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Article

Chemical Profile Determination and Quantitative Analysis of Components in Oryeong-san Using UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS

by
Seol Jang
1,
Ami Lee
1,2 and
Youn-Hwan Hwang
1,2,*
1
KM Convergence Research Division, Korea Institute of Oriental Medicine, Yuseong-daero 1672, Yuseong-gu, Daejeon 34054, Republic of Korea
2
Korean Convergence Medical Science Major, KIOM School, University of Science & Technology (UST), Yuseong-gu, Daejeon 34054, Republic of Korea
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(9), 3685; https://doi.org/10.3390/molecules28093685
Submission received: 5 April 2023 / Revised: 19 April 2023 / Accepted: 20 April 2023 / Published: 24 April 2023
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Abstract

:
In this study, a method to both qualitatively and quantitively analyze the components of Oryeong-san (ORS), which is composed of five herbal medicines (Alisma orientale Juzepzuk, Polyporus umbellatus Fries, Atractylodes japonica Koidzumi, Poria cocos Wolf, and Cinnamomum cassia Presl) and is prescribed in traditional Oriental medicine practices, was established for the first time. First, ORS components were profiled using ultra-high-performance liquid chromatography/quadrupole Orbitrap mass spectrometry, and 19 compounds were clearly identified via comparison against reference standard compounds. Subsequently, a quantitative method based on ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry was established to simultaneously measure the identified compounds. Nineteen compounds were accurately quantified using the multiple-reaction-monitoring mode and used to analyze the sample; we confirmed that coumarin was the most abundant compound. The method was validated, achieving good linearity (R2 ≤ 0.9991), recovery (RSD, 0.11–3.15%), and precision (RSD, 0.35–9.44%). The results suggest that this method offers a strategy for accurately and effectively determining the components of ORS, and it can be used for quality assessment and management.

1. Introduction

Oryeong-san (ORS; also known as Wulingsan in China and Gorei-san in Japan) is a traditional Korean prescription manufactured in Sanghanron; it is composed of five herbal medicines (Alisma orientale Juzepzuk, Polyporus umbellatus Fries, Atractylodes japonica Koidzumi, Poria cocos Wolf, and Cinnamomum cassia Presl at the ratio 5:3:3:3:2) [1,2]. ORS is prescribed to promote diuresis, reduce edema, and improve water metabolism in the body [3,4]. In addition, it is widely used in the treatment of renal diseases, and has been reported to improve renal functioning through antihypertensive and antidiabetic effects [5,6,7]. Clinical studies have also reported that ORS is effective in preventing calcium oxalate nephrolithiasis [8]. In addition, ORS was found to reduce gastrointestinal adverse reactions in patients taking selective serotonin reuptake inhibitors (SSRIs), and it was proven to reduce hematoma in patients with chronic subdural hematoma (CSDH) [9,10]. Traditional Oriental medicines (TOMs), which include prescriptions such as ORS, are widely used to prevent diseases, owing to their efficacy and low toxicity [11]. When evaluating the quality of TOMs, the components of various herbal medicines are selected as evaluation indicators; however, these approaches primarily focus on individual herbal medicines. If prescriptions are composed of two or more herbal medicines, their chemical properties may differ from those of a single herbal medicine; this makes it difficult to accurately reflect their characteristics in prescription quality evaluations [12,13,14].
According to Korean Pharmacopoeia [15], the quality control of each herbal medicine and ORS except C. cassia is mainly managed by thin-layer chromatographic analysis. The five individual herbal medicine components in ORS have been reported as follows: triterpenoids (e.g., alisol A) from A. orientale [16], steroids (e.g., polyporusterone A) from P. umbellatus [17], sesquiterpenoids (e.g., atractyloside A) from A. japonica [18], triterpenoids (e.g., 16α-hydroxytrametenolic acid) from P. cocos [19], coumarins (e.g., coumarin), and flavonoids (e.g., procyanidin B1) from C. cassia [20]. In addition, studies have simultaneously determined the components of ORS using high-performance liquid chromatography and liquid chromatography–mass spectrometry (LC-MS) for ORS quality control [1,21,22]. However, these studies are limited to the quantitative analysis of several major components or the screening-based qualitative analysis of all components. Therefore, it is necessary to establish an appropriate analytical method that can simultaneously identify components and determine their contents to facilitate accurate quality control of the ORS.
LC-MS methods are widely used to identify and characterize the chemical compositions of various TOMs, as well as their related preparations [23,24,25]. These methods can facilitate comprehensive chemical profiling; in particular, mass spectrometry using an Orbitrap analyzer offers ion information with low mass errors, thereby facilitating rapid component identification [14]. In addition, triple-quadrupole mass spectrometry (TQ-MS/MS) using multiple-reaction monitoring (MRM) is a highly sensitive and powerful quantitative method offering high throughput [26,27].
Therefore, in this study, an analysis method based upon ultra-high-performance liquid chromatography/quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) was established to identify the components of ORS, and 19 compounds were identified. For the simultaneous quantitative analysis of the identified compounds, an ultra-performance liquid chromatography coupled with a triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS) analysis method using the MRM mode was established and applied for content analysis.

