety (C20H26O11) lost from the precursor ion [38,39]. Jatrorrhizine, palmatine, and berberine exhibited precursor ions [M]+ at 338.1, 352.1, and 336.1, respectively. Furthermore, the product ions for all three compounds showed characteristic m/z values of 322.1, 336.1, and 320.1, respectively, owing to [M – CH3 – H]+ [40]. Coptisine exhibited a precursor ion at m/z 320.0 and a product ion at m/z 292.1, which resulted in the observed loss of the CO group [41]. Ginsenoside Re, Rg1, and Rb1 formed product ions via the loss of HCOOH from the precursor ion. Therefore, ginsenoside Re and Rg1 exhibited [M – H]− ions at m/z 945.6 and 799.5, respectively, whereas Rb1 showed [M – 2H]2− ions at m/z 553.3 [42,43]. 6-Gingerol was formed as a product ion of [M + H–H2O–C6H12O]+ at m/z 177.1 owing to the loss and rearrangement of the neutral alkyl moiety from the precursor ion [44]. Additionally, 6-shogaol cleaved the bond between the carbonyl moiety and the aromatic ring from the precursor ion [M + H]+ formed at m/z 277.1, resulting in the formation of a product ion at m/z 137.0 [45].
The contents of 22 compounds in BHSST samples were measured using this established quantitative analysis method. Among the measured compounds, two flavonoids, baicalin and wogonoside, exhibited the highest content. These two flavonoids are the main components of S. baicalensis, and they have been previously reported as the most abundant components in BHSST [21]. Additionally, liquiritin apioside and glycyrrhizic acid, derived from G. uralensis, were detected in larger amounts compared to other components. Our results indicate that the components of BHSST were accurately quantified in actual samples analyzed via the validated UHPLC-TQ-MS/MS method. The established quantitative analytical method can therefore be applied in BHSST component analysis and quality control.

5. Conclusions

In this study, an UHPLC-Q-Orbitrap-MS method was applied for the qualitative evaluation of BHSST, and 42 compounds were identified. The characterized components were quantified using the MRM mode of UHPLC-TQ-MS/MS, and a rapid method capable of simultaneously quantifying the 22 compounds was established. The established method was validated for its linearity, precision, and accuracy and was proven effective for the analysis of real samples. Our method can provide comprehensive information on the composition of BHSST by revealing its chemical profile, while enabling simultaneous quantitative evaluation. In addition, it can provide reliable data for quality control of BHSST and related preparations. These results can contribute to evaluating the pharmaceutical effects of each component of BHSST in in vivo research or identifying the mechanisms of interaction. In the future, based on this study, there is a need to evaluate the actual clinical application potential of BHSST in various disease models, and further research is needed to systematically analyze the interaction network between each compound to better understand various pharmaceutical effects and mechanisms.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr12081563/s1, Figure S1: The MS2 spectrum of 42 chemical compounds in BHSST via UHPLC-Q-Orbitrap-MS.

