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. 2024 Aug 27;17(9):1130. doi: 10.3390/ph17091130

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

Yu Jin Kim 1,*, Seol Jang 1, Youn-Hwan Hwang 1,*
Editors: Andrew P Bowman1, Rob J Vreeken1
PMCID: PMC11435331  PMID: 39338295

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.

Keywords: Sayeok-tang, UPLC-Q-Orbitrap-MS, UPLC-TQ-MS/MS, multiple reaction monitoring, quality control

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.

Table 1.

Phytochemicals identified in SYT via UPLC-Q-Orbitrap-MS analysis.

No. RT (min) Precursor Ion (m/z) Error (ppm) Formula MS/MS Fragments (m/z) Identifications
Calculated Estimated Adduct
1 4.40 394.2595 394.2588 M + H 1.8300 C22H35NO5 394.2594, 376.2486, 238.1674 Karacolidine
2 4.82 486.2708 486.2698 M + H 2.2282 C24H39NO9 468.2529, 454.2444, 436.2336 Mesaconine
3 4.91 424.2701 424.2694 M + H 1.6264 C23H37NO6 424.2702, 406.2595, 388.2481 Senbusine A
4 5.01 378.2646 378.2639 M + H 1.8437 C22H35NO4 360.2533, 243.3279, 127.9954 Karacoline
5 5.15 500.2866 500.2854 M + H 2.4208 C25H41NO9 420.2416, 402.2276, 276.1242 Aconine
6 5.19 358.2385 358.2377 M + H 2.2174 C22H31NO3 358.2383, 340.2278, 191.1758 Napellonine
7 5.45 593.1526 593.1512 M − H 2.4596 C27H30O15 473.1106, 383.0779, 353.0671 Vicenin-2
8 5.47 470.2759 470.2748 M + H 2.2091 C24H39NO8 470.2759, 438.2494, 310.1442 Hypaconine
9 5.52 454.2809 454.2799 M + H 2.2339 C24H39NO7 454.2809, 436.2684 Fuziline
10 5.76 438.2860 438.2850 M + H 2.3529 C24H39NO6 438.2859, 420.2757, 388.2509 Bullatine B
11 5.87 563.1420 563.1406 M − H 2.3756 C26H28O14 503.1196, 443.0996, 353.0671 Schaftoside
12 5.99 417.1189 417.1180 M + H 2.0521 C21H20O9 416.2454, 255.0655, 137.0236 Daidzin
13 6.18 422.2907 422.2901 M + H 1.4456 C24H39NO5 422.2909, 390.2650, 258.0841 Talatisamine
14 6.69 419.1343 419.1337 M + H 1.5457 C21H22O9 257.0812, 147.0446, 137.0237 Neoliquiritin
15 6.72 549.1624 549.1614 M − H 1.8092 C26H30O13 255.0665, 135.0076, 119.0491 Liquiritin apioside
16 6.85 417.1198 417.1191 M − H 1.5367 C21H22O9 255.0665, 135.0076, 119.0490 Liquiritin
17 6.99 193.0502 193.0506 M − H −2.2201 C10H10O4 178.0264, 149.0598, 134.0362 Ferulic acid
18 7.07 477.1046 477.1038 M + HCO2 1.6019 C21H20O10 431.0991, 269.0459, 255.0665 Genistin
19 8.31 549.1626 549.1614 M − H 2.2537 C26H30O13 255.0664, 151.0390, 135.0075 Isoliquiritin apioside
20 8.57 590.2971 590.2960 M + H 1.8433 C31H43NO10 558.2698, 540.2626, 105.0343 Benzoylmesaconine
21 8.60 417.1199 417.1180 M − H 4.4697 C21H22O9 297.0777, 255.0664, 135.0076 Isoliquiritin
22 8.92 431.1345 431.1337 M + H 1.9274 C22H22O9 269.0812 Ononin
23 9.01 285.0774 285.0768 M − H 1.7962 C16H14O5 285.0771, 270.0537, 150.0312 Licochalcone B
24 9.18 604.3130 604.3116 M + H 2.2642 C32H45NO10 554.2754, 501.9368, 269.0811 Benzoylaconine
25 9.25 257.0814 257.0808 M + H 2.2596 C15H12O4 239.0709, 147.0445, 137.0237 Liquiritigenin
26 9.56 574.3024 574.3011 M + H 2.3841 C31H43NO9 574.3021, 542.2756, 147.0821 Benzoylhypacoitine
27 9.79 616.3131 616.3116 M + H 2.4182 C33H45NO10 488.3347, 411.4201, 313.6526 Hypaconitine
28 10.65 339.1600 339.1591 M + H 2.6039 C21H22O4 215.1073, 163.0758, 137.0601 Licochalcone A
29 10.72 271.0606 271.0601 M + H 1.8738 C15H10O5 229.0865, 153.0186, 121.0290 Genistein
30 10.76 271.0618 271.0612 M − H 2.2065 C15H12O5 177.0184, 151.0026, 119.0489 Naringenin
31 10.79 271.0971 271.0965 M + H 2.1799 C16H14O4 229.0865, 153.0186, 121.0290 Echinatin
32 12.00 257.0813 257.0808 M + H 1.6661 C15H12O5 239.0707, 147.0444, 137.0237 Isoliquiritigenin
33 12.50 269.0813 269.0808 M + H 1.7052 C16H12O4 254.0571, 237.0545, 137.0595 Formononetin
34 13.09 821.3982 821.3965 M − H 2.0494 C42H62O16 776.1565, 351.0583, 193.0348 Glycyrrhizic acid
35 14.52 277.1804 277.1798 M − H2O + H 1.9400 C17H26O4 177.0914, 145.0652, 137.0601 6-Gingerol
36 16.95 325.1440 325.1434 M + H 1.7606 C20H20O4 189.0914, 149.0601, 123.0446 Glabridin
37 17.31 305.2118 305.2111 M − H2O + H 2.1976 C19H30O4 177.0914, 145.0652, 137.0601 8-Gingerol
38 17.69 277.1804 277.1798 M + H 1.9442 C17H24O3 137.0601 6-Shogaol
39 18.40 398.2547 398.2537 M + NH4 2.3977 C21H32O6 261.1853, 163.0757, 137.0601 Diacetoxy-6-gingerdiol
40 19.06 471.3479 471.3469 M + H 2.0789 C30H46O4 267.0661, 235.1690, 189.1646 Glycyrrhetinic acid
41 19.06 373.2356 373.2349 M + Na 1.8654 C21H34O4 218.1184, 159.0420, 129.0550 10-Gingerol
42 19.39 305.2118 305.2111 M + H 2.2017 C19H28O3 137.0600 8-Shogaol
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Figure 1.