2. Results and Discussion

2.1. Identification of Compounds in ORS via UHPLC-Q-Orbitrap-MS

Qualitative analysis was performed to identify the chemical compounds in ORS; to this end, UHPLC-Q-Orbitrap-MS was used. The chemical compounds in ORS were identified by comparing the retention times and mass spectra of the reference standard compounds, and 19 compounds were identified. The base peak chromatograms of the ORS in both positive and negative ion modes are shown in Figure 1, and detailed information regarding the identified compounds is listed in Table 1. Injection peak was found around 1.5 min, and this peak may be composed of the various eluents of sugars, amino acids, and so on (Figure 1). The apparent peak at 10.4 min, which were only found in UV chromatogram, was not sufficient to identify a certain phytochemical with low intensity and less MS2 fragmentation pattern (Figure 1, upper panel). In this regard, the quantitative analysis of ORS was performed except for the above-mentioned peaks.
Among the chemical compounds identified in ORS, 16 compounds were identical to those reported in previous studies; these are as follows: alisol A, alisol A 24-acetate, alisol B, alisol B 23-acetate, alisol C, and alisol C 23-acetate from A. orientale; polyporusterone A from P. umbellatus; atractyloside A, atractylenolide Ⅰ, atractylenolide Ⅱ, and atractylenolide Ⅲ from A. japonica; 16α-hydroxytrametenolic acid, 3-O-acetyl-16α- hydroxytrametenolic acid, and pachymic acid from P. cocos; and procyanidin B2 and coumarin from C. cassia [5,22,28]. As such, some studies on the pharmacological activities of the compounds identified in ORS have been reported. It has been reported that procyanidin B1, procyanidin B2, and rosavin have antioxidant activities, and coumarin and polyporusterone A have various activities, such as anti-inflammatory, antioxidant, and anticancer activities [17,29,30,31]. Atractyloside A, a compound of A. japonica, and other compounds were also found to have anti-inflammatory activity [18]. In addition, studies have reported that pachymic acid and other compounds that are components of P. cocos, and alisol A and other compounds of A. orientale, have anti-inflammatory and anticancer effects [32,33].