Author Contributions

Conceptualization, Y.-H.H.; investigation, S.J.; writing—original draft preparation, S.J.; writing—review and editing, S.J. and Y.-H.H.; funding acquisition, Y.-H.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Korea Institute of Oriental Medicine (grant number KSN2213020).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Processes 12 01563 g001
Figure 1. Base peak ion chromatograms of the BHSST extract in positive (A) and negative (B) ion modes obtained via UHPLC-Q-Orbitrap-MS. The ID numbers of the various types of phytochemicals are listed in Table 1.
Figure 1. Base peak ion chromatograms of the BHSST extract in positive (A) and negative (B) ion modes obtained via UHPLC-Q-Orbitrap-MS. The ID numbers of the various types of phytochemicals are listed in Table 1.
Processes 12 01563 g001
Processes 12 01563 g002
Figure 2. UHPLC-TQ-MS/MS chromatograms of the mixed standard solution (A) and BHSST extract (B) with their MRM modes. The ID numbers of the chemical compounds are presented in Table 2.
Figure 2. UHPLC-TQ-MS/MS chromatograms of the mixed standard solution (A) and BHSST extract (B) with their MRM modes. The ID numbers of the chemical compounds are presented in Table 2.
Processes 12 01563 g002
Table 1. Analysis conditions of UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS.
Table 1. Analysis conditions of UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS.
UHPLCQ-Orbitrap-MSTQ-MS/MS
ParameterConditionParameterConditionParameterCondition
ColumnAcquity BEH C18 column
(100 × 2.1 mm, 1.7 µm)		Ion modePositive/
Negative		Ion modePositive/
Negative
Column temp.40 °CIon sourceESIIon sourceESI
Injection volume3.0 µLScan modeFull MS-ddMS2Capillary voltage3500 V (pos)
Sample temp.4 °CScan range100–1500 m/z3000 V (neg)
Mobile phase A0.1% Formic acid in waterCapillary temp.320 °CGas temp.130 °C
Mobile phase BAcetonitrileSpray voltage3.8 kVNebulizer gas25 psi
Flow rate0.25 mL/minAUX gas10 auDrying gas flow11 L/min
Gradient
program		Time (min)B (%)Sheath gas40 auNozzle voltage500 V (pos)
03Collision energy25 eV1500 V (neg)
13MS resolution70,000SoftwareMassHunter v.10.1
215MS/MS resolution17,500
1350SoftwareXcalibur v.3.0
20100Tracefinder v. 3.2
23100
23.53
27.53
Table 2. Optimized parameters for MRM of analytes and internal standards for UHPLC-TQ-MS/MS analysis.
Table 2. Optimized parameters for MRM of analytes and internal standards for UHPLC-TQ-MS/MS analysis.
No.Compound NameRt
(min)		Precursor
Ion
(m/z)		Product
Ion
(m/z)		Fragmentor
(V)		Collision
Energy (V)		Cell
Accelerator (V)		Polarity
9Rutin5.23609.1300.1166404Negative
10Liquiritin apioside5.48549.2255.1166344Negative
11Scutellarin5.58463.0287.0166224Positive
12Liquiritin5.59417.2255.1166184Negative
13Acteoside5.64623.2161.0166404Negative
16Jatrorrhizine6.74338.1322.1166304Positive
19Coptisine6.82320.0292.1166304Positive
20Isoliquiritin apioside7.05549.1255.1166304Negative
21Isoliquiritin7.37417.0255.1166184Negative
22Baicalin7.58447.1271.0166224Positive
23Ginsenoside Re7.52991.6945.6166264Negative
24Ginsenoside Rg17.52845.5799.5166264Negative
26Palmatine7.69352.1336.1166344Positive
27Berberine7.81336.1320.1166344Positive
29Wogonoside9.08461.1285.1166224Positive
31Ginsenoside Rb110.32599.3553.3166144Negative
32Baicalein10.60271.0123.0166344Positive
35Glycyrrhizic acid11.86821.4351.0166404Negative
36Wogonin12.67285.1270.0166264Positive
38Oroxylin A13.22285.0270.0166264Positive
396-Gingerol13.24277.1177.1166104Positive
416-Shogaol16.50277.1137.0166224Positive
IS1Warfarin13.64309.0163.0166144Positive
IS2Warfarin13.64307.0250.0166224Negative
