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Article

Qualitative and Quantitative Analysis of Phytochemicals in Sayeok-Tang via UPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS

by
Yu Jin Kim
*,
Seol Jang
and
Youn-Hwan Hwang
*
KM Convergence Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2024, 17(9), 1130; https://doi.org/10.3390/ph17091130
Submission received: 9 July 2024 / Revised: 23 August 2024 / Accepted: 26 August 2024 / Published: 27 August 2024
(This article belongs to the Special Issue Advances in Mass Spectrometry Metrology in Pharmaceutical Sciences)
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Abstract

:
Sayeok-tang (SYT) is a traditional herbal formula comprising three medicinal herbs: Glycyrrhiza uralensis, Zingiber officinale, and Aconitum carmichaeli. Several studies have employed liquid chromatography-mass spectrometry (LC-MS) to qualitatively analyze the components and metabolites of SYT in vitro and in vivo; however, studies on quantitative analysis of SYT, which is important for quality control, are absent or limited to only a few components. In this study, ultrahigh-performance liquid chromatography coupled with quadrupole (UPLC-Q)-Orbitrap-MS was used to screen the phytochemicals of SYT, revealing a total of 42 compounds. Among them, 24 compounds were simultaneously quantified within 20 min via UPLC-TQ-MS/MS in the multiple reaction monitoring mode. The developed analytical method was validated for its linearity (r2 ≥ 0.9992), precision (0.36–2.96%), accuracy (−6.52–4.64%), and recovery (94.39–119.07%) for all analytes, exhibiting acceptable results. The validated method was applied in the analysis of SYT extracts, and the 24 compounds were quantified in the range of 0.004–6.882 mg/g (CV ≤ 3.746%). Among them, liquiritin apioside (6.870–6.933 mg/g), glycyrrhizic acid (5.418–5.540 mg/g), and liquiritin (1.303–1.331 mg/g) from G. uralensis were identified as the relatively abundant compounds. The presented validated analytical method is highly promising for the comprehensive quality control of SYT, offering fast, highly sensitive, and reliable analysis.

1. Introduction

Sayeok-tang (SYT), known as Shigyaku-to in Japan and Sini-tang in China, is a traditional herbal formula of Shang Han Lun, comprising three medicinal herbs: Glycyrrhiza uralensis, Zingiber officinale, and Aconitum carmichaeli [1]. Previous studies have shown that SYT is effective in treating cardiovascular diseases, including the improvement of early ventricular remodeling and cardiac function in heart failure following myocardial infarction [2,3,4,5]. Clinical studies on the therapeutic effects of SYT on ischemia/reperfusion injury in patients with acute myocardial infarction and on angina pectoris in coronary artery disease have also been reported [6,7]. SYT has also been applied to improve lung injury caused by sepsis through various mechanisms. SYT ameliorates the symptoms and pathology associated with sepsis, such as pulmonary histopathological lesions in cecal ligation and puncture mice models by modulating gut microbiota [8] and improves sepsis-induced acute lung injury by regulating the ACE2-Ang (1–7)-Mas axis and inhibiting the mitogen-activated protein kinase signaling pathway [9]. Additionally, SYT has been shown to possess anti-inflammatory and antioxidant properties that attenuate acute lung injury induced by E. coli in mice [10]. A previous study predicted the association between SYT and ulcerative colitis (UC) through network pharmacology analysis and revealed the pharmacological effects of SYT on UC using rats with UC [11]. Although the various experimental and clinical efficacies of SYT are known, few studies report analytical methods for quality control of SYT.
The quality of herbal medicines contained in herbal formulas varies depending on various environmental factors; therefore, quality control is important to ensure their safety and efficacy. In recent years, ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) has become a powerful tool for chemical profiling of natural products [12]. In particular, UPLC coupled with quadrupole Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) has been widely used to screen and identify phytochemicals in complex herbal samples owing to its excellent analytical sensitivity and specificity compared to other techniques, being ideal for identifying compounds by obtaining accurate molecular mass and multistage MSn fragment ions of analytes [13,14,15]. Currently, UPLC coupled with triple quadrupole mass spectrometry (UPLC-TQ-MS/MS) has become a promising tool for simultaneous analysis of multiple target compounds in complex mixtures at low concentrations due to its high sensitivity and fast resolution [16,17]. The multiple reaction monitoring (MRM) mode of TQ-MS/MS is a rapid and highly sensitive analytical method that can selectively identify and quantify target compounds in complex mixtures by rapidly screening the transitions from specific precursor ions to product ions [17,18]. In addition, it is frequently applied to quantitative analysis in various research fields because it provides very low detection and quantitation limits without considering peak overlap interference [19,20,21]. Even though several studies have reported the qualitative analysis of the components and metabolites of SYT in vitro and in vivo using liquid chromatography-mass spectrometry (LC-MS), studies on quantitative analysis of SYT, which is important for quality control, are absent or limited to only a few components [22,23,24,25].
Therefore, in this study, a UPLC-Q-Orbitrap-MS method was applied to screen and characterize 42 phytochemicals of SYT by comparing retention times and MS information with reference standards. In addition, simultaneous quantification of 24 phytochemicals in SYT was performed using a validated UPLC-TQ-MS/MS method in the MRM mode, enabling rapid, sensitive, and high-throughput analysis. This study offers an efficient and reliable analytical method being a valuable tool for the comprehensive quality control of SYT.