Figure 1

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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.

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.

Table 2.

Optimized MRM parameters for the 24 compounds in SYT extracts.

No. Compound RT (min) Molecular Weight Polarity MRM Transition (m/z) Collision Energy (V)
1 Karacoline 3.91 377.5 Positive 378.2 → 360.2 30
2 Fuziline 4.39 453.6 Positive 454.3 → 436.3 34
3 Bullatine B 4.60 437.6 Positive 438.3 → 420.3 30
4 Talatisamine 5.04 421.6 Positive 422.3 → 390.2 30
5 Liquiritin apioside 5.65 550.5 Negative 549.2 → 255.1 34
6 Neoliquiritin 5.65 418.4 Positive 419.1 → 257.1 10
7 Liquiritin 5.79 418.4 Negative 417.2 → 255.0 18
8 Isoliquiritin apioside 7.24 550.5 Negative 549.1 → 255.1 30
9 Benzoylmesaconine 7.44 589.7 Positive 590.3 → 105.0 40
10 Isoliquiritin 7.58 418.4 Negative 417.0 → 255.1 18
11 Ononin 7.87 430.4 Positive 431.1 → 269.1 18
12 Benzoylaconine 8.06 603.7 Positive 604.3 → 105.0 40
13 Liquiritigenin 8.30 256.3 Positive 257.0 → 137.0 26
14 Echinatin 9.78 270.3 Positive 271.1 → 121.0 26
15 Genistein 9.80 270.2 Positive 271.0 → 91.1 40
16 Isoliquiritigenin 11.09 256.3 Positive 257.0 → 137.0 22
17 Formononetin 11.52 268.3 Positive 269.0 → 197.0 40
18 Glycyrrhizic acid 11.98 822.9 Negative 821.4 → 351.0 40
19 6-Gingerol 13.52 294.4 Positive 277.1 → 177.1 10
20 Glabridin 16.01 324.4 Positive 325.1 → 189.1 14
21 8-Gingerol 16.35 322.4 Positive 305.2 → 177.1 10
22 6-Shogaol 16.74 276.4 Positive 277.1 → 137.1 10
23 Diacetoxy-6-gingerdiol 17.44 380.5 Positive 398.2 → 137.0 30
24 8-Shogaol 18.45 304.4 Positive 305.1 → 137.0 14
IS Warfarin 13.98 307.1 Positive 309.0 → 163.0 14
IS Warfarin 13.98 307.1 Negative 307.0 → 250.0 22
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Figure 2.

