Simultaneous Determination of 12 Marker Components in Yeonkyopaedok-san Using HPLC–PDA and LC–MS/MS

Abstract

Yeonkyopaedok-san is a traditional Korean medicine used in the early treatment of boils. In the present study, its 12 marker components for quality control were determined using high-performance liquid chromatography (HPLC) with photodiode array detection and ultra-performance liquid chromatography–mass spectrometry with tandem mass spectrometry (UPLC–MS/MS). The investigated 12 marker components of Yeonkyopaedok-san were as follows: 3-caffeoylquinic acid, cimifugin 7-glucoside, liquiritin apioside, ferulic acid, narirutin, 5-O-methylvisammioside, naringin, neohesperidin, oxypeucedanin hydrate, arctigenin, glycyrrhizic acid, and 6-gingerol. The analytical column used for the separation of the 12 marker analytes in Yeonkyopaedok-san was a Waters SunFire C18 column (4.6 mm × 250 mm, 5 μm). The two mobile phases used were 0.1% (v/v) aqueous formic acid and 0.1% (v/v) formic acid in acetonitrile. In the UPLC–MS/MS analysis, all components were separated using a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The two mobile phases used were 0.1% (v/v) aqueous formic acid and acetonitrile. The coefficient of determination of the calibration curves in both analysis systems showed good linearity (>0.99). The amounts of the 12 marker components in Yeonkyopaedok-san determined using HPLC–photodiode array detection and UPLC–MS/MS analyses were found to be 0.14–9.00 mg/g and 2.35–853.11 μg/g, respectively.

1. Introduction

Traditional Korean medicine (TKM), traditional Chinese medicine (TCM), and Kampo medicine (KM) generally use chemically active complexes composed of two or more medicinal herbs. They have been used for thousands of years for the prevention or treatment of various diseases. However, scientific evidence pertaining to the safety and standardization of TKM, TCM, and KM is lacking; this information is now required. Yeonkyopaedok-san (YPS), also known as Lian Qiao Bai Du San in Chinese, is a TKM. It is made up of extracts from the following 14 herbs: Forsythiae Fructus, Lonicerae Flos, Schizonepetae Spica, Saposhnikoviae Radix, Osterici seu Notopterygii Radix et Rhizoma, Araliae Continentalis Radix, Bupleuri Radix, Cnidii Rhizoma, Aurantii Fructus Immaturus, Platycodonis Radix, Poria Sclerotium, Glycyrrhizae Radix et Rhizoma, Menthae Herba, and Zingiberis Rhizoma Recens [1].

As reported in the Dongeuibogam (representative source of the Korean medicine), YPS has been used in the initial treatment of boils. It has anti-inflammatory effects (i.e., for treatment of colds, allergies, and dermatitis) and antiatherogenic effects [1,2,3]. Recently, Chen et al. [4,5] reported a high-performance liquid chromatography (HPLC)–diode array detection and micellar electrokinetic chromatography assays for quality control of Lianqiao Baidu pills (LBP). In China, LBP is used for the treatment of diseases similar to those treated by YPS in Korea [4,5]. However, LBP is a completely different herbal formulation, consisting of 19 herbal medicines [6]. Although some studies on the efficacy of the TKM and YPS have been reported, there are no documented standardization studies which include a quality evaluation.

The chemical composition of the main 14 raw materials constituting YPS is as follows: lignans (e.g., arctigenin (ARC) and pinoresinol) from Forsythiae Fructus [7,8], phenylpropanoids (e.g., chlorogenic acid) from Lonicerae Flos [9], monoterpenoids (e.g., pulegone) from Schizonepetae Spica [10], flavonoids (e.g., cimifugin) and chromones (e.g., cimifugin 7-glucoside (CFG) and 5-O-methylvisammioside (5-MVS)) from Saposhnikoviae Radix [11], coumarins (e.g., oxypeucedanin hydrate (OPDH) and imperatorin) from Osterici seu Notopterygii Radix et Rhizoma [12], diterpenoids (e.g., kaurenoic acid and continentalic acid) from Araliae Continentalis Radix [13], triterpenoid saponins (e.g., saikosaponin A) from Bupleuri Radix [14], phenylpropanoids (e.g., ferulic acid (FA)) from Cnidii Rhizoma [15], flavonoids (e.g., naringin (NAG)) and flavonoid glucosides (e.g., NAG, and neohesperidin (NHES)) from Aurantii Fructus Immaturus [16], triterpenoid saponins (e.g., platycodin D and platycodin D2) from Platycodonis Radix [17], triterpenoids (e.g., polyporenic acid C and pachymic acid) from Poria Sclerotium [18], triterpenoid saponins (e.g., glycyrrhizinic acid) and flavonoids (e.g., liquiritin apioside) from Glycyrrhizae Radix et Rhizoma [19], monoterpenoids (e.g., menthone and menthol) from Menthae Herba [20], and phenols (e.g., 6-, 8, and 10-gingerol) from Zingiberis Rhizoma Recens [21].