2. Results and Discussion

2.1. Qualitative Analysis of SYT

SYT extracts were analyzed via UPLC-Q-Orbitrap-MS to identify the phytochemicals attributed to the three herbal medicines: G. uralensis, Z. officinale, and A. carmichaeli [26]. The different compounds were separated within 20 min using an Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) with gradient elution of 0.1% (v/v) aqueous formic acid and acetonitrile. Both the positive and negative ESI modes were used to acquire MS spectra. A total of 42 compounds, including vicenin-2, schaftoside, daidzin, neoliquiritin, liquiritin apioside, liquiritin, ferulic acid, genistin, isoliquiritin apioside, isoliquiritin, ononin, licochalcone B, liquiritigenin, licochalcone A, genistein, naringenin, echinatin, isoliquiritigenin, formononetin, glycyrrhizic acid, glabridin, and glycyrrhetinic acid from G. uralensis [27], 6-gingerol, 8-gingerol, 6-shogaol, diacetoxy-6-gingerdiol, 10-gingerol, and 8-shogaol from Z. officinale [28], and karacolidine, mesaconine, senbusine A, karacoline, aconine, napellonine, hypaconine, fuziline, bullatine B, talatisamine, benzoylmesaconine, benzoylaconine, benzoylhypacoitine, and hypaconitine from A. carmichaeli [29,30,31], were identified by comparing their retention times, precursor ions, and MS/MS fragments to those of reference standards. The characteristics of all the identified compounds in SYT based on MS data are summarized in Table 1. Alkaloids from A. carmichaeli and phenols from Z. officinale were clearly detected in the positive ion mode, whereas the compounds from G. uralensis were ionized in similar proportions in the positive and negative ion modes. The LC chromatogram at 250 nm and base peak chromatograms in the positive and negative ion modes of SYT extracts are presented in Figure 1.

2.2. Quantitative Analysis

To quantify the 24 phytochemicals identified in the SYT extracts, UPLC-TQ-MS/MS analysis was performed in dynamic MRM mode optimized for each analyte, and all analytes were detected within 20 min under 0.1% (v/v) aqueous formic acid-acetonitrile gradient conditions. The MRM mode of TQ-MS/MS is an ideal method for selectively identifying and quantifying compounds in complex mixtures by rapidly screening for transitions from specific precursor ions to product ions [18]. The optimized MRM parameters for each of the 24 compounds and internal standards (IS), including ionization mode, MRM transitions, and collision energy, are summarized in Table 2. The retention times, precursor ions, and product ions of each analyte were compared to those of reference standards. Most analytes were detected in the positive ion mode, while five analytes, liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid, were more suitably ionized in the negative ion mode. The MRM chromatograms of the analytes in the positive or negative ion modes are shown in Figure 2.
The MS fragmentation patterns from the precursor ions to the dominant product ions were confirmed through UPLC-TQ-MS/MS analysis in the dynamic MRM mode. The six Aconitum alkaloids, karacoline, fuziline, bullatine B, talatisamine, benzoylmesaconine, and benzoylaconine, exhibited protonated molecular ions [M + H]+ at m/z 378.2, 454.3, 438.3, 422.3, 590.3, and 604.3, respectively. Karacoline, fuziline, and bullatine B lost a water molecule (18 Da) from their precursor ions to form [M + H − H2O]+ ions at m/z 360.2, 436.3, and 420.3, respectively [30,31,32]. Talatisamine generated a fragment ion [M + H − CH3OH]+ at m/z 390.2 by losing a methanol molecule (32 Da) from the precursor ion. Benzoylmesaconine and benzoylaconine generated a product ion at m/z 105.0, corresponding to the benzoyl group [33]. Among the 13 constituents of G. uralensis, five compounds, liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid, exhibited [M − H]− ions at m/z 549.2, 417.2, 549.1, 417.0, and 821.4, respectively. Liquiritin and isoliquiritin generated [M − H − Glc]− ions at m/z 255.0 and 255.1, respectively, which resulted from the loss of glucose (162 Da). In the case of liquiritin apioside and isoliquiritin apioside, a fragment ion [M − H − Api − Glc]− was produced at m/z 255.1 by losing an apiosyl glucoside from the precursor ion. Glycyrrhizic acid produced a fragment ion [2GluA − H]− at m/z 351.0, indicating the loss of two glucuronic acids [34]. In the positive ion mode, protonated molecular ions [M + H]+ of the remaining eight compounds from G. uralensis were observed. For neoliquiritin and ononin, the precursor ions at m/z 419.1 and 431.1 eliminated a glucose molecule (162 Da) to generate fragment ions [M + H − Glc]+ at m/z 257.1 and 269.1, respectively. Liquiritigenin and isoliquiritigenin exhibited [M + H]+ ions at m/z 257.0 and had the same fragment ions [M + H − C8H8O]+ at m/z 137.0 [35,36]. The precursor ion [M + H]+ of formononetin observed at m/z 269.0 subsequently underwent several fragmentations, including loss of CH4 (16 Da) and 2CO (56 Da), to generate a specific fragment ion [M + H − C3H4O2]+ at m/z 197.0 [37]. The fragment ions of echinatin at m/z 121.0 and genistein at m/z 91.1 were generated from the precursor ions [M + H]+ at m/z 271.1 and 271.0, respectively [38,39]. Regarding glabridin, a characteristic fragment ion [M + H − C8H8O2]+ was identified at m/z 189, generated by a Retro-Diels-Alder reaction from the precursor ion at m/z 325.1 [M + H]+ [40,41]. The precursor ions of 6-gingerol and 8-gingerol in the form [M + H − H2O]+ were identified at m/z 277.1 and 305.2, respectively, while the [M + H − H2O − C6H12O]+ and [M + H − H2O − C8H16O]+ fragment ions were generated at m/z 177.1, respectively, by the loss of the neutral alkyl moiety and rearrangement [42]. Diacetoxy-6-gingerdiol exhibited an m/z 398.2 [M + NH4]+ and fragment ion at m/z 137.0. Regarding 6-shogaol and 8-shogaol, the precursor ions [M + H]+ were observed at m/z 277.1 and 305.1, respectively, and the fragment ions [M + H − C9H16O]+ and [M + H − C11H20O]+ were produced at m/z 137.1 and 137.0, respectively [28].