2.2. Quantitative Analysis of Compounds in ORS Using UPLC-TQ-MS/MS

The UPLC-TQ-MS/MS method (in the MRM mode) was used to quantify the compounds in ORS, and 19 compounds were simultaneously detected within 20 min. The MRM mode allows for highly specific and sensitive analyses [34]. A single standard solution of each analyte was injected to investigate the ion pairs (consisting of precursor and product ions in both positive and negative ion modes). Atractyloside A was determined in the negative ion mode, and all remaining compounds were determined in the positive ion mode. For all analytes (including the internal standard, IS), the MRM parameters (including the selected MRM pairs and collision energies) were optimized (the details are listed in Table 2). The chromatograms of the 19 compounds and IS, as obtained in the MRM mode, are shown in Figure 2.
The same precursor ion (m/z 579.1) was selected for procyanidins B1 and B2, and a characteristic fragment ion was produced at m/z 127.0; this was selected as the product ion for each compound [35]. Umbelliferone and coumarin generated precursor ions in the form of [M + H]+ at m/z 163.0 and m/z 147.1, respectively; both compounds formed product ions in the form of [M + H − 2CO]+. Umbelliferone formed a product ion at m/z 107.0, and coumarin formed a product ion at m/z 91.1 as a result of the loss of both CO2 (m/z 44) and C (m/z 12) [36,37,38]. The precursor ions of alisol A and alisol A 24-acetate were formed at m/z 473.3 and m/z 515.3, respectively, in the form of [M + H − H2O]+; however, in the case of the product ion, alisol A was formed at m/z 383.3 in the form of [M + H − H2O − C4H10O2]+, and alisol A 24-acetate was formed at m/z 497.3 in the form of [M + H − 2H2O]+. Similarly, alisol B produced a precursor ion at m/z 455.4 in the form of [M + H − H2O]+ and a product ion at m/z 383.3 in the form of [M + H − H2O − C4H8O]+. In addition, the precursor ions of alisol C and alisol C 23-acetate were produced in the form of [M + H]+ at m/z 487.3 and m/z 529.3, respectively, and alisol C formed a product ion in the form of [M + H − C4H8O]+ at m/z 415.3. For alisol C 23-acetate, the loss of HAc at C-23 and the loss of H2O (18 Da) occurred simultaneously to generate a product ion in the form of [M + H − Hac − H2O]+ at m/z 451.3 [39,40]. The lactone components, atractylenolide I, II, and III, produced precursor ions in the form of [M + H]+ at m/z 231.0, m/z 233.1, and m/z 249.2, respectively. Atractylenolide I and atractylenolide II produced product ions in the form of [M + H − H2O − CO]+ at m/z 185.1 and m/z 187.1, respectively, via the simultaneous loss of H2O and CO groups. Unlike the other two compounds, atractylenolide III formed a product ion in the form of [M + H − H2O]+ at m/z 231.1, owing to the loss of H2O molecules [41,42]. 16α-hydroxytrametenolic acid and 3-O-acetyl-16α-hydroxytrametenolic acid generated precursor ions at m/z 455.4 and m/z 497.3, respectively, in the form of [M + H − H2O]+; both compounds formed product ions at m/z 437.3: 16α-hydroxytrametenolic acid in the form of [M + H − 2H2O]+ and 3-O-acetyl-16α-hydroxytrametenolic acid in the form of [M + H − H2O − CH3COOH]+, respectively [43]. Similarly, pachymic acid generated a precursor ion in the form of [M + H − 2H2O]+ at m/z 511.3 and then formed a product ion in the form of [M + H − H2O − CH3COOH]+ at m/z 451.3, owing to the loss of the AcOH moiety [44].

2.3. Method Validation

Calibration curves for all analytes were plotted against the concentrations of the standard solutions, and the linearity of the analytical method was evaluated using the correlation coefficient of each calibration curve. The correlation coefficient of the calibration curve exhibited good linearity (>0.9991) within the test range. The lower limit of quantitation (LLOQ) was within the range of 0.01–0.20 ng/mL for all compounds. Therefore, it was confirmed that the established method provides a sensitive quantitative analysis of ORS compounds. Detailed information for the regression equation, correlation coefficient (R2), linear range, and LLOQ is listed in Table 3.
Recovery tests were performed by spiking mixed standard solutions of three different concentrations with a known quantity of the sample. As shown in Table 4, the recovery values of the 19 compounds were 89.32–110.32%, and the relative standard deviation (RSD) was less than 3.15, indicating that the accuracy of the quantification method was good.
The precision and accuracy were validated by mixing standard solutions at three concentrations, as shown in Table 5. Intra- and inter-day tests were performed by analyzing a sample prepared six times within the same day and over three consecutive days, respectively. The precision of the method was expressed in terms of the RSD; the intra-day value was less than 7.32%, the inter-day value was less than 9.44%, and the accuracy of the intra- and inter-day values varies as 89.17–112.34% and 88.86–110.20%, respectively.
These results indicate that the established analytical method based on UPLC-TQ-MS/MS can accurately and efficiently quantify the 19 constituent compounds of ORS.

2.4. Sample Analysis

The established UPLC-TQ-MS/MS method was used to simultaneously determine 19 compounds in 3 batches of ORS. The contents of all compounds, as obtained from the calibration curves calculated via the IS method, are shown in Table 6.
Among the 19 compounds measured, the coumarin content was highest (at 7.391–7.683 ng/g) in the 3 batches of ORS; these results resemble those of previously reported studies [2]. In addition to coumarin, the contents of alisol B 23-acetate, atractyloside A, and atractylenolide III were higher in ORS than in the other compounds. These four compounds are derived from C. cassia, A. orientale, and A. japonica (three of the single-herb medicines constituting ORS) and they have been reported as major compounds in previous studies into these individual herbal medicines [18,20,45]. The UPLC-TQ-MS/MS method established in our study was successfully applied to simultaneously quantify the 19 compounds, demonstrating the method’s suitability for component analysis of ORS.