Figure 2

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Multiple reaction monitoring (MRM) chromatograms of the 24 compounds in the (A) SYT extracts and (B) standard mixture.

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.

Table 3.

Regression equations, linear ranges, correlation coefficients, LODs, and LOQs of the 24 compounds present in SYT.

No. Compound Linear Range
(ng/mL)		 Regression Equation
(y = ax + b) a 		Correlation Coefficient (r2) LOD b
(ng/mL)		 LOQ c
(ng/mL)
1 Karacoline 0.024–6.25 y = 0.246822x − 0.001428 0.9995 0.024 0.071
2 Fuziline 0.024–6.25 y = 0.365585x − 0.002038 0.9994 0.068 0.207
3 Bullatine B 0.049–12.5 y = 0.175573x − 0.002382 0.9993 0.051 0.154
4 Talatisamine 0.024–6.25 y = 0.317425x − 0.001548 0.9997 0.017 0.051
5 Liquiritin apioside 3.125–800 y = 0.149587x − 0.041386 0.9999 2.753 8.341
6 Neoliquiritin 0.781–200 y = 0.038745x − 0.005777 0.9993 1.337 4.050
7 Liquiritin 1.563–400 y = 0.373646x − 0.036446 0.9998 1.670 5.059
8 Isoliquiritin apioside 0.195–50 y = 0.168865x − 0.002181 0.9997 0.169 0.513
9 Benzoylmesaconine 0.098–25 y = 0.075469x − 0.001463 0.9995 0.150 0.456
10 Isoliquiritin 0.195–50 y = 0.212640x − 0.002329 0.9995 0.198 0.601
11 Ononin 0.781–200 y = 0.294159x − 0.009772 0.9995 0.915 2.772
12 Benzoylaconine 0.024–6.25 y = 0.018718x − 0.000024 0.9995 0.023 0.070
13 Liquiritigenin 0.049–12.5 y = 0.120708x − 0.000840 0.9995 0.080 0.242
14 Echinatin 0.024–6.25 y = 0.645024x − 0.001478 0.9995 0.037 0.113
15 Genistein 0.024–6.25 y = 0.062600x − 0.000374 0.9994 0.024 0.072
16 Isoliquiritigenin 0.012–3.125 y = 0.125080x + 0.000054 0.9997 0.007 0.020
17 Formononetin 0.012–3.125 y = 0.613307x − 0.001759 0.9995 0.028 0.084
18 Glycyrrhizic acid 3.125–800 y = 0.061782x − 0.013360 0.9997 5.165 15.651
19 6-Gingerol 0.781–200 y = 0.099518x − 0.015199 0.9992 1.033 3.131
20 Glabridin 0.049–12.5 y = 0.192746x − 0.000652 0.9997 0.059 0.180
21 8-Gingerol 0.049–12.5 y = 0.071919x + 0.000108 0.9995 0.047 0.144
22 6-Shogaol 0.098–25 y = 0.275032x − 0.004930 0.9994 0.138 0.417
23 Diacetoxy-6-gingerdiol 0.049–12.5 y = 0.389618x − 0.002943 0.9997 0.032 0.098
24 8-Shogaol 0.024–6.25 y = 0.106655x − 0.000004 0.9997 0.027 0.080
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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.