2.3. Method Validation for Quantitative Analysis

The linearity, limits of detection (LOD) and quantification (LOQ), precision, accuracy, and recovery were evaluated to validate the developed analytical method. The calibration curves for each analyte were linear over a wide concentration range and observed appropriate results without weighting compared to using weighting factors such as 1/x, 1/x2, 1/y, or 1/y2. The correlation coefficients are within the acceptable limits (r2 ≥ 0.9992). The LODs and LOQs of the 24 analytes ranged from 0.007–5.165 ng/mL and 0.020–15.651 ng/mL, respectively. The linear ranges, regression equations, correlation coefficient values, LODs, and LOQs of the 24 compounds are listed in Table 3. Precision was expressed as the coefficient of variation (CV) (%) of the observed concentration values for six replicates of the reference standards at three concentration levels (low, medium, and high). The intra- and inter-day precisions of the 24 compounds were less than 2.54% and 2.96%, respectively, and the accuracies, expressed as the relative error (RE) (%), ranged from −6.52 to 4.37% and −5.41 to 4.64%, respectively (Table 4). Recovery tests were performed by adding the standard solutions of the 24 compounds at three different concentrations (low, medium, and high) to the original sample of known concentration (Table 5). The recovery (%) of all analytes ranged from 94.39 to 119.07% (CV ≤ 4.75%). These verified results demonstrate that the established UPLC-TQ-MS/MS method exhibits acceptable linearity, sensitivity, precision, accuracy, and recovery and is suitable for the quantitative analysis of 24 phytochemicals in SYT.

2.4. Quantification of 24 Phytochemicals in SYT

The validated UPLC-TQ-MS/MS method in MRM mode was subsequently applied to the quantitative analysis of 24 phytochemicals in three batches of SYT samples. The contents of the 24 compounds were measured in the range of 0.004 to 6.882 mg/g (CV ≤ 3.746%) based on the calibration curve, and the average contents of each batch for all analytes are presented in Table 6. Among these compounds, liquiritin apioside (6.870–6.933 mg/g), glycyrrhizic acid (5.418–5.540 mg/g), and liquiritin (1.303–1.331 mg/g) from G. uralensis were relatively abundant in all three batches of SYT samples.
Several researchers have reported in previous studies that the contents of the components in the three herbal medicines of SYT vary depending on seasonal and geographical factors [43,44,45,46,47]. The content and composition of SYT ingredients may be influenced by environmental changes, geographical location, soil conditions, and harvest time. These influence factors can affect the overall quality and efficacy of herbal medicines [48,49]. Although we have developed and validated fast and sensitive UPLC-MS-based methods for the quality control in SYT, the evaluation of its phytochemical diversity and complexity considering various influence factors were not included in this study. In this regard, further studies are required to investigate various seasonality or to compare with other blends coming from geographical locations with different characteristics. Therefore, our precise and sensitive analytical methods can provide sufficiently valuable and helpful information for investigating various subsequent studies of SYT quality control.

3. Materials and Methods

3.1. Materials and Reagents

The three herbal medicines included in SYT, Glycyrrhiza uralensis, Zingiber officinale, and Aconitum carmichaeli, were purchased from the herbal medicine market Kwangmyungdang Pharmaceutical (Ulsan, Republic of Korea), and the voucher specimens were deposited at the KM Convergence Research Division of the Korea Institute of Oriental Medicine (Daejeon, Republic of Korea). The 42 reference standards (purity ≥ 95%) used in the qualitative analysis of SYT were purchased from TargetMol (Boston, MA, USA). The 24 reference standards (purity ≥ 98%), karacoline, fuziline, bullatine B, talatisamine, liquiritin apioside, neoliquiritin, liquiritin, isoliquiritin apioside, benzoylmesaconine, isoliquiritin, ononin, benzoylaconine, liquiritigenin, echinatin, genistein, isoliquiritigenin, formononetin, glycyrrhizic acid, 6-gingerol, glabridin, 8-gingerol, 6-shogaol, diacetoxy-6-gingerdiol, and 8-shogaol were purchased from ChemFaces Biochemical (Wuhan, China) and used for quantitative analysis. Warfarin was used as IS and was obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol, water, acetonitrile, and formic acid (LC-MS grade) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

3.2. Preparation of Standard Solutions

The 24 reference standards and warfarin (IS) were each prepared at a concentration of 1.0 mg/mL in methanol. These stock solutions were then further diluted with methanol to obtain a series of standard solutions for the calibration curves and method validation. The concentration of IS was consistently fixed at 5.0 ng/mL in all standard solutions.

