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. 2023 Apr 24;28(9):3685. doi: 10.3390/molecules28093685

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

Seol Jang 1, Ami Lee 1,2, Youn-Hwan Hwang 1,2,*
Editor: Wilfried Rozhon
PMCID: PMC10180092  PMID: 37175095

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.

Keywords: Oryeong-san, UHPLC-Q-Orbitrap-MS, UPLC-TQ-MS/MS, quality control

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.

Figure 1.

Figure 1

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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.

Table 1.

Characterization of chemical constituents of ORS using UHPLC-Q-Orbitrap-MS.

No. Identification Rt
(min)		 Formula Adduct Predicted
(m/z)		 Measured
(m/z)		 Error
(ppm)		 MS/MS
(m/z)
1 Atractyloside A 4.95 C21H36O10 [M + HCO2]− 493.2290 493.2293 −0.02 447.2245, 285.1714,
89.0229
2 Procyanidin B1 5.05 C30H26O12 [M − H]− 577.1351 577.1352 −0.34 407.0771, 289.0720
125.0230
3 Procyanidin B2 5.62 C30H26O12 [M − H]− 577.1351 577.1351 −0.44 407.0774, 289.0721
125.0231
4 Umbelliferone 7.12 C9H6O3 [M + H]+ 163.0390 163.0388 −0.84 163.0388
5 Rosavin 7.40 C20H28O10 [M + HCO2]− 473.1664 473.1661 −1.23 293.0878, 89.0228
6 Coumarin 9.09 C9H6O2 [M + H]+ 147.0441 147.0440 −0.44 147.0439, 103.0546
7 Polyporusterone A 12.02 C28H46O6 [M + H]+ 479.3365 479.3365 −0.40 95.0861
8 Alisol C 15.16 C30H46O5 [M + H]+ 487.3418 487.3417 −0.18 415.2840
9 Atractylenolide III 15.79 C15H20O3 [M + H]+ 249.1485 249.1483 −0.88 231.1379, 163.0753
10 Alisol C 23-acetate 17.07 C32H48O6 [M + H]+ 529.3524 529.3526 0.44 451.3205
11 Atractylenolide II 17.69 C15H20O2 [M + H]+ 233.1536 233.1535 −0.57 233.1535, 215.1431
187.1481, 151.0754
12 Alisol A 18.12 C30H50O5 [M + HCO2]− 535.3640 535.3643 −0.01 471.3499
13 16α-Hydroxytrametenolic acid 18.13 C30H48O4 [M + H]+ 473.3625 473.3625 −0.03 437.3433, 295.2415
14 Atractylenolide I 18.79 C15H18O2 [M + H]+ 231.1380 231.1379 −0.27 231.1379
15 Alisol A 24-acetate 18.93 C32H52O6 [M + HCO2]− 577.3746 577.3748 −0.02 169.0408, 59.0122
16 Alisol B 19.82 C30H48O4 [M + HCO2]− 517.3535 517.3535 −0.48 241.4872, 100.0714
17 3-O-Acetyl-16α-hydroxytrametenolic acid 20.42 C32H50O5 [M + H]+ 515.3731 515.3730 −0.09 437.3416, 295.2421
133.0860, 89.0603
18 Alisol B 23-acetate 20.62 C32H50O5 [M + H]+ 515.3731 515.3729 −0.33 339.2672, 151.1116
97.0653
19 Pachymic acid 20.82 C33H52O5 [M − H]− 527.3742 527.3740 −0.90 527.3741
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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.

Table 2.

Optimized MRM parameters of the 19 compounds in the UPLC-TQ-MS/MS.