In this study, a simultaneous analysis of the 12 marker compounds for a quality assessment of YPS was carried out using HPLC–photodiode array detection (PDA) and an ultra-performance liquid chromatography–mass spectrometry with tandem mass spectrometry (UPLC–MS/MS). The investigated 12 marker compounds were as follows: 3-caffeoylquinic acid (3-CQA, Lonicerae Flos); CFG and 5-MVS (Saposhnikoviae Radix); liquiritin apioside (LIQA) and glycyrrhizic acid (GA) (Glycyrrhizae Radix et Rhizoma); FA (Cnidii Rhizoma); narirutin (NAR), NAG, and NHES (Aurantii Fructus Immaturus); OPDH (Osterici seu Notopterygii Radix et Rhizoma); ARC (Forsythiae Fructus); and 6-gingerol (6-GIN, Zingiberis Rhizoma Recens).

2. Materials and Methods

2.1. Plant Materials

Table S1 lists the 14 crude plant materials constituting YPS. They were purchased from Kwangmyungdang Medicinal Herbs (KMH; Ulsan, Korea) in November 2017. Dr. Seung-Yeol Oh, president of KMH, confirmed the origin of each ingredient based on “The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine” [22]. The voucher specimen (2017KE59–1~2017KE59–15) was deposited at the Herbal Medicine Research Division, Korea Institute of Oriental Medicine.

2.2. Chemicals and Reagents

The reference standard compounds were obtained from the following suppliers: 3-CQA (PubChem CID: 1794427, 99.6%), Acros Organics (Pittsburgh, PA, USA); CFG (PubChem CID: 14034912, 99.4%), ChemNorm Biotech Co., Ltd. (Wuhan, China); LIQA (PubChem CID: 10076238, ≥98.0%), NHES (PubChem CID: 232990, 98.4%), and ARC (PubChem CID: 64981, 99.4%), Shanghai Sunny Biotech (Shanghai, China); FA (PubChem CID: 445858, 98.0%), GA (PubChem CID: 14982, ≥99.0%), and 6-GIN (PubChem CID: 442793, 99.3%), Wako Chemicals (Osaka, Japan); NAR (PubChem CID: 442431, 99.5%) and 5-MVS (PubChem CID: 21670038, 99.9%), Biopurify Phytochemicals (Chengdu, China); NAG (PubChem CID: 442428, 95.0%), Merck KGaA (Darmstadt, Germany); and OPDH (PubChem CID: 17536, 98.0%), ChemFaces Biochemical Co., Ltd. (Wuhan, China). The chemical structures of these compounds are shown in Figure S1.

The HPLC-grade solvents like methanol, acetonitrile, and water, required for preparation of test solution and quantitative analysis, were purchased from JT Baker (Phillipsburg, NJ, USA). Formic acid (ACS reagent-grade, 98.0–100.0%) was purchased from Merck KGaA.

2.3. Preparation of the YPS Water Decoction

For the preparation of the YPS water decoction, 14 herbal medicines (Forsythiae Fructus, Lonicerae Flos, Schizonepetae Spica, Saposhnikoviae Radix, Osterici seu Notopterygii Radix et Rhizoma, Araliae Continentalis Radix, Bupleuri Radix, Cnidii Rhizoma, Aurantii Fructus Immaturus, Platycodonis Radix, Poria Sclerotium, Glycyrrhizae Radix et Rhizoma, Menthae Herba (each 368.5 g), and Zingiberis Rhizoma Recens (210.1 g)) were mixed, followed by the addition of distilled water (50 L). Then, using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, Korea), extraction was carried out for 2 h at 100 °C under pressure (98 kPa). Pretreatment of the extract was performed according to a previously reported method [23]. The extract was then filtered and lyophilized to obtain a freeze-dried powder sample (1150.0 g, 23.0%).

2.4. Preparations of Sample and Standard Solutions for HPLC Analysis

A sample solution for the simultaneous quantitative analysis of the 12 marker components (3-CQA, CFG, LIQA, FA, NAR, 5-MVS, NAG, NHES, OPDH, ARC, GA, and 6-GIN) was prepared as follows: 100 mg of lyophilized YPS sample was added to 10 mL of 70% methanol and then extracted for 60 min at room temperature using a Branson 8510 ultrasonicator (Denbury, CT, USA). The extracted solution was filtered through a 0.2 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA) before sample injection for HPLC analysis.