3. Materials and Methods

3.1. Materials and Reagents

The reference standards used in this study were atractyloside A, procyanidin_B1, procyanidin B2, umbelliferon, rosavin, coumarin, alisol C, atractylenolide III, alisol C 23-acetate, atractylenolide II, alisol A, 16α-hydroxytrametenolic acid, atractylenolide I, alisol A 24-acetate, alisol B, 3-O-acetyl-16α-hydroxytrametenolic acid, alisol B 23-acetate, and pachymic acid; these were purchased from ChemFaces (Wuhan, China). Polyporusterone A was purchased from Chem-Norm Biotech (Wuhan, China), and warfarin, an IS, was purchased from Sigma-Aldrich (St. Louis, MO, USA). All reference standards were used with a purity of 98% or higher. MS-grade methanol, acetonitrile, water, and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The five herbal medicines (A. orientale, P. umbellatus, A. japonica, P. cocos, and P. cocos) were purchased from Kwangmyungdang Pharmaceutical (Ulsan, Republic of Korea). Each raw herbal medicine was deposited at the KM Convergence Research Division of the Korea Institute of Oriental Medicine (specimen No. TDC-01, TDC-04, and TDC-06–08).

3.2. Preparation of ORS

ORS extracts were prepared using the method described in a previous study [46]. In total, 5 herbal medicines were combined in the ratios shown in Table 7, and 10 times their total weight of water was added, followed by reflux extraction at 100 °C for 3 h. The extracted water was then filtered and concentrated under reduced pressure using a rotary evaporator. The concentrated aqueous extract was freeze-dried, and the obtained powder sample (yield: 20.3%) was used for analyses.

3.3. Preparation of Standard and Sample Solutions

Nineteen standard compounds and one IS (warfarin) were individually dissolved in methanol to prepare stock solutions. The stock solutions were prepared by mixing aliquots for each solution. Working solutions containing 19 compounds were diluted in methanol to prepare a set of appropriate concentrations. The IS concentration was kept consistent in each sample at 5 ng/mL. Quality control (QC) samples were used for method validation and prepared at high, medium, and low concentrations using the method described above. ORS powder (20 mg) was extracted using methanol in an ultrasonic bath for 30 min. The extract was centrifuged at 12,500 rpm for 15 min and analyzed.

3.4. UHPLC-Q-Orbitrap-MS Conditions for Qualitative Analysis

The UHPLC-Q-Orbitrap-MS method was performed on a Dionex UltiMate 3000 system equipped with a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The analytical conditions for determining the chemical compounds in the ORS matched those of previously reported analytical methods [46]. In brief, the separation was performed on an C18 column (100 × 2.1 mm, 1.7 µm, Acquity BEH C18, Waters, Milford, MA, USA), and a mobile phase (a gradient mixture of 0.1% formic acid in water (A) and acetonitrile (B)) was used. All samples were analyzed in the positive and negative ion conversion modes, and the mass scan was performed in the range 100–1500 m/z. Full scan and MS/MS scan data were acquired at resolutions of 70,000 full width at half maximum and 17,500 in both positive and negative modes, respectively. Xcalibur v.3.0 (Thermo Fisher Scientific, Waltham, MA, USA) was used to acquire and analyze the data.

3.5. UPLC-TQ-MS/MS Conditions for Quantitative Analysis

Quantitative analyses were conducted using an Agilent 1290 Infinity II system interfaced with an Agilent 6495C triple-quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Ionization was performed using a jet-stream electrospray ionization source. The operating conditions for chromatographic separation and mass spectrometric detection were determined using previously reported methods [46]. MRM was performed to quantify the 19 ORS compounds, and the MRM transitions and collision energy values were optimized for each compound (Table 2). All MRM data were acquired and processed using the Agilent MassHunter workstation quantitative analysis software (version 10.1).