No. Compound Rt
(min)		 MW MRM Transition
(m/z)		 Collision
Energy (V)
1 Atractyloside A 3.63 448.5 493.2 → 447.2 14
2 Procyanidin B1 3.68 578.5 579.1 → 127.0 30
3 Procyanidin B2 4.09 578.5 579.1 → 127.0 30
4 Umbelliferone 5.67 162.1 163.0 → 107.0 22
5 Rosavin 5.84 428.4 446.2 → 117.0 14
6 Coumarin 7.55 146.1 147.1 → 91.1 26
7 Polyporusterone A 10.34 478.7 479.3 → 95.1 30
8 Alisol C 13.48 486.7 487.3 → 415.3 18
9 Atractylenolide III 14.18 248.3 249.2 → 231.1 10
10 Alisol C 23-acetate 15.59 528.7 529.3 → 451.3 18
11 Atractylenolide II 16.28 232.3 233.1 → 187.1 14
12 Alisol A 16.62 490.7 473.3 → 383.3 10
13 16α-Hydroxytrametenolic acid 16.71 472.7 455.4 → 437.3 18
14 Atractylenolide I 17.40 230.3 231.0 → 185.1 18
15 Alisol A 24-acetate 17.50 532.8 515.3 → 497.3 10
16 Alisol B 18.41 472.7 455.4 → 383.3 10
17 3-O-Acetyl-16α-hydroxytrametenolic acid 19.06 514.7 497.3 → 437.3 18
18 Alisol B 23-acetate 19.24 514.7 497.4 → 201.1 22
19 Pachymic acid 19.45 528.8 511.3 → 451.3 18
IS1 Warfarin 13.64 307.1 309.0 → 163.0 14
IS2 Warfarin 13.64 307.1 307.0 → 250.0 22
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Figure 2.

Figure 2

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UPLC-TQ-MS/MS chromatograms in MRM mode for (A) mixed-reference solution and (B) ORS extract.

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.

Table 3.

Calibration curves, linear range, and lower limit of quantification (LLOQ) for the 19 compounds.

No. Compound Calibration Curves R2 Linear Range
(ng/mL)		 LLOQ (ng/mL)
1 Atractyloside A y = 1.5739x + 0.0418 0.9995 0.10–25.00 0.10
2 Procyanidin B1 y = 0.2186x − 0.0094 0.9991 0.10–12.50 0.10
3 Procyanidin B2 y = 0.2277x − 0.0083 0.9994 0.10–12.50 0.10
4 Umbelliferone y = 3.7074x − 0.0012 0.9996 0.01–3.13 0.01
5 Rosavin y = 2.4065x − 0.0108 0.9993 0.02–3.13 0.02
6 Coumarin y = 3.9901x + 0.1995 0.9994 0.10–50.00 0.10
7 Polyporusterone A y = 0.9290x − 0.0015 0.9998 0.10–25.00 0.10
8 Alisol C y = 1.2274x − 0.0099 0.9993 0.05–6.25 0.05
9 Atractylenolide III y = 2.9593x − 0.0390 0.9996 0.10–25.00 0.10
10 Alisol C 23-acetate y = 5.9883x + 0.0233 0.9992 0.01–3.13 0.01
11 Atractylenolide II y = 7.2963x + 0.0235 0.9995 0.01–3.13 0.01
12 Alisol A y = 1.4905x − 0.0296 0.9995 0.10–25.00 0.10
13 16α-Hydroxytrametenolic acid y = 0.6745x − 0.0043 0.9991 0.02–3.13 0.02
14 Atractylenolide I y = 3.8661x + 0.0091 0.9993 0.01–3.13 0.01
15 Alisol A 24-acetate y = 1.2214x − 0.0693 0.9993 0.20–25.00 0.20
16 Alisol B y = 0.2495x − 0.0075 0.9992 0.20–25.00 0.20
17 3-O-Acetyl-16α-hydroxytrametenolic acid y = 0.5743x − 0.0036 0.9994 0.02–3.13 0.02
18 Alisol B 23-acetate y = 0.5967x + 0.0013 0.9992 0.05–12.50 0.05
19 Pachymic acid y = 1.1861x − 0.0156 0.9991 0.05–6.25 0.05
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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.

Table 4.

Recovery of the 19 compounds in ORS.