Table 3. Characterization of chemical compounds in BHSST via UHPLC-Q-Orbitrap-MS.
Table 3. Characterization of chemical compounds in BHSST via UHPLC-Q-Orbitrap-MS.
No.Rt
(min)		IdentificationFormulaAdductMeasured
Mass (m/z)		Error
(ppm)		MS2 Fragment
(m/z)
11.26Maltose *C12H22O11[M − H]−341.1088−0.305889.0228, 71.0122, 59.0122
22.24Leucine *C6H13NO2[M + H]+132.10211.181286.0972
33.80PhenylalanineC9H11NO2[M + H]+166.08630.3551120.0811
45.09Darendoside AC19H28O11[M − H]−431.1556−0.5575125.0229, 101.0228, 57.0329
55.51Vicenin-2 *C27H30O15[M − H]−593.15140.4016473.1086, 353.0664
65.86Magnoflorine *C20H24NO4[M]+342.1698−0.4374342.1700, 297.1122
75.98Schaftoside *C26H28O14[M − H]−563.1406−0.0089443.0982, 383.0774, 353.0665
86.09Daidzin *C21H20O9[M + H]+417.1177−0.6549255.0659
96.54Rutin *C27H30O16[M − H]−609.14620.2138300.0272, 223.0607
106.77Liquiritin apioside *C26H30O13[M − H]−549.1613−0.0803255.0660
116.89Scutellarin *C21H18O12[M + H]+463.0869−0.4015287.0553
126.91Liquiritin *C21H22O9[M − H]−417.11910.0734255.0660
137.00Acteoside *C29H36O15[M − H]−623.1981−0.1298161.0233
147.37Isoacteoside *C29H36O15[M − H]−623.1981−0.0319161.0233
157.70Acanthoside B *C28H36O13[M − H]−579.20840.1105417.1552, 181.0496
168.10Jatrorrhizine *C20H20NO4[M]+338.1384−0.9265338.1385, 336.1230
178.17Scutellarin methylester *C22H20O12[M − H]−475.0880−0.3300299.0559, 113.0228
188.19Epiberberine *C20H18NO4[M]+336.1228−0.5853336.1230
198.24Coptisine *C19H14NO4[M]+320.0916−0.3631320.0917
208.36Isoliquiritin apiosideC26H30O13[M − H]−549.1613−0.0803255.0660
218.68Isoliquiritin *C21H22O9[M − H]−417.1190−0.2924255.0659
228.89Baicalin *C21H18O11[M + H]+447.0919−0.6063271.0600
238.91Ginsenoside Re *C48H82O18[M + HCO2]−991.5478−0.534945.5434, 637.4317, 113.0229, 101.0228
248.96Ginsenoside Rg1 *C42H72O14[M + HCO2]−845.4902−0.2799113.0229, 101.0228, 71.0121
259.02Ononin *C22H22O9[M + H]+431.1336−0.0546269.0807
269.21Palmatine *C21H22NO4[M]+352.1541−0.7873352.1543
279.26Berberine *C20H18NO4[M]+336.1229−0.4038336.1230
289.32Liquiritigenin *C15H12O4[M − H]−255.0662−0.2229255.0652, 135.0073, 119.0487
2910.41Wogonoside *C22H20O11[M + H]+461.1076−0.4435285.0757
3011.46Ginsenoside Rf *C42H72O14[M + HCO2]−845.49050.0810113.0229, 101.0228, 71.0121
3111.69Ginsenoside Rb1 *C54H92O23[M + HCO2]−1153.6008−0.26871107.5956, 179.0551, 101.0228
3211.90Baicalein *C15H10O5[M + H]+271.06030.6353271.0599
3312.02Ginsenoside Rc *C53H90O22[M + HCO2]−1123.5902−0.32931077.5838, 149.0442, 89.0227
3412.56Formononetin *C16H12O4[M + H]+269.08090.3442269.0808
3513.15Glycyrrhizic acid *C42H62O16[M − H]−821.39650.0431351.0574, 193.0341
3614.01Wogonin *C16H12O5[M + H]+285.0757−0.1259285.0757
3714.13Chrysin *C15H10O4[M − H]−253.0505−0.4274253.0504
3814.53Oroxylin A *C16H12O5[M + H]+285.0757−0.2329285.0757
3914.626-Gingerol *C17H26O4[M]+294.18301.3355150.0677, 137.0598
4017.01Glabridin *C20H20O4[M − H]−323.12900.2701323.1287, 135.0438
4117.756-Shogaol *C17H24O3[M + H]+277.1798−0.1477137.0598
4218.24Hederagenin *C30H48O4[M − H]−471.3478−0.3631471.3475
* Compared with a reference standard.
Table 4. Linear regression data and LLOQ of the 22 analytes in BHSST for quantitative analysis method validation.
Table 4. Linear regression data and LLOQ of the 22 analytes in BHSST for quantitative analysis method validation.
No.Compound NameRegression EquationR2Linear Range
(ng/mL)		LLOQ
(ng/mL)
9Rutiny = 0.0891x − 0.0003930.99920.02–6.250.02
10Liquiritin apiosidey = 0.1956x − 0.0074610.99940.20–50.000.20
11Scutellariny = 0.0107x + 0.0000350.99930.02–6.250.02
12Liquiritiny = 0.3732x − 0.0060840.99940.10–25.000.10
13Acteosidey = 0.1284x − 0.0025490.99920.05–12.500.05