3.3. Extraction of SYT

SYT (228 g), containing a mixture of the three herbal medicines Glycyrrhiza uralensis, Zingiber officinale, and Aconitum carmichaeli in a ratio of 1:1.5:0.75, was extracted via refluxing with distilled water at 100 °C for 3 h. The extract solution was filtered, concentrated using a rotary evaporator system under vacuum, and freeze-dried to obtain a powdered extract (57.72 g, 25.32%). The powdered SYT extract was dissolved in methanol at a concentration of 50 μg/mL, filtered through a syringe filter (0.2 μm pore size), and used as a sample solution for analysis.

3.4. UPLC-Q-Orbitrap-MS Conditions

Qualitative analysis of SYT was performed using a Dionex UltiMate 3000 system connected to a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source according to the previously reported methods [50]. The phytochemicals in SYT were identified by gradient elution of 0.1% (v/v) aqueous formic acid and acetonitrile on an Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) maintained at 40 °C. MS analysis was conducted with an ESI source in both the positive and negative modes and MS spectra were acquired at a normalized collision energy of 25 eV in full MS-ddMS2 mode over a scan range of 100–1500 m/z. The source parameters were set as follows: ion spray voltage, 3.8 kV; capillary temperature, 320 °C; sheath gas pressure, 40 arbitrary units (au); auxiliary gas pressure, 10 au; Slens RF level, 60; and resolution, 70,000 (full MS) and 17,500 (ddMS2). All data were processed using Thermo Xcalibur v.3.0 and Tracefinder v.3.2 (Thermo Fisher Scientific, Bremen, Germany).

3.5. UPLC-TQ-MS/MS Conditions

Quantitative analysis of the 24 compounds in SYT was performed with an Agilent 1290 Infinity II UPLC system equipped with a 6495C triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a jet-stream ESI source. The 24 compounds were separated on an Acquity BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) maintained at 40 °C by gradient elution of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B) using the following method: 3% B for 0–1 min, 3–15% B for 1–2 min, 15–50% B for 2–13 min, 50–100% B for 13–20 min, and 100% B for 20–23 min at a flow rate of 0.25 mL/min. The mass spectrometer was operated in the dynamic MRM mode, and the MRM data were collected in the positive or negative ion mode depending on the optimal ionization conditions for each compound. The ESI source conditions involved a drying gas temperature of 130 °C, drying gas flow of 11 L/min, nebulizer pressure of 25 psi, sheath gas temperature of 400 °C, sheath gas flow of 12 L/min, capillary voltage of 3500 V (positive) and 3000 V (negative), and nozzle voltage of 500 V (positive) and 1500 V (negative). Agilent MassHunter Workstation v.10.1 software (Agilent Technologies, Santa Clara, CA, USA) was used for all data acquisition and processing.

3.6. Validation of the UPLC-TQ-MS/MS Method

Calibration curves of the 24 reference standards were established from the peak areas of standard solutions at nine different concentration levels, and the linear relationships between the peak area (y) and corresponding concentration (x, ng/mL) of each standard were expressed via the regression equation (y = ax + b). Standard solutions were measured five times repeatedly to obtain the calibration curves. The LOD and LOQ for the 24 compounds were calculated using the slope of the calibration curve and the standard deviation (SD) of the intercept as follows: LOD = 3.3 × (SD of the response/slope of the calibration curve) and LOQ = 10 × (SD of the response/slope of the calibration curve). To assess precision, three standard solutions containing low, medium, and high concentrations of each standard were analyzed repeatedly (n = 6) in one day and three consecutive days to measure the intra- and inter-day variation. Precision was expressed as CV (%) of the measured concentration values and calculated using the following formula: CV (%) = (SD/Mean) × 100. Accuracy was represented by RE (%) and calculated as follows: RE (%) = (observed concentration − expected concentration)/expected concentration × 100. Recovery tests were performed by spiking standard solutions of three different concentrations (low, medium, and high) into samples of known concentration. The recovery (%) was calculated according to the following equation: recovery (%) = (found concentration − original concentration)/spiked concentration × 100.

4. Conclusions

The phytochemicals of SYT were studied via UPLC-Q-Orbitrap-MS analyses, and a total of 42 compounds were identified in the positive and negative ESI modes. The qualitative analysis results, including retention time and MS data, were compared with those of reference standards. Within 20 min, 24 compounds were simultaneously quantified in the MRM mode using the optimized UPLC-TQ-MS/MS method. The method was validated for its linearity, precision, accuracy, and recovery, exhibiting acceptable results and confirming that the established analytical method is suitable for quantifying the components of SYT. Our study offers a valuable tool for the comprehensive quality control of SYT.