No. Compound Spiked Concentration
(ng/mL)		 Measured Concentration
(ng/mL)		 Recovery
(%)		 RSD
(%)
1 Atractyloside A 10.671 10.160 95.21 1.09
4.421 4.349 98.39 0.90
2.858 2.891 101.17 1.67
2 Procyanidin B1 4.351 3.998 91.89 1.18
1.226 1.245 101.60 1.29
0.445 0.468 105.29 1.13
3 Procyanidin B2 4.458 3.982 89.32 0.91
1.333 1.255 94.11 2.38
0.552 0.550 99.60 1.33
4 Umbelliferone 1.051 1.011 96.20 0.81
0.270 0.272 100.93 1.30
0.075 0.069 92.96 1.73
5 Rosavin 1.223 1.215 99.39 1.09
0.442 0.438 99.09 0.76
0.246 0.238 96.82 1.64
6 Coumarin 11.404 10.354 90.79 0.97
5.154 5.686 110.32 0.47
3.592 3.713 103.37 0.91
7 Polyporusterone A 8.429 7.874 93.41 1.02
2.179 2.154 98.86 0.66
0.617 0.568 92.02 0.61
8 Alisol C 2.322 2.281 98.22 1.20
0.759 0.756 99.55 1.25
0.369 0.363 98.44 0.63
9 Atractylenolide III 10.491 10.006 95.37 0.83
4.241 4.164 98.19 1.08
2.678 2.629 98.14 0.70
10 Alisol C 23-acetate 2.312 2.359 102.03 0.56
1.531 1.590 103.90 0.73
1.335 1.456 109.02 0.99
11 Atractylenolide II 1.482 1.463 98.71 2.57
0.700 0.716 102.23 1.98
0.505 0.537 106.31 3.15
12 Alisol A 8.927 8.475 94.94 0.44
2.677 2.521 94.21 0.51
1.114 1.050 94.28 0.87
13 16α-Hydroxytrametenolic acid 1.067 1.058 99.18 1.32
0.286 0.280 97.95 1.53
0.091 0.084 92.76 3.07
14 Atractylenolide I 1.115 1.036 92.90 1.81
0.334 0.334 99.95 1.47
0.139 0.146 105.17 0.48
15 Alisol A 24-acetate 8.635 7.766 89.94 0.11
2.385 2.214 92.84 0.65
0.822 0.776 94.43 0.43
16 Alisol B 8.966 8.620 96.14 1.17
2.716 2.517 92.67 1.71
1.153 1.055 91.51 2.49
17 3-O-Acetyl-16α-hydroxytrametenolic acid 1.094 1.069 97.66 1.06
0.313 0.308 98.29 0.65
0.118 0.107 90.68 2.20
18 Alisol B 23-acetate 6.990 6.709 95.98 1.05
3.865 3.890 100.65 1.56
3.084 3.171 102.83 1.30
19 Pachymic acid 2.186 2.092 95.68 1.36
0.624 0.603 96.66 1.11
0.233 0.227 97.36 2.88
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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.

Table 5.

Precision and accuracy of the 19 compounds in ORS.

No. Compound Concentration
(ng/mL)		 Intra-Day Inter-Day
Precision
(%)		 Accuracy
(%)		 Precision
(%)		 Accuracy
(%)
1 Atractyloside A 16.67 1.39 105.13 3.01 102.02
4.17 2.46 105.61 3.97 101.06
1.04 2.42 108.63 7.03 101.57
2 Procyanidin B1 8.33 1.73 96.24 2.53 96.19
2.08 7.32 91.29 0.40 90.99
0.52 1.72 94.25 9.44 97.56
3 Procyanidin B2 8.33 1.43 96.55 1.99 98.25
2.08 1.52 89.43 1.82 88.86
0.52 4.31 92.04 0.97 91.15
4 Umbelliferone 2.08 1.14 104.94 1.01 106.14
0.52 0.77 102.78 2.25 105.50
0.13 0.87 95.94 4.41 95.53
5 Rosavin 2.08 0.72 96.17 4.09 98.06
0.52 1.35 95.38 2.95 96.38
0.13 0.98 99.75 3.51 98.32
6 Coumarin 16.67 0.44 103.09 3.35 107.23
4.17 1.52 108.42 1.66 107.39
1.04 0.95 108.28 2.40 106.47
7 Polyporusterone A 16.67 0.65 107.98 6.53 105.20
4.17 0.59 104.15 5.62 102.74
1.04 0.97 93.65 3.49 97.52
8 Alisol C 4.17 1.23 101.40 0.35 101.56
1.04 1.36 104.58 1.65 103.01
0.26 1.28 104.09 4.13 103.30
9 Atractylenolide III 16.67 1.36 109.94 2.78 106.52
4.17 0.77 106.08 1.32 104.49
1.04 0.64 90.99 1.70 92.68
10 Alisol C 23-acetate 2.08 0.93 108.62 6.55 104.88
0.52 0.55 108.14 3.27 106.80
0.13 1.14 98.57 1.79 99.63
11 Atractylenolide II 2.08 1.33 109.62 3.45 106.47
0.52 3.25 112.34 2.87 110.20
0.13 2.62 99.07 2.11 98.06
12 Alisol A 16.67 0.85 106.82 3.93 103.79
4.17 0.96 102.06 4.29 99.09
1.04 0.94 92.37 2.65 94.02
13 16α-Hydroxytrametenolic acid 2.08 2.29 100.72 9.09 101.95
0.52 2.67 89.17 6.92 93.24
0.13 3.91 98.91 3.27 97.03
14 Atractylenolide I 2.08 0.62 107.66 1.80 105.46
0.52 1.14 109.94 4.81 107.22
0.13 2.57 93.70 1.52 95.15
15 Alisol A 24-acetate 16.67 0.76 111.72 6.84 108.29
4.17 0.83 99.21 5.50 97.96
1.04 1.52 93.60 3.05 94.06
16 Alisol B 16.67 1.48 109.18 5.11 106.96
4.17 1.33 101.20 4.18 99.03
1.04 1.23 94.37 1.12 93.73
17 3-O-Acetyl-16α-hydroxytrametenolic acid 2.08 1.16 93.66 3.90 96.65
0.52 1.75 101.84 5.62 95.74
0.13 1.09 100.28 2.29 98.15
18 Alisol B 23-acetate 8.33 1.06 110.55 7.93 106.71
2.08 3.74 111.31 8.95 106.38
0.52 2.38 92.11 1.83 93.99
19 Pachymic acid 4.17 1.56 95.04 7.22 98.00
1.04 0.98 94.44 3.02 92.49
0.26 1.04 95.46 2.37 98.11
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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.