16Jatrorrhiziney = 0.5496x − 0.0043750.99930.02–6.250.02
19Coptisiney = 0.2028x − 0.0042680.99910.05–12.500.05
20Isoliquiritin apiosidey = 0.2073x − 0.0015450.99940.05–12.500.05
21Isoliquiritiny = 0.3112x − 0.0015850.99940.02–6.250.02
22Baicaliny = 0.0106x + 0.0011080.99931.56–400.001.56
23Ginsenoside Rey = 0.2309x − 0.0020480.99920.05–12.500.05
24Ginsenoside Rg1y = 0.0690x − 0.0017110.99920.10–25.000.10
26Palmatiney = 0.5096x − 0.0216450.99910.10–25.000.10
27Berberiney = 0.3658x − 0.0375720.99900.20–50.000.20
29Wogonosidey = 0.0123x − 0.0051800.99901.56–400.001.56
31Ginsenoside Rb1y = 0.2998x − 0.0067130.99930.10–25.000.10
32Baicaleiny = 0.0120x − 0.0000400.99900.05–12.500.05
35Glycyrrhizic acidy = 0.0805x − 0.0073550.99910.39–100.000.39
36Wogoniny = 0.6316x − 0.0015520.99920.02–6.250.02
38Oroxylin Ay = 0.1985x − 0.0013330.99960.02–6.250.02
396-Gingeroly = 0.0621x − 0.0007170.99930.05–12.500.05
416-Shogaoly = 0.1483x − 0.0005040.99920.02–6.250.02
Table 5. Recovery test of the 22 analytes in BHSST for quantitative analysis method validation.
Table 5. Recovery test of the 22 analytes in BHSST for quantitative analysis method validation.
No.Compound NameRecovery (%)
Low LevelMedium LevelHigh Level
Added
(ng/mL)		MeanRSD
(%)		Added
(ng/mL)		MeanRSD
(%)		Added
(ng/mL)		MeanRSD
(%)
9Rutin0.8194.883.431.33103.132.342.3799.931.73
10Liquiritin apioside17.0894.910.7721.2591.811.1929.5896.201.03
11Scutellarin1.0193.392.801.53106.491.072.57102.513.40
12Liquiritin4.6597.381.436.7398.460.9110.90103.770.53
13Acteoside2.2492.362.213.2899.122.255.36103.523.60
16Jatrorrhizine1.42100.081.251.94100.270.902.98100.132.18
19Coptisine2.6696.041.173.7095.690.845.7992.260.37
20Isoliquiritin apioside2.6895.281.333.7296.942.045.81107.332.18
21Isoliquiritin0.9696.121.281.48104.952.302.52106.231.59
22Baicalin97.1394.941.00130.46102.590.80197.13101.220.72
23Ginsenoside Re2.6695.331.943.70100.322.055.78103.620.74
24Ginsenoside Rg15.17100.631.117.25100.860.8611.42101.431.46
26Palmatine4.5597.663.026.6397.310.6210.8097.192.77
27Berberine14.5694.831.1318.7297.630.9327.06105.440.95
29Wogonoside114.8692.540.64148.1996.341.28214.8699.211.28
31Ginsenoside Rb14.9998.381.407.07104.241.2711.24110.540.65
32Baicalein2.5097.193.563.55108.860.445.63102.401.43
35Glycyrrhizic acid23.0793.111.4631.4199.031.8948.07101.140.52
36Wogonin1.0292.330.961.54101.541.562.58105.800.78
38Oroxylin A1.1293.051.701.6495.021.102.6891.321.00
396-Gingerol2.7994.451.063.8391.901.435.9197.501.17
416-Shogaol0.8294.581.381.34100.262.122.3899.370.62
Table 6. Precision and accuracy of the 22 analytes in BHSST for quantitative analysis method validation.
Table 6. Precision and accuracy of the 22 analytes in BHSST for quantitative analysis method validation.
No.Compound NameConcentration
(ng/mL)		Precision (RSD, %)Accuracy (%)
IntradayInterdayIntradayInterday
9Rutin4.171.805.53110.26104.14
1.041.502.64102.9399.99
0.261.202.7194.0194.63
10Liquiritin apioside33.331.585.97110.77103.67
8.330.824.24105.55100.93
2.081.291.07102.83103.91
11Scutellarin4.171.538.00112.25104.66
1.042.821.32101.3799.85
0.263.194.0891.3595.04
12Liquiritin16.671.125.27109.75103.53
4.170.503.64104.67100.47
1.041.393.73100.93103.91
13Acteoside8.331.072.51108.43107.36
2.081.041.45103.12104.74
0.521.048.0893.47101.33
16Jatrorrhizine4.170.823.59102.8599.29
1.042.134.77102.2896.94
0.261.845.1198.1394.13
19Coptisine8.330.421.1599.7598.73
2.081.422.0497.3695.12
0.520.732.8893.1791.33
20Isoliquiritin apioside8.330.637.21111.90103.33
2.081.135.26108.42102.21
0.521.881.39100.09101.48
21Isoliquiritin4.170.755.47110.60104.10