3.6. Method Validation of Quantitative Analysis

The UPLC-TQ-MS/MS method used to quantitatively analyze the 19 ORS compounds was validated using linearity, recovery, precision, and accuracy parameters [47]. Linearity was evaluated by constructing a calibration curve for each compound, using the peak area ratio between the analyte concentration and IS. The LLOQ was defined as the lowest concentration in the standard curve that could be measured with acceptable accuracy and precision, and the limit of quantification (LOQ) was determined at a signal to noise (S/N) ratio of 10. To investigate the recovery, different concentrations (high, medium, and low) for each compound were added to the ORS samples. Recovery was evaluated by comparing the spiked and detected quantities of each analyte. Intra- and inter-day variations were performed to evaluate the method precision. QC samples were prepared at three concentration levels, and precision was determined using the relative standard deviation calculated from the measured concentrations. To evaluate intra-day precision, QC samples were measured six times within one day; to evaluate inter-day precision, the same samples were measured on three consecutive days.

4. Conclusions

In this study, the qualitative and quantitative analysis methods UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS, respectively, were applied to comprehensively analyze ORS components. Chemical profiling of ORS was performed using the developed UHPLC-Q-Orbitrap-MS analysis method, and 19 compounds were identified via comparison with reference standard compounds. The 19 identified compounds were simultaneously quantified within 20 min using an established MRM-mode quantitative analysis method; this was successfully applied for practical sample analysis. These results facilitate the qualitative and quantitative analysis of ORS constituents, in turn facilitating the accurate chemical identification and simultaneous determination of compounds. It also aids in the routine analysis of ORS and the identification of biologically active substances, suggesting that it can be effectively applied for overall quality control.

Author Contributions

Conceptualization, Y.-H.H.; investigation, S.J., A.L. and Y.-H.H.; 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: KSN2212020).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflict of interest.