Table 6.

Contents of the 19 compounds in 3 batches of ORS.

No. Compound ORS-1 ORS-2 ORS-3
Mean
(ng/g)		 SD CV
(%)		 Mean
(ng/g)		 SD CV
(%)		 Mean
(ng/g)		 SD CV
(%)
1 Atractyloside A 3.697 0.057 1.549 3.511 0.060 1.722 3.699 0.043 1.171
2 Procyanidin B1 0.265 0.004 1.320 0.263 0.001 0.349 0.264 0.004 1.514
3 Procyanidin B2 0.550 0.013 2.396 0.525 0.010 1.933 0.554 0.020 3.542
4 Umbelliferone 0.022 0.000 2.100 0.023 0.001 2.503 0.023 0.001 2.341
5 Rosavin 0.276 0.004 1.530 0.264 0.006 2.403 0.280 0.001 0.226
6 Coumarin 7.611 0.066 0.873 7.391 0.100 1.348 7.683 0.082 1.070
7 Polyporusterone A 0.239 0.004 1.528 0.231 0.003 1.222 0.242 0.004 1.574
8 Alisol C 0.395 0.004 1.112 0.382 0.007 1.843 0.400 0.006 1.390
9 Atractylenolide III 3.208 0.090 2.811 3.117 0.050 1.601 3.277 0.056 1.701
10 Alisol C 23-acetate 2.540 0.016 0.645 2.437 0.015 0.606 2.573 0.028 1.079
11 Atractylenolide II 0.749 0.009 1.256 0.726 0.015 2.056 0.770 0.014 1.857
12 Alisol A 1.027 0.013 1.226 0.994 0.009 0.871 1.045 0.014 1.353
13 16α-Hydroxytrametenolic acid 0.070 0.003 3.847 0.068 0.003 4.264 0.070 0.003 4.123
14 Atractylenolide I 0.108 0.003 2.979 0.104 0.002 1.737 0.108 0.004 3.866
15 Alisol A 24-acetate 0.689 0.004 0.539 0.680 0.008 1.150 0.694 0.005 0.690
16 Alisol B 1.120 0.036 3.225 1.078 0.018 1.647 1.134 0.024 2.095
17 3-O-Acetyl-16α-hydroxytrametenolic acid 0.102 0.001 1.420 0.099 0.002 1.923 0.101 0.005 4.674
18 Alisol B 23-acetate 4.406 0.092 2.084 4.181 0.226 5.403 4.433 0.071 1.602
19 Pachymic acid 0.212 0.003 1.316 0.206 0.005 2.328 0.215 0.004 2.037
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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.

Table 7.

Composition of ORS.

Scientific Name Scientific Name Weight Ratio
Alismatis Rhizoma Alisma orientale Juzepzuk 5.0
Polyporus Polyporus umbellatus Fries 3.0
Atractylodis Rhizoma Alba Atractylodes japonica Koidzumi 3.0
Poria Sclerotium Poria cocos Wolf 3.0
Cinnamomi Cortex Cinnamomum cassia Presl 2.0
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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.

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.

Funding Statement

This research was funded by the Korea Institute of Oriental Medicine (grant number: KSN2212020).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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

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