No. Compound Conc.
(ng/mL)		 Intra-Day (n = 6) Inter-Day (n = 6)
Observed Conc. (ng/mL) CV a (%) RE b (%) Observed Conc. (ng/mL) CV (%) RE (%)
1 Karacoline 0.52 0.53 0.94 1.07 0.53 1.23 1.17
2.08 2.10 0.77 0.76 2.09 1.08 0.49
4.17 4.14 1.07 −0.71 4.04 2.29 −3.00
2 Fuziline 0.52 0.52 0.87 −0.20 0.53 1.34 1.27
2.08 2.09 0.67 0.36 2.14 2.42 2.80
4.17 4.07 1.39 −2.40 4.06 2.10 −2.55
3 Bullatine B 1.04 1.05 0.83 0.35 1.05 0.84 0.87
4.17 4.20 0.48 0.74 4.29 2.50 2.92
8.33 8.14 1.02 −2.29 8.02 2.06 −3.77
4 Talatisamine 0.52 0.52 1.11 −0.04 0.52 0.95 0.43
2.08 2.09 0.39 0.30 2.13 1.95 2.30
4.17 4.02 0.60 −3.41 4.03 0.98 −3.37
5 Liquiritin apioside 66.67 65.93 0.47 −1.10 66.03 0.91 −0.95
266.67 262.40 0.71 −1.60 261.14 0.73 −2.07
533.33 538.76 0.76 1.02 537.34 0.94 0.75
6 Neoliquiritin 16.67 16.73 0.56 0.35 16.72 0.73 0.32
66.67 68.17 1.00 2.26 69.76 2.27 4.64
133.33 129.65 0.50 −2.77 127.83 1.61 −4.13
7 Liquiritin 33.33 33.10 1.04 −0.70 33.26 1.74 −0.21
133.33 134.85 0.62 1.14 132.80 1.42 −0.40
266.67 270.51 1.27 1.44 270.41 1.33 1.40
8 Isoliquiritin apioside 4.17 4.16 0.69 −0.11 4.20 1.34 0.76
16.67 16.50 0.57 −0.97 16.56 0.74 −0.62
33.33 33.33 0.99 −0.02 33.81 1.41 1.42
9 Benzoylmesaconine 2.08 2.09 0.43 0.20 2.11 1.14 1.35
8.33 8.48 0.88 1.75 8.53 1.80 2.30
16.67 16.45 0.74 −1.30 15.90 2.93 −4.58
10 Isoliquiritin 4.17 4.16 0.40 −0.08 4.15 0.93 −0.52
16.67 16.77 1.03 0.59 16.63 0.96 −0.20
33.33 33.46 1.05 0.39 34.36 2.28 3.07
11 Ononin 16.67 16.88 0.36 1.29 16.87 2.19 1.24
66.67 69.58 0.89 4.37 68.93 1.89 3.40
133.33 135.54 1.03 1.65 134.66 1.32 1.00
12 Benzoylaconine 0.52 0.53 1.29 1.30 0.53 1.06 1.30
2.08 2.17 0.88 4.31 2.16 1.45 3.65
4.17 4.14 0.61 −0.54 4.07 1.89 −2.30
13 Liquiritigenin 1.04 1.05 0.67 0.79 1.06 1.41 1.99
4.17 4.23 0.58 1.53 4.35 2.26 4.45
8.33 8.22 1.12 −1.41 8.09 1.71 −2.95
14 Echinatin 0.52 0.51 0.81 −1.54 0.52 1.28 −0.95
2.08 2.06 1.30 −0.93 2.08 1.76 −0.10
4.17 4.03 1.77 −3.38 4.09 2.25 −1.93
15 Genistein 0.52 0.52 1.43 −0.89 0.52 1.67 −0.90
2.08 2.11 1.15 1.38 2.12 1.53 1.84
4.17 4.03 0.90 −3.16 4.06 1.90 −2.54
16 Isoliquiritigenin 0.26 0.27 1.02 2.26 0.27 1.91 3.03
1.04 1.06 1.60 1.28 1.08 2.96 3.96
2.08 2.04 0.81 −2.17 2.02 2.66 −2.90
17 Formononetin 0.26 0.26 0.99 1.28 0.26 1.30 1.44
1.04 1.06 0.94 1.98 1.07 1.45 2.87
2.08 2.07 0.51 −0.75 2.02 2.04 −2.87
18 Glycyrrhizic acid 66.67 65.20 1.30 −2.19 65.97 1.72 −1.04
266.67 257.30 2.54 −3.51 260.61 1.98 −2.27
533.33 522.38 0.83 −2.05 526.07 1.42 −1.36
19 6-Gingerol 16.67 15.97 0.76 −4.21 16.25 1.67 −2.47
66.67 66.26 0.74 −0.61 66.88 0.98 0.33
133.33 126.84 1.69 −4.87 126.13 2.36 −5.41
20 Glabridin 1.04 1.02 1.26 −2.46 1.01 1.73 −3.26
4.17 4.19 0.93 0.66 4.18 1.73 0.31
8.33 8.07 0.91 −3.18 8.03 2.16 −3.65
21 8-Gingerol 1.04 1.04 0.51 −0.02 1.05 1.10 0.79
4.17 4.20 0.92 0.77 4.30 2.39 3.17
8.33 8.11 0.59 −2.72 7.96 2.14 −4.52
22 6-Shogaol 2.08 2.06 0.67 −0.96 2.10 1.99 0.87
8.33 8.53 1.38 2.38 8.52 1.49 2.21
16.67 16.10 1.53 −3.41 16.02 1.44 −3.88
23 Diacetoxy-6-gingerdiol 1.04 1.04 0.73 −0.19 1.05 1.35 0.35
4.17 4.22 1.17 1.17 4.28 1.65 2.80
8.33 8.02 1.50 −3.82 8.04 1.66 −3.47
24 8-Shogaol 0.52 0.52 0.91 −0.29 0.52 0.85 0.32
2.08 2.11 0.47 1.30 2.12 1.03 1.71
4.17 3.89 1.06 −6.52 4.01 2.85 −3.78
Open in a new tab