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Molecules 28 03685 g001 550
Figure 1. UHPLC-Q-Orbitrap-MS base peak ion chromatograms of ORS extract. The ID numbers of the various types of phytochemicals are listed in Table 1.
Figure 1. UHPLC-Q-Orbitrap-MS base peak ion chromatograms of ORS extract. The ID numbers of the various types of phytochemicals are listed in Table 1.
Molecules 28 03685 g001
Molecules 28 03685 g002 550
Figure 2. UPLC-TQ-MS/MS chromatograms in MRM mode for (A) mixed-reference solution and (B) ORS extract.
Figure 2. UPLC-TQ-MS/MS chromatograms in MRM mode for (A) mixed-reference solution and (B) ORS extract.
Molecules 28 03685 g002
Table
Table 1. Characterization of chemical constituents of ORS using UHPLC-Q-Orbitrap-MS.
Table 1. Characterization of chemical constituents of ORS using UHPLC-Q-Orbitrap-MS.
No.IdentificationRt
(min)		FormulaAdductPredicted
(m/z)		Measured
(m/z)		Error
(ppm)		MS/MS
(m/z)
1Atractyloside A4.95C21H36O10[M + HCO2]−493.2290493.2293−0.02447.2245, 285.1714,
89.0229
2Procyanidin B15.05C30H26O12[M − H]−577.1351577.1352−0.34407.0771, 289.0720
125.0230
3Procyanidin B25.62C30H26O12[M − H]−577.1351577.1351−0.44407.0774, 289.0721
125.0231
4Umbelliferone7.12C9H6O3[M + H]+163.0390163.0388−0.84163.0388
5Rosavin7.40C20H28O10[M + HCO2]−473.1664473.1661−1.23293.0878, 89.0228
6Coumarin9.09C9H6O2[M + H]+147.0441147.0440−0.44147.0439, 103.0546
7Polyporusterone A12.02C28H46O6[M + H]+479.3365479.3365−0.4095.0861
8Alisol C15.16C30H46O5[M + H]+487.3418487.3417−0.18415.2840
9Atractylenolide III15.79C15H20O3[M + H]+249.1485249.1483−0.88231.1379, 163.0753
10Alisol C 23-acetate17.07C32H48O6[M + H]+529.3524529.35260.44451.3205
11Atractylenolide II17.69C15H20O2[M + H]+233.1536233.1535−0.57233.1535, 215.1431
187.1481, 151.0754
12Alisol A18.12C30H50O5[M + HCO2]−535.3640535.3643−0.01471.3499
1316α-Hydroxytrametenolic acid18.13C30H48O4[M + H]+473.3625473.3625−0.03437.3433, 295.2415
14Atractylenolide I18.79C15H18O2[M + H]+231.1380231.1379−0.27231.1379
15Alisol A 24-acetate18.93C32H52O6[M + HCO2]−577.3746577.3748−0.02169.0408, 59.0122
16Alisol B19.82C30H48O4[M + HCO2]−517.3535517.3535−0.48241.4872, 100.0714
173-O-Acetyl-16α-hydroxytrametenolic acid20.42C32H50O5[M + H]+515.3731515.3730−0.09437.3416, 295.2421
133.0860, 89.0603
18Alisol B 23-acetate20.62C32H50O5[M + H]+515.3731515.3729−0.33339.2672, 151.1116
97.0653
19Pachymic acid20.82C33H52O5[M − H]−527.3742527.3740−0.90527.3741
Table
Table 2. Optimized MRM parameters of the 19 compounds in the UPLC-TQ-MS/MS.
Table 2. Optimized MRM parameters of the 19 compounds in the UPLC-TQ-MS/MS.
No.CompoundRt
(min)		MWMRM Transition
(m/z)		Collision
Energy (V)
1Atractyloside A3.63448.5493.2 → 447.214
2Procyanidin B13.68578.5579.1 → 127.030
3Procyanidin B24.09578.5579.1 → 127.030
4Umbelliferone5.67162.1163.0 → 107.022
5Rosavin5.84428.4446.2 → 117.014
6Coumarin7.55146.1147.1 → 91.126
7Polyporusterone A10.34478.7479.3 → 95.130
8Alisol C13.48486.7487.3 → 415.318
9Atractylenolide III14.18248.3249.2 → 231.110
10Alisol C 23-acetate15.59528.7529.3 → 451.318
11Atractylenolide II16.28232.3233.1 → 187.114
12Alisol A16.62490.7473.3 → 383.310
1316α-Hydroxytrametenolic acid16.71472.7455.4 → 437.318
14Atractylenolide I17.40230.3231.0 → 185.118
15Alisol A 24-acetate17.50532.8515.3 → 497.310
16Alisol B18.41472.7455.4 → 383.310
173-O-Acetyl-16α-hydroxytrametenolic acid19.06514.7497.3 → 437.318
18Alisol B 23-acetate19.24514.7497.4 → 201.122
19Pachymic acid19.45528.8511.3 → 451.318
IS1Warfarin13.64307.1309.0 → 163.014
IS2Warfarin13.64307.1307.0 → 250.022
Table
Table 3. Calibration curves, linear range, and lower limit of quantification (LLOQ) for the 19 compounds.
Table 3. Calibration curves, linear range, and lower limit of quantification (LLOQ) for the 19 compounds.
No.CompoundCalibration CurvesR2Linear Range
(ng/mL)		LLOQ (ng/mL)