1.041.962.06103.08100.77
0.266.454.8399.19105.02
22Baicalin266.670.608.90110.47100.78
66.670.355.35104.3399.05
16.670.753.5499.1397.18
23Ginsenoside Re8.330.985.06110.27104.22
2.082.724.11107.39102.54
0.524.972.77109.41106.12
24Ginsenoside Rg116.671.065.75111.14104.22
4.171.742.66104.24101.20
1.043.481.67100.17101.18
26Palmatine16.671.115.02105.18100.01
4.170.861.0795.0294.77
1.040.611.7595.2693.52
27Berberine33.330.506.84106.7898.97
8.330.441.1294.2093.79
2.081.373.4497.0893.64
29Wogonoside266.670.644.22102.3097.56
66.671.983.5299.7295.83
16.671.383.4292.7894.75
31Ginsenoside Rb116.670.893.35110.83106.99
4.171.192.82106.66103.42
1.042.122.76103.24105.03
32Baicalein8.330.428.38109.11101.09
2.081.444.58101.7496.64
0.520.903.16102.34105.00
35Glycyrrhizic acid66.671.246.10111.73104.39
16.670.745.97108.92102.22
4.173.157.9893.12102.56
36Wogonin4.172.116.70108.71100.95
1.042.162.6798.5795.64
0.262.072.4390.7492.42
38Oroxylin A4.170.747.92106.9298.68
1.041.081.8598.1697.18
0.261.362.8293.9895.55
396-Gingerol8.330.666.13104.5797.87
2.080.501.1192.2993.43
0.521.242.3397.3094.78
416-Shogaol4.170.724.52107.11102.34
1.042.370.4698.9898.71
0.262.024.3698.58100.19
Table 7. Contents of the 22 analytes in 3 batches of BHSST.
Table 7. Contents of the 22 analytes in 3 batches of BHSST.
No.CompoundBHSST-1BHSST-2BHSST-3
Mean
(mg/g)		SDCV
(%)		Mean
(mg/g)		SDCV
(%)		Mean
(mg/g)		SDCV
(%)
9Rutin0.1250.0053.600.1270.0075.260.1260.0053.93
10Liquiritin apioside5.3060.0330.615.3000.0440.845.2570.0510.96
11Scutellarin0.2560.0124.570.2630.0114.310.2630.0041.58
12Liquiritin1.0020.0040.411.0030.0100.990.9990.0111.15
13Acteoside0.5950.0132.100.6010.0111.890.5960.0152.55
16Jatrorrhizine0.4590.0122.650.4320.0061.450.4370.0051.21
19Coptisine0.5830.0152.650.5680.0071.190.5710.0061.01
20Isoliquiritin apioside0.6550.0111.620.6240.0132.070.6130.0081.25
21Isoliquiritin0.1710.0042.450.1700.0063.600.1670.0052.99
22Baicalin35.1570.5211.4834.8480.3881.1134.6610.4011.16
23Ginsenoside Re0.5580.0081.520.5540.0061.160.5510.0040.69
24Ginsenoside Rg11.0800.0060.581.1320.0060.561.1100.0060.54
26Palmatine0.8310.0070.840.8140.0081.030.8130.0101.21
27Berberine2.7490.0652.352.6540.0411.552.6780.0421.58
29Wogonoside28.9190.6572.2728.8180.5982.0728.8930.5101.76
31Ginsenoside Rb11.1200.0080.751.1120.0080.681.0910.0070.61
32Baicalein0.6260.0111.800.6300.0071.100.6170.0121.97
35Glycyrrhizic acid4.8690.0591.204.8980.0581.194.8940.0501.01
36Wogonin0.1860.0042.140.1830.0010.810.1830.0010.78
38Oroxylin A0.2040.0125.770.2020.0094.400.2050.0062.69
396-Gingerol0.6240.0091.500.6100.0071.150.6060.0040.64
416-Shogaol0.1010.0010.820.0980.0011.470.1000.0011.32
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Jang, S.; Hwang, Y.-H. Qualitative and Quantitative Analysis of Banhasasim-Tang Using UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS. Processes 2024, 12, 1563. https://doi.org/10.3390/pr12081563

AMA Style

Jang S, Hwang Y-H. Qualitative and Quantitative Analysis of Banhasasim-Tang Using UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS. Processes. 2024; 12(8):1563. https://doi.org/10.3390/pr12081563

Chicago/Turabian Style

Jang, Seol, and Youn-Hwan Hwang. 2024. "Qualitative and Quantitative Analysis of Banhasasim-Tang Using UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS" Processes 12, no. 8: 1563. https://doi.org/10.3390/pr12081563

APA Style

Jang, S., & Hwang, Y.-H. (2024). Qualitative and Quantitative Analysis of Banhasasim-Tang Using UHPLC-Q-Orbitrap-MS and UHPLC-TQ-MS/MS. Processes, 12(8), 1563. https://doi.org/10.3390/pr12081563

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