Author Contributions

Conceptualization, Y.-H.H.; investigation, Y.J.K. and S.J.; writing—original draft preparation, Y.J.K.; writing—review and editing, Y.J.K. and Y.-H.H.; supervision, 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.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Pharmaceuticals 17 01130 g001
Figure 1. LC chromatogram and base peak chromatograms in the positive and negative ion modes of SYT extracts confirmed by UPLC-Q-Orbitrap-MS. Information on each compound corresponding to each number is presented in Table 1.
Figure 1. LC chromatogram and base peak chromatograms in the positive and negative ion modes of SYT extracts confirmed by UPLC-Q-Orbitrap-MS. Information on each compound corresponding to each number is presented in Table 1.
Pharmaceuticals 17 01130 g001
Pharmaceuticals 17 01130 g002
Figure 2. Multiple reaction monitoring (MRM) chromatograms of the 24 compounds in the (A) SYT extracts and (B) standard mixture.
Figure 2. Multiple reaction monitoring (MRM) chromatograms of the 24 compounds in the (A) SYT extracts and (B) standard mixture.
Pharmaceuticals 17 01130 g002
Table 1. Phytochemicals identified in SYT via UPLC-Q-Orbitrap-MS analysis.
Table 1. Phytochemicals identified in SYT via UPLC-Q-Orbitrap-MS analysis.
No.RT (min)Precursor Ion (m/z)Error (ppm)FormulaMS/MS Fragments (m/z)Identifications
CalculatedEstimatedAdduct
14.40394.2595394.2588M + H1.8300C22H35NO5394.2594, 376.2486, 238.1674Karacolidine
24.82486.2708486.2698M + H2.2282C24H39NO9468.2529, 454.2444, 436.2336Mesaconine
34.91424.2701424.2694M + H1.6264C23H37NO6424.2702, 406.2595, 388.2481Senbusine A
45.01378.2646378.2639M + H1.8437C22H35NO4360.2533, 243.3279, 127.9954Karacoline
55.15500.2866500.2854M + H2.4208C25H41NO9420.2416, 402.2276, 276.1242Aconine
65.19358.2385358.2377M + H2.2174C22H31NO3358.2383, 340.2278, 191.1758Napellonine
75.45593.1526593.1512M − H2.4596C27H30O15473.1106, 383.0779, 353.0671Vicenin-2
85.47470.2759470.2748M + H2.2091C24H39NO8470.2759, 438.2494, 310.1442Hypaconine
95.52454.2809454.2799M + H2.2339C24H39NO7454.2809, 436.2684Fuziline
105.76438.2860438.2850M + H2.3529C24H39NO6438.2859, 420.2757, 388.2509Bullatine B
115.87563.1420563.1406M − H2.3756C26H28O14503.1196, 443.0996, 353.0671Schaftoside
125.99417.1189417.1180M + H2.0521C21H20O9416.2454, 255.0655, 137.0236Daidzin
136.18422.2907422.2901M + H1.4456C24H39NO5422.2909, 390.2650, 258.0841Talatisamine
146.69419.1343419.1337M + H1.5457C21H22O9257.0812, 147.0446, 137.0237Neoliquiritin
156.72549.1624549.1614M − H1.8092C26H30O13255.0665, 135.0076, 119.0491Liquiritin apioside
166.85417.1198417.1191M − H1.5367C21H22O9255.0665, 135.0076, 119.0490Liquiritin
176.99193.0502193.0506M − H−2.2201C10H10O4178.0264, 149.0598, 134.0362Ferulic acid
187.07477.1046477.1038M + HCO21.6019C21H20O10431.0991, 269.0459, 255.0665Genistin
198.31549.1626549.1614M − H2.2537C26H30O13255.0664, 151.0390, 135.0075Isoliquiritin apioside
208.57590.2971590.2960M + H1.8433C31H43NO10558.2698, 540.2626, 105.0343Benzoylmesaconine
218.60417.1199417.1180M − H4.4697C21H22O9297.0777, 255.0664, 135.0076Isoliquiritin
228.92431.1345431.1337M + H1.9274C22H22O9269.0812Ononin
239.01285.0774285.0768M − H1.7962C16H14O5285.0771, 270.0537, 150.0312Licochalcone B
249.18604.3130604.3116M + H2.2642C32H45NO10554.2754, 501.9368, 269.0811Benzoylaconine
259.25257.0814257.0808M + H2.2596C15H12O4239.0709, 147.0445, 137.0237Liquiritigenin
269.56574.3024574.3011M + H2.3841C31H43NO9574.3021, 542.2756, 147.0821Benzoylhypacoitine
279.79616.3131616.3116M + H2.4182C33H45NO10488.3347, 411.4201, 313.6526Hypaconitine
2810.65339.1600339.1591M + H2.6039C21H22O4215.1073, 163.0758, 137.0601Licochalcone A
2910.72271.0606271.0601M + H1.8738C15H10O5229.0865, 153.0186, 121.0290Genistein
3010.76271.0618271.0612M − H2.2065C15H12O5177.0184, 151.0026, 119.0489Naringenin
3110.79271.0971271.0965M + H2.1799C16H14O4229.0865, 153.0186, 121.0290Echinatin
3212.00257.0813257.0808M + H1.6661C15H12O5239.0707, 147.0444, 137.0237Isoliquiritigenin
3312.50269.0813269.0808M + H1.7052C16H12O4254.0571, 237.0545, 137.0595Formononetin