A standard solution of each reference standard compound was prepared at a concentration of 1000 μg/mL using methanol or methanol:DMSO (1:1) solution. Prepared stock solutions were stored in a refrigerator (4 °C) until required for use.

2.5. HPLC Apparatus and Conditions for Simultaneous Analysis of the 12 Marker Components

The analytical method used for YPS analysis was a modification of the HPLC method used in earlier research [24]. Briefly, a HPLC system for simultaneous analysis was used, i.e., a Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) equipped with PDA detector and the LC Solution software (Version 1.24, SP1) for data analysis and system control. Other analytical conditions are given in Table S2.

2.6. Validation of the HPLC Analytical Method

The optimized assay using HPLC was tested and validated with respect to parameters such as linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and precision, according to the International Conference on Harmonisation guidance for Q2B Validation of Analytical Procedures [25]. Briefly, linearity was evaluated by the coefficient of determination (r²), whereas the LOD and LOQ were calculated using the respective equations LOD = 3.3 × σ/S and LOQ = 10 × σ/S (where σ is the standard deviation of the y-intercept and S is the slope of the calibration curve). Accuracy was assessed by the recovery test and calculated using Equation (1).

$$\mathrm{Recovery}(\%)=\frac{\left(\mathrm{found}\mathrm{amount}-\mathrm{original}\mathrm{amount}\right)}{\mathrm{spiked}\mathrm{amound}}\times 100$$

(1)

Precision was assessed by the relative standard deviation (RSD) values throughout the intra- and inter- day precision and repeatability.

$$\mathrm{RSD}(\%)=\frac{\mathrm{standard}\mathrm{deviation}\left(\mathrm{SD}\right)}{\mathrm{mean}}\times 100$$

(2)

The system suitability of the assay was evaluated using the parameters such as capacity factor (k′), selectivity factor (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf) [26].

2.7. UPLC–MS/MS Apparatus and Conditions for Quantification of the 12 Marker Components

The apparatus used in the UPLC–MS/MS system for the quantification of the 12 marker components in YPS included an ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) with a Xevo TQ-S micro triple quadrupole mass spectrometer (Waters) equipped with an electrospray ionization (ESI) interface. The MassLynx software (version 4.2, Waters) was used to carry out the data acquisition and processing. The analytical column used for chromatographic separation was a Waters ACQUITY UPLC BEH C₁₈ column (2.1 mm × 100 mm, 1.7 μm). Two mobile phases were used: 0.1% (v/v) aqueous formic acid (solvent A) and acetonitrile (solvent B). The mobile phase was eluted at 0.3 mL/min. The gradient conditions used were as follows: 10% solvent B at initial to 1.0 min, 10–40% solvent B at 1.0 to 6.0 min, 40–95% solvent B at 6.0 to 10.0 min, 95% solvent B at 10.0 to 15.0 min, 95–10% solvent B at 15.0 to 15.1 min, and 10% solvent B at 15.1–18.0 min. The volume of autosampler injection for the quantification of all analytes was 2 μL, and the temperature of the column and sample tray were maintained at 40 °C and 5 °C, respectively. The 12 marker components in a MS system were detected in positive and negative ion mode using an ESI interface. Quantification was conducted using the multiple reaction monitoring (MRM) mode. Capillary voltage, source temperature, desolvation temperature, desolvation gas flow, and cone gas flow were set at 1.2 kV, 150 °C, 450 °C, 800 L/h, and 50 L/h, respectively. Apart from these common MS parameters, other optimized analytical parameters, such as collision energy, cone voltage, and the transition required for the analysis of each analyte are displayed in Table S3.

3. Results and Discussion

3.1. Optimization of HPLC Chromatographic Conditions

The 12 marker components in YPS decoction (3-CQA, CFG, LIQA, FA, NAR, 5-MVS, NAG, NHES, OPDH, ARC, GA, and 6-GIN) were efficiently set to the separation conditions using a SunFire C₁₈ column (250 mm × 4.6 mm, 5 μm) maintained at 40 °C and a mobile phase system of distilled water–acetonitrile and formic acid. Using the established assay, all analytes were separated within 50 min, with Rs values ≥4.16 (Figure 1).

3.2. Method Validation for Quantitative Analysis by HPLC

In the established HPLC analytical method, the values of the system suitability parameters for the 12 marker components, namely, k′, α, N, Rs, and Tf were 4.02–14.44, 1.02–1.34, 2.7624792 × 10⁵–1.36925323 × 10⁶, 4.16–50.58, and 1.09–1.29, respectively (Table S4). The calibration curve of each analyte was determined by plotting the peak area (y) versus the corresponding concentration (x) using the standard solution, in the different concentration levels (

Table 1. Linear range, regression equation, r², LODs, and LOQs for marker compounds using HPLC–PDA (n = 3).