a CV: coefficient of variation. b RE: relative error.

Table 5.

Recovery data for the 24 compounds in SYT.

No. Compound Original Conc.
(ng/mL)		 Spiked Conc. (ng/mL) Observed Conc. (ng/mL) Recovery (%) a CV (%)
1 Karacoline 0.52 0.26 0.79 105.16 2.94
1.04 1.67 110.24 2.48
4.17 5.27 114.05 2.11
2 Fuziline 0.34 0.26 0.64 114.55 3.37
1.04 1.50 111.37 2.43
4.17 5.00 111.91 1.64
3 Bullatine B 0.71 0.52 1.29 110.77 2.69
2.08 3.02 110.58 1.89
8.33 10.19 113.67 1.55
4 Talatisamine 0.29 0.26 0.59 112.84 2.28
1.04 1.49 114.57 2.88
4.17 5.13 116.01 2.08
5 Liquiritin apioside 101.62 33.33 135.51 101.67 1.47
133.33 257.50 116.91 2.28
533.33 730.29 117.87 1.57
6 Neoliquiritin 16.43 8.33 25.81 112.54 2.73
33.33 54.18 113.24 3.19
133.33 173.47 117.78 1.58
7 Liquiritin 20.26 16.67 37.49 103.37 2.70
66.67 95.60 113.00 1.43
266.67 328.27 115.50 1.24
8 Isoliquiritin apioside 5.95 2.08 8.12 104.34 2.78
8.33 15.55 115.26 1.98
33.33 45.32 118.11 0.60
9 Benzoylmesaconine 1.53 1.04 2.75 117.07 2.87
4.17 6.19 111.96 2.71
16.67 20.52 113.94 1.43
10 Isoliquiritin 2.86 2.08 5.03 103.98 3.13
8.33 12.52 115.96 1.74
33.33 41.96 117.31 1.03
11 Ononin 10.92 8.33 20.63 116.47 1.68
33.33 50.20 117.82 0.91
133.33 169.68 119.07 0.43
12 Benzoylaconine 0.81 0.26 1.08 102.68 4.75
1.04 1.92 106.26 2.38
4.17 5.36 109.00 1.67
13 Liquiritigenin 1.02 0.52 1.57 105.70 2.73
2.08 3.31 110.19 3.35
8.33 10.53 114.15 1.59
14 Echinatin 0.11 0.26 0.39 108.10 3.70
1.04 1.24 108.17 2.78
4.17 4.61 108.14 2.58
15 Genistein 0.10 0.26 0.41 118.22 1.86
1.04 1.25 110.53 2.35
4.17 4.65 109.37 2.53
16 Isoliquiritigenin 0.12 0.13 0.26 107.60 1.42
0.52 0.69 109.15 2.06
2.08 2.46 112.06 1.47
17 Formononetin 0.08 0.13 0.24 117.86 3.19
0.52 0.67 112.19 2.64
2.08 2.35 108.77 0.63
18 Glycyrrhizic acid 92.53 33.33 123.99 94.39 4.13
133.33 242.88 112.76 2.13
533.33 682.41 110.60 0.82
19 6-Gingerol 14.48 8.33 22.84 100.39 4.27
33.33 49.39 104.74 4.56
133.33 168.54 115.55 2.26
20 Glabridin 0.56 0.52 1.06 96.84 3.81
2.08 2.78 106.63 2.53
8.33 9.97 112.97 2.71
21 8-Gingerol 0.96 0.52 1.47 96.63 2.62
2.08 3.12 103.40 2.43
8.33 9.96 107.99 2.51
22 6-Shogaol 1.69 1.04 2.68 94.88 1.33
4.17 6.08 105.44 3.12
16.67 20.26 111.43 1.47
23 Diacetoxy-6-gingerdiol 0.35 0.52 0.94 112.04 2.67
2.08 2.64 109.82 2.43
8.33 9.65 111.58 2.07
24 8-Shogaol 0.31 0.26 0.57 101.39 1.17
1.04 1.48 112.77 1.28
4.17 5.09 114.80 1.85
Open in a new tab