3413.09821.3982821.3965M − H2.0494C42H62O16776.1565, 351.0583, 193.0348Glycyrrhizic acid
3514.52277.1804277.1798M − H2O + H1.9400C17H26O4177.0914, 145.0652, 137.06016-Gingerol
3616.95325.1440325.1434M + H1.7606C20H20O4189.0914, 149.0601, 123.0446Glabridin
3717.31305.2118305.2111M − H2O + H2.1976C19H30O4177.0914, 145.0652, 137.06018-Gingerol
3817.69277.1804277.1798M + H1.9442C17H24O3137.06016-Shogaol
3918.40398.2547398.2537M + NH42.3977C21H32O6261.1853, 163.0757, 137.0601Diacetoxy-6-gingerdiol
4019.06471.3479471.3469M + H2.0789C30H46O4267.0661, 235.1690, 189.1646Glycyrrhetinic acid
4119.06373.2356373.2349M + Na1.8654C21H34O4218.1184, 159.0420, 129.055010-Gingerol
4219.39305.2118305.2111M + H2.2017C19H28O3137.06008-Shogaol
Table 2. Optimized MRM parameters for the 24 compounds in SYT extracts.
Table 2. Optimized MRM parameters for the 24 compounds in SYT extracts.
No.CompoundRT (min)Molecular WeightPolarityMRM Transition (m/z)Collision Energy (V)
1Karacoline3.91377.5Positive378.2 → 360.230
2Fuziline4.39453.6Positive454.3 → 436.334
3Bullatine B4.60437.6Positive438.3 → 420.330
4Talatisamine5.04421.6Positive422.3 → 390.230
5Liquiritin apioside5.65550.5Negative549.2 → 255.134
6Neoliquiritin5.65418.4Positive419.1 → 257.110
7Liquiritin5.79418.4Negative417.2 → 255.018
8Isoliquiritin apioside7.24550.5Negative549.1 → 255.130
9Benzoylmesaconine7.44589.7Positive590.3 → 105.040
10Isoliquiritin7.58418.4Negative417.0 → 255.118
11Ononin7.87430.4Positive431.1 → 269.118
12Benzoylaconine8.06603.7Positive604.3 → 105.040
13Liquiritigenin8.30256.3Positive257.0 → 137.026
14Echinatin9.78270.3Positive271.1 → 121.026
15Genistein9.80270.2Positive271.0 → 91.140
16Isoliquiritigenin11.09256.3Positive257.0 → 137.022
17Formononetin11.52268.3Positive269.0 → 197.040
18Glycyrrhizic acid11.98822.9Negative821.4 → 351.040
196-Gingerol13.52294.4Positive277.1 → 177.110
20Glabridin16.01324.4Positive325.1 → 189.114
218-Gingerol16.35322.4Positive305.2 → 177.110
226-Shogaol16.74276.4Positive277.1 → 137.110
23Diacetoxy-6-gingerdiol17.44380.5Positive398.2 → 137.030
248-Shogaol18.45304.4Positive305.1 → 137.014
ISWarfarin13.98307.1Positive309.0 → 163.014
ISWarfarin13.98307.1Negative307.0 → 250.022
Table 3. Regression equations, linear ranges, correlation coefficients, LODs, and LOQs of the 24 compounds present in SYT.
Table 3. Regression equations, linear ranges, correlation coefficients, LODs, and LOQs of the 24 compounds present in SYT.
No.CompoundLinear Range
(ng/mL)		Regression Equation
(y = ax + b) a		Correlation Coefficient (r2)LOD b
(ng/mL)		LOQ c
(ng/mL)
1Karacoline0.024–6.25y = 0.246822x − 0.0014280.99950.0240.071
2Fuziline0.024–6.25y = 0.365585x − 0.0020380.99940.0680.207
3Bullatine B0.049–12.5y = 0.175573x − 0.0023820.99930.0510.154
4Talatisamine0.024–6.25y = 0.317425x − 0.0015480.99970.0170.051
5Liquiritin apioside3.125–800y = 0.149587x − 0.0413860.99992.7538.341
6Neoliquiritin0.781–200y = 0.038745x − 0.0057770.99931.3374.050
7Liquiritin1.563–400y = 0.373646x − 0.0364460.99981.6705.059
8Isoliquiritin apioside0.195–50y = 0.168865x − 0.0021810.99970.1690.513
9Benzoylmesaconine0.098–25y = 0.075469x − 0.0014630.99950.1500.456
10Isoliquiritin0.195–50y = 0.212640x − 0.0023290.99950.1980.601
11Ononin0.781–200y = 0.294159x − 0.0097720.99950.9152.772
12Benzoylaconine0.024–6.25y = 0.018718x − 0.0000240.99950.0230.070
13Liquiritigenin0.049–12.5y = 0.120708x − 0.0008400.99950.0800.242
14Echinatin0.024–6.25y = 0.645024x − 0.0014780.99950.0370.113
15Genistein0.024–6.25y = 0.062600x − 0.0003740.99940.0240.072
16Isoliquiritigenin0.012–3.125y = 0.125080x + 0.0000540.99970.0070.020
17Formononetin0.012–3.125y = 0.613307x − 0.0017590.99950.0280.084
18Glycyrrhizic acid3.125–800y = 0.061782x − 0.0133600.99975.16515.651
196-Gingerol0.781–200y = 0.099518x − 0.0151990.99921.0333.131
20Glabridin0.049–12.5y = 0.192746x − 0.0006520.99970.0590.180
218-Gingerol0.049–12.5y = 0.071919x + 0.0001080.99950.0470.144
226-Shogaol0.098–25y = 0.275032x − 0.0049300.99940.1380.417
23Diacetoxy-6-gingerdiol0.049–12.5y = 0.389618x − 0.0029430.99970.0320.098