1Atractyloside Ay = 1.5739x + 0.04180.99950.10–25.000.10
2Procyanidin B1y = 0.2186x − 0.00940.99910.10–12.500.10
3Procyanidin B2y = 0.2277x − 0.00830.99940.10–12.500.10
4Umbelliferoney = 3.7074x − 0.00120.99960.01–3.130.01
5Rosaviny = 2.4065x − 0.01080.99930.02–3.130.02
6Coumariny = 3.9901x + 0.19950.99940.10–50.000.10
7Polyporusterone Ay = 0.9290x − 0.00150.99980.10–25.000.10
8Alisol Cy = 1.2274x − 0.00990.99930.05–6.250.05
9Atractylenolide IIIy = 2.9593x − 0.03900.99960.10–25.000.10
10Alisol C 23-acetatey = 5.9883x + 0.02330.99920.01–3.130.01
11Atractylenolide IIy = 7.2963x + 0.02350.99950.01–3.130.01
12Alisol Ay = 1.4905x − 0.02960.99950.10–25.000.10
1316α-Hydroxytrametenolic acidy = 0.6745x − 0.00430.99910.02–3.130.02
14Atractylenolide Iy = 3.8661x + 0.00910.99930.01–3.130.01
15Alisol A 24-acetatey = 1.2214x − 0.06930.99930.20–25.000.20
16Alisol By = 0.2495x − 0.00750.99920.20–25.000.20
173-O-Acetyl-16α-hydroxytrametenolic acidy = 0.5743x − 0.00360.99940.02–3.130.02
18Alisol B 23-acetatey = 0.5967x + 0.00130.99920.05–12.500.05
19Pachymic acidy = 1.1861x − 0.01560.99910.05–6.250.05
Table
Table 4. Recovery of the 19 compounds in ORS.
Table 4. Recovery of the 19 compounds in ORS.
No.CompoundSpiked Concentration
(ng/mL)		Measured Concentration
(ng/mL)		Recovery
(%)		RSD
(%)
1Atractyloside A10.67110.16095.211.09
4.4214.34998.390.90
2.8582.891101.171.67
2Procyanidin B14.3513.99891.891.18
1.2261.245101.601.29
0.4450.468105.291.13
3Procyanidin B24.4583.98289.320.91
1.3331.25594.112.38
0.5520.55099.601.33
4Umbelliferone1.0511.01196.200.81
0.2700.272100.931.30
0.0750.06992.961.73
5Rosavin1.2231.21599.391.09
0.4420.43899.090.76
0.2460.23896.821.64
6Coumarin11.40410.35490.790.97
5.1545.686110.320.47
3.5923.713103.370.91
7Polyporusterone A8.4297.87493.411.02
2.1792.15498.860.66
0.6170.56892.020.61
8Alisol C2.3222.28198.221.20
0.7590.75699.551.25
0.3690.36398.440.63
9Atractylenolide III10.49110.00695.370.83
4.2414.16498.191.08
2.6782.62998.140.70
10Alisol C 23-acetate2.3122.359102.030.56
1.5311.590103.900.73
1.3351.456109.020.99
11Atractylenolide II1.4821.46398.712.57
0.7000.716102.231.98
0.5050.537106.313.15
12Alisol A8.9278.47594.940.44
2.6772.52194.210.51
1.1141.05094.280.87
1316α-Hydroxytrametenolic acid1.0671.05899.181.32
0.2860.28097.951.53
0.0910.08492.763.07
14Atractylenolide I1.1151.03692.901.81
0.3340.33499.951.47
0.1390.146105.170.48
15Alisol A 24-acetate8.6357.76689.940.11
2.3852.21492.840.65
0.8220.77694.430.43
16Alisol B8.9668.62096.141.17
2.7162.51792.671.71
1.1531.05591.512.49
173-O-Acetyl-16α-hydroxytrametenolic acid1.0941.06997.661.06
0.3130.30898.290.65
0.1180.10790.682.20
18Alisol B 23-acetate6.9906.70995.981.05
3.8653.890100.651.56
3.0843.171102.831.30
19Pachymic acid2.1862.09295.681.36
0.6240.60396.661.11
0.2330.22797.362.88
Table
Table 5. Precision and accuracy of the 19 compounds in ORS.
Table 5. Precision and accuracy of the 19 compounds in ORS.
No.CompoundConcentration
(ng/mL)		Intra-DayInter-Day
Precision
(%)		Accuracy
(%)		Precision
(%)		Accuracy
(%)
1Atractyloside A16.671.39105.133.01102.02
4.172.46105.613.97101.06
1.042.42108.637.03101.57
2Procyanidin B18.331.7396.242.5396.19
2.087.3291.290.4090.99
0.521.7294.259.4497.56
3Procyanidin B28.331.4396.551.9998.25
2.081.5289.431.8288.86
0.524.3192.040.9791.15
4Umbelliferone2.081.14104.941.01106.14
0.520.77102.782.25105.50
0.130.8795.944.4195.53
5Rosavin2.080.7296.174.0998.06
0.521.3595.382.9596.38
0.130.9899.753.5198.32
6Coumarin16.670.44103.093.35107.23
4.171.52108.421.66107.39
1.040.95108.282.40106.47
7Polyporusterone A16.670.65107.986.53105.20
4.170.59104.155.62102.74
1.040.9793.653.4997.52
8Alisol C4.171.23101.400.35101.56
1.041.36104.581.65103.01
0.261.28104.094.13103.30
9Atractylenolide III16.671.36109.942.78106.52
4.170.77106.081.32104.49
1.040.6490.991.7092.68
10Alisol C 23-acetate2.080.93108.626.55104.88