a Recovery (%) = (Observed concentration − Original concentration)/Spiked concentration × 100.

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.

Table 6.

Contents of the 24 compounds in SYT extracts.

No. Compound Batch 1 Batch 2 Batch 3
Mean ± SD
(mg/g) 		CV (%) Mean ± SD
(mg/g) 		CV (%) Mean ± SD
(mg/g) 		CV (%)
1 Karacoline 0.027 ± 0.001 2.429 0.025 ± 0.001 2.842 0.025 ± 0.001 3.213
2 Fuziline 0.018 ± 0.000 2.179 0.018 ± 0.000 1.057 0.018 ± 0.000 1.356
3 Bullatine B 0.039 ± 0.001 2.080 0.037 ± 0.001 1.489 0.037 ± 0.001 3.156
4 Talatisamine 0.014 ± 0.000 1.936 0.013 ± 0.000 1.862 0.013 ± 0.000 1.852
5 Liquiritin apioside 6.882 ± 0.051 0.746 6.933 ± 0.064 0.923 6.870 ± 0.055 0.802
6 Neoliquiritin 0.885 ± 0.010 1.125 0.824 ± 0.017 2.063 0.814 ± 0.021 2.554
7 Liquiritin 1.303 ± 0.010 0.782 1.324 ± 0.023 1.709 1.331 ± 0.015 1.093
8 Isoliquiritin apioside 0.396 ± 0.005 1.237 0.399 ± 0.005 1.232 0.395 ± 0.004 1.125
9 Benzoylmesaconine 0.080 ± 0.001 1.555 0.077 ± 0.001 1.848 0.076 ± 0.002 2.829
10 Isoliquiritin 0.180 ± 0.002 1.322 0.182 ± 0.002 1.292 0.183 ± 0.002 1.081
11 Ononin 0.637 ± 0.013 2.030 0.599 ± 0.014 2.410 0.599 ± 0.010 1.628
12 Benzoylaconine 0.042 ± 0.000 1.133 0.038 ± 0.001 3.608 0.039 ± 0.001 3.746
13 Liquiritigenin 0.056 ± 0.001 1.695 0.054 ± 0.001 1.499 0.054 ± 0.001 1.941
14 Echinatin 0.006 ± 0.000 2.574 0.006 ± 0.000 2.992 0.006 ± 0.000 2.908
15 Genistein 0.008 ± 0.000 1.668 0.008 ± 0.000 1.894 0.008 ± 0.000 2.455
16 Isoliquiritigenin 0.006 ± 0.000 1.751 0.005 ± 0.000 3.293 0.005 ± 0.000 2.738
17 Formononetin 0.004 ± 0.000 1.441 0.004 ± 0.000 2.809 0.004 ± 0.000 2.144
18 Glycyrrhizic acid 5.540 ± 0.106 1.919 5.418 ± 0.157 2.891 5.505 ± 0.101 1.833
19 6-Gingerol 0.686 ± 0.006 0.929 0.649 ± 0.008 1.204 0.651 ± 0.013 2.073
20 Glabridin 0.031 ± 0.001 2.176 0.031 ± 0.001 2.103 0.031 ± 0.001 2.387
21 8-Gingerol 0.058 ± 0.001 1.848 0.056 ± 0.002 2.719 0.057 ± 0.001 1.376
22 6-Shogaol 0.097 ± 0.002 1.746 0.095 ± 0.001 1.391 0.095 ± 0.002 1.879
23 Diacetoxy-6-gingerdiol 0.020 ± 0.000 0.848 0.020 ± 0.000 0.472 0.020 ± 0.000 0.691
24 8-Shogaol 0.017 ± 0.000 2.243 0.017 ± 0.000 1.802 0.016 ± 0.000 1.834
Open in a new tab

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.

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.

Funding Statement

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

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

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Data Availability Statement

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

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