248-Shogaol0.024–6.25y = 0.106655x − 0.0000040.99970.0270.080
a y = ax + b, y indicates peak area and x indicates concentration (ng/mL). b LOD: 3.3 × (standard deviation (SD) of the response/slope of the calibration curve). c LOQ: 10 × (SD of the response/slope of the calibration curve).
Table 4. Precision and accuracy data for the 24 compounds in SYT.
Table 4. Precision and accuracy data for the 24 compounds in SYT.
No.CompoundConc.
(ng/mL)		Intra-Day (n = 6)Inter-Day (n = 6)
Observed Conc. (ng/mL)CV a (%)RE b (%)Observed Conc. (ng/mL)CV (%)RE (%)
1Karacoline0.520.530.941.070.531.231.17
2.082.100.770.762.091.080.49
4.174.141.07−0.714.042.29−3.00
2Fuziline0.520.520.87−0.200.531.341.27
2.082.090.670.362.142.422.80
4.174.071.39−2.404.062.10−2.55
3Bullatine B1.041.050.830.351.050.840.87
4.174.200.480.744.292.502.92
8.338.141.02−2.298.022.06−3.77
4Talatisamine0.520.521.11−0.040.520.950.43
2.082.090.390.302.131.952.30
4.174.020.60−3.414.030.98−3.37
5Liquiritin apioside66.6765.930.47−1.1066.030.91−0.95
266.67262.400.71−1.60261.140.73−2.07
533.33538.760.761.02537.340.940.75
6Neoliquiritin16.6716.730.560.3516.720.730.32
66.6768.171.002.2669.762.274.64
133.33129.650.50−2.77127.831.61−4.13
7Liquiritin33.3333.101.04−0.7033.261.74−0.21
133.33134.850.621.14132.801.42−0.40
266.67270.511.271.44270.411.331.40
8Isoliquiritin apioside4.174.160.69−0.114.201.340.76
16.6716.500.57−0.9716.560.74−0.62
33.3333.330.99−0.0233.811.411.42
9Benzoylmesaconine2.082.090.430.202.111.141.35
8.338.480.881.758.531.802.30
16.6716.450.74−1.3015.902.93−4.58
10Isoliquiritin4.174.160.40−0.084.150.93−0.52
16.6716.771.030.5916.630.96−0.20
33.3333.461.050.3934.362.283.07
11Ononin16.6716.880.361.2916.872.191.24
66.6769.580.894.3768.931.893.40
133.33135.541.031.65134.661.321.00
12Benzoylaconine0.520.531.291.300.531.061.30
2.082.170.884.312.161.453.65
4.174.140.61−0.544.071.89−2.30
13Liquiritigenin1.041.050.670.791.061.411.99
4.174.230.581.534.352.264.45
8.338.221.12−1.418.091.71−2.95
14Echinatin0.520.510.81−1.540.521.28−0.95
2.082.061.30−0.932.081.76−0.10
4.174.031.77−3.384.092.25−1.93
15Genistein0.520.521.43−0.890.521.67−0.90
2.082.111.151.382.121.531.84
4.174.030.90−3.164.061.90−2.54
16Isoliquiritigenin0.260.271.022.260.271.913.03
1.041.061.601.281.082.963.96
2.082.040.81−2.172.022.66−2.90
17Formononetin0.260.260.991.280.261.301.44
1.041.060.941.981.071.452.87
2.082.070.51−0.752.022.04−2.87
18Glycyrrhizic acid66.6765.201.30−2.1965.971.72−1.04
266.67257.302.54−3.51260.611.98−2.27
533.33522.380.83−2.05526.071.42−1.36
196-Gingerol16.6715.970.76−4.2116.251.67−2.47
66.6766.260.74−0.6166.880.980.33
133.33126.841.69−4.87126.132.36−5.41
20Glabridin1.041.021.26−2.461.011.73−3.26
4.174.190.930.664.181.730.31
8.338.070.91−3.188.032.16−3.65
218-Gingerol1.041.040.51−0.021.051.100.79
4.174.200.920.774.302.393.17
8.338.110.59−2.727.962.14−4.52
226-Shogaol2.082.060.67−0.962.101.990.87
8.338.531.382.388.521.492.21
16.6716.101.53−3.4116.021.44−3.88
23Diacetoxy-6-gingerdiol1.041.040.73−0.191.051.350.35
4.174.221.171.174.281.652.80
8.338.021.50−3.828.041.66−3.47
248-Shogaol0.520.520.91−0.290.520.850.32
2.082.110.471.302.121.031.71
4.173.891.06−6.524.012.85−3.78
a CV: coefficient of variation. b RE: relative error.
Table 5. Recovery data for the 24 compounds in SYT.
Table 5. Recovery data for the 24 compounds in SYT.
No.CompoundOriginal Conc.
(ng/mL)		Spiked Conc. (ng/mL)Observed Conc. (ng/mL)Recovery (%) aCV (%)
1Karacoline0.520.260.79105.162.94
1.041.67110.242.48
4.175.27114.052.11
2Fuziline0.340.260.64114.553.37
1.041.50111.372.43
4.175.00111.911.64
3Bullatine B0.710.521.29110.772.69
2.083.02110.581.89
8.3310.19113.671.55
4Talatisamine0.290.260.59112.842.28
1.041.49114.572.88
4.175.13116.012.08
5Liquiritin apioside101.6233.33135.51101.671.47
133.33257.50116.912.28
533.33730.29117.871.57
6Neoliquiritin16.438.3325.81112.542.73
33.3354.18113.243.19
133.33173.47117.781.58
7Liquiritin20.2616.6737.49103.372.70
66.6795.60113.001.43
266.67328.27115.501.24
8Isoliquiritin apioside5.952.088.12104.342.78
8.3315.55115.261.98
33.3345.32118.110.60
9Benzoylmesaconine1.531.042.75117.072.87
4.176.19111.962.71