0.520.55108.143.27106.80
0.131.1498.571.7999.63
11Atractylenolide II2.081.33109.623.45106.47
0.523.25112.342.87110.20
0.132.6299.072.1198.06
12Alisol A16.670.85106.823.93103.79
4.170.96102.064.2999.09
1.040.9492.372.6594.02
1316α-Hydroxytrametenolic acid2.082.29100.729.09101.95
0.522.6789.176.9293.24
0.133.9198.913.2797.03
14Atractylenolide I2.080.62107.661.80105.46
0.521.14109.944.81107.22
0.132.5793.701.5295.15
15Alisol A 24-acetate16.670.76111.726.84108.29
4.170.8399.215.5097.96
1.041.5293.603.0594.06
16Alisol B16.671.48109.185.11106.96
4.171.33101.204.1899.03
1.041.2394.371.1293.73
173-O-Acetyl-16α-hydroxytrametenolic acid2.081.1693.663.9096.65
0.521.75101.845.6295.74
0.131.09100.282.2998.15
18Alisol B 23-acetate8.331.06110.557.93106.71
2.083.74111.318.95106.38
0.522.3892.111.8393.99
19Pachymic acid4.171.5695.047.2298.00
1.040.9894.443.0292.49
0.261.0495.462.3798.11
Table
Table 6. Contents of the 19 compounds in 3 batches of ORS.
Table 6. Contents of the 19 compounds in 3 batches of ORS.
No.CompoundORS-1ORS-2ORS-3
Mean
(ng/g)		SDCV
(%)		Mean
(ng/g)		SDCV
(%)		Mean
(ng/g)		SDCV
(%)
1Atractyloside A3.6970.0571.5493.5110.0601.7223.6990.0431.171
2Procyanidin B10.2650.0041.3200.2630.0010.3490.2640.0041.514
3Procyanidin B20.5500.0132.3960.5250.0101.9330.5540.0203.542
4Umbelliferone0.0220.0002.1000.0230.0012.5030.0230.0012.341
5Rosavin0.2760.0041.5300.2640.0062.4030.2800.0010.226
6Coumarin7.6110.0660.8737.3910.1001.3487.6830.0821.070
7Polyporusterone A0.2390.0041.5280.2310.0031.2220.2420.0041.574
8Alisol C0.3950.0041.1120.3820.0071.8430.4000.0061.390
9Atractylenolide III3.2080.0902.8113.1170.0501.6013.2770.0561.701
10Alisol C 23-acetate2.5400.0160.6452.4370.0150.6062.5730.0281.079
11Atractylenolide II0.7490.0091.2560.7260.0152.0560.7700.0141.857
12Alisol A1.0270.0131.2260.9940.0090.8711.0450.0141.353
1316α-Hydroxytrametenolic acid0.0700.0033.8470.0680.0034.2640.0700.0034.123
14Atractylenolide I0.1080.0032.9790.1040.0021.7370.1080.0043.866
15Alisol A 24-acetate0.6890.0040.5390.6800.0081.1500.6940.0050.690
16Alisol B1.1200.0363.2251.0780.0181.6471.1340.0242.095
173-O-Acetyl-16α-hydroxytrametenolic acid0.1020.0011.4200.0990.0021.9230.1010.0054.674
18Alisol B 23-acetate4.4060.0922.0844.1810.2265.4034.4330.0711.602
19Pachymic acid0.2120.0031.3160.2060.0052.3280.2150.0042.037
Table
Table 7. Composition of ORS.
Table 7. Composition of ORS.
Scientific NameScientific NameWeight Ratio
Alismatis RhizomaAlisma orientale Juzepzuk5.0
PolyporusPolyporus umbellatus Fries3.0
Atractylodis Rhizoma AlbaAtractylodes japonica Koidzumi3.0
Poria SclerotiumPoria cocos Wolf3.0
Cinnamomi CortexCinnamomum cassia Presl2.0
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Jang, S.; Lee, A.; Hwang, Y.-H. Chemical Profile Determination and Quantitative Analysis of Components in Oryeong-san Using UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS. Molecules 2023, 28, 3685. https://doi.org/10.3390/molecules28093685

AMA Style

Jang S, Lee A, Hwang Y-H. Chemical Profile Determination and Quantitative Analysis of Components in Oryeong-san Using UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS. Molecules. 2023; 28(9):3685. https://doi.org/10.3390/molecules28093685

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Jang, Seol, Ami Lee, and Youn-Hwan Hwang. 2023. "Chemical Profile Determination and Quantitative Analysis of Components in Oryeong-san Using UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS" Molecules 28, no. 9: 3685. https://doi.org/10.3390/molecules28093685

APA Style

Jang, S., Lee, A., & Hwang, Y. -H. (2023). Chemical Profile Determination and Quantitative Analysis of Components in Oryeong-san Using UHPLC-Q-Orbitrap-MS and UPLC-TQ-MS/MS. Molecules, 28(9), 3685. https://doi.org/10.3390/molecules28093685

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