16.6720.52113.941.43
10Isoliquiritin2.862.085.03103.983.13
8.3312.52115.961.74
33.3341.96117.311.03
11Ononin10.928.3320.63116.471.68
33.3350.20117.820.91
133.33169.68119.070.43
12Benzoylaconine0.810.261.08102.684.75
1.041.92106.262.38
4.175.36109.001.67
13Liquiritigenin1.020.521.57105.702.73
2.083.31110.193.35
8.3310.53114.151.59
14Echinatin0.110.260.39108.103.70
1.041.24108.172.78
4.174.61108.142.58
15Genistein0.100.260.41118.221.86
1.041.25110.532.35
4.174.65109.372.53
16Isoliquiritigenin0.120.130.26107.601.42
0.520.69109.152.06
2.082.46112.061.47
17Formononetin0.080.130.24117.863.19
0.520.67112.192.64
2.082.35108.770.63
18Glycyrrhizic acid92.5333.33123.9994.394.13
133.33242.88112.762.13
533.33682.41110.600.82
196-Gingerol14.488.3322.84100.394.27
33.3349.39104.744.56
133.33168.54115.552.26
20Glabridin0.560.521.0696.843.81
2.082.78106.632.53
8.339.97112.972.71
218-Gingerol0.960.521.4796.632.62
2.083.12103.402.43
8.339.96107.992.51
226-Shogaol1.691.042.6894.881.33
4.176.08105.443.12
16.6720.26111.431.47
23Diacetoxy-6-gingerdiol0.350.520.94112.042.67
2.082.64109.822.43
8.339.65111.582.07
248-Shogaol0.310.260.57101.391.17
1.041.48112.771.28
4.175.09114.801.85
a Recovery (%) = (Observed concentration − Original concentration)/Spiked concentration × 100.
Table 6. Contents of the 24 compounds in SYT extracts.
Table 6. Contents of the 24 compounds in SYT extracts.
No.CompoundBatch 1Batch 2Batch 3
Mean ± SD
(mg/g)		CV (%)Mean ± SD
(mg/g)		CV (%)Mean ± SD
(mg/g)		CV (%)
1Karacoline0.027 ± 0.0012.4290.025 ± 0.0012.8420.025 ± 0.0013.213
2Fuziline0.018 ± 0.0002.1790.018 ± 0.0001.0570.018 ± 0.0001.356
3Bullatine B0.039 ± 0.0012.0800.037 ± 0.0011.4890.037 ± 0.0013.156
4Talatisamine0.014 ± 0.0001.9360.013 ± 0.0001.8620.013 ± 0.0001.852
5Liquiritin apioside6.882 ± 0.0510.7466.933 ± 0.0640.9236.870 ± 0.0550.802
6Neoliquiritin0.885 ± 0.0101.1250.824 ± 0.0172.0630.814 ± 0.0212.554
7Liquiritin1.303 ± 0.0100.7821.324 ± 0.0231.7091.331 ± 0.0151.093
8Isoliquiritin apioside0.396 ± 0.0051.2370.399 ± 0.0051.2320.395 ± 0.0041.125
9Benzoylmesaconine0.080 ± 0.0011.5550.077 ± 0.0011.8480.076 ± 0.0022.829
10Isoliquiritin0.180 ± 0.0021.3220.182 ± 0.0021.2920.183 ± 0.0021.081
11Ononin0.637 ± 0.0132.0300.599 ± 0.0142.4100.599 ± 0.0101.628
12Benzoylaconine0.042 ± 0.0001.1330.038 ± 0.0013.6080.039 ± 0.0013.746
13Liquiritigenin0.056 ± 0.0011.6950.054 ± 0.0011.4990.054 ± 0.0011.941
14Echinatin0.006 ± 0.0002.5740.006 ± 0.0002.9920.006 ± 0.0002.908
15Genistein0.008 ± 0.0001.6680.008 ± 0.0001.8940.008 ± 0.0002.455
16Isoliquiritigenin0.006 ± 0.0001.7510.005 ± 0.0003.2930.005 ± 0.0002.738
17Formononetin0.004 ± 0.0001.4410.004 ± 0.0002.8090.004 ± 0.0002.144
18Glycyrrhizic acid5.540 ± 0.1061.9195.418 ± 0.1572.8915.505 ± 0.1011.833
196-Gingerol0.686 ± 0.0060.9290.649 ± 0.0081.2040.651 ± 0.0132.073
20Glabridin0.031 ± 0.0012.1760.031 ± 0.0012.1030.031 ± 0.0012.387
218-Gingerol0.058 ± 0.0011.8480.056 ± 0.0022.7190.057 ± 0.0011.376
226-Shogaol0.097 ± 0.0021.7460.095 ± 0.0011.3910.095 ± 0.0021.879
23Diacetoxy-6-gingerdiol0.020 ± 0.0000.8480.020 ± 0.0000.4720.020 ± 0.0000.691
248-Shogaol0.017 ± 0.0002.2430.017 ± 0.0001.8020.016 ± 0.0001.834
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Kim, Y.J.; Jang, S.; Hwang, Y.-H. Qualitative and Quantitative Analysis of Phytochemicals in Sayeok-Tang via UPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS. Pharmaceuticals 2024, 17, 1130. https://doi.org/10.3390/ph17091130

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Kim YJ, Jang S, Hwang Y-H. Qualitative and Quantitative Analysis of Phytochemicals in Sayeok-Tang via UPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS. Pharmaceuticals. 2024; 17(9):1130. https://doi.org/10.3390/ph17091130

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Kim, Yu Jin, Seol Jang, and Youn-Hwan Hwang. 2024. "Qualitative and Quantitative Analysis of Phytochemicals in Sayeok-Tang via UPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS" Pharmaceuticals 17, no. 9: 1130. https://doi.org/10.3390/ph17091130

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