Compound	Linear Range (μg/mL)	Regression Equation a $\mathit{y}=\mathbf{a}\mathit{x}+\mathbf{b}$	r2	LOD (μg/mL)	LOQ (μg/mL)
3-CQA	1.56–100.00	y = 3.168268 × 104x − 2.279203 × 104	0.9986	0.19	0.57
CFG	0.78–50.00	y = 1.589998 × 104x − 3.69893 × 104	0.9993	0.13	0.41
LIQA	1.56–100.00	y = 1.435196 × 104x − 6.7471 × 103	0.9991	0.16	0.49
FA	0.78–50.00	y = 7.557159 × 104x − 1766964 × 104	0.9991	0.09	0.26
NAR	0.78–50.00	y = 1.698577 × 104x − 4.07642 × 103	0.9990	0.05	0.16
5-MVS	0.78–50.00	y = 1.850794 × 104x − 4.67958 × 103	0.9990	0.11	0.32
NAG	2.34–150.00	y = 1.779584 × 104x − 1.182341 × 104	0.9991	0.09	0.27
NHES	1.56–100.00	y = 2.215239 × 104x − 1.007198 × 104	0.9991	0.10	0.31
OPDH	0.78–50.00	y = 2.949819 × 104x − 6.27553 × 103	0.9994	0.15	0.47
ARC	0.78–50.00	y = 1.019838 × 104x − 2.73577 × 103	0.9989	0.16	0.49
GA	1.56–100.00	y = 8.69885 × 103x − 3.5352 × 102	0.9993	0.20	0.60
6-GIN	0.78–50.00	y = 8.57485 × 103x − 2.32865 × 103	0.9990	0.06	0.18

^ay: peak area (mAU) of compounds; x: concentration (μg/mL) of compounds.

). The linearity of these components was ≥0.99. The results showed good linearity. The LOD and LOQ ranges were calculated to be 0.05–0.20 μg/mL and 0.16–0.60 μg/mL, respectively (Table 1). The accuracy (recovery) test of the marker components was conducted using the standard addition method, and the recoveries averaged 92.30–101.30% with the RSD (%) values of <3.00 (

Table 2. Recovery test for the assay of 12 components in YPS using HPLC–PDA.

Compound	Original Amount (μg/mL)	Spiked Amount (μg/mL)	Found Amount (μg/mL)	Recovery (%)	SD	RSD (%)
3-CQA	44.98	8.00	52.65	95.87	1.44	1.51
		20.00	64.02	95.16	2.85	2.99
		40.00	82.68	94.24	0.56	0.60
CFG	5.55	1.00	6.50	95.28	2.35	2.47
		2.00	7.52	98.62	2.00	2.02
		4.00	9.49	98.61	1.37	1.39
LIQA	30.28	6.00	36.05	96.14	1.59	1.65
		15.00	44.73	96.33	1.61	1.67
		30.00	59.70	98.05	0.91	0.93
FA	3.38	1.00	4.36	98.22	1.77	1.80
		2.00	5.28	94.85	0.68	0.72
		4.00	7.29	97.85	1.43	1.47
NAR	11.81	2.00	13.83	101.22	1.35	1.34
		5.00	16.81	100.14	1.87	1.87
		10.00	21.85	100.41	0.60	0.60
5-MVS	6.01	1.00	6.98	97.34	2.38	2.45
		2.00	7.96	97.66	1.63	1.67
		4.00	9.83	95.58	2.53	2.65
NAG	44.07	8.00	52.09	100.32	2.90	2.89
		20.00	63.74	98.39	0.95	0.97
		40.00	84.25	100.46	1.26	1.25
NHES	31.45	6.00	37.06	93.48	1.33	1.42
		15.00	45.51	93.71	0.33	0.35
		30.00	59.14	92.30	0.97	1.05
OPDH	6.35	1.00	7.31	96.39	1.42	1.47
		2.00	8.26	95.73	1.29	1.34
		4.00	10.26	97.69	0.36	0.37
ARC	9.17	2.00	11.16	99.56	2.80	2.81
		5.00	14.08	98.38	1.44	1.46
		10.00	18.80	96.38	1.04	1.08
GA	29.28	6.00	34.91	93.76	1.11	1.19
		15.00	43.30	93.41	1.05	1.13
		30.00	56.99	92.36	0.67	0.72
6-GIN	1.14	1.00	2.15	101.30	1.31	1.29
		2.00	3.12	99.26	2.44	2.45
		4.00	5.11	99.29	2.49	2.51

). Furthermore, the RSD (%) values of repeatability and intra- and inter-day precisions showed good precision within 4.79% (

Table 3. Precision assay for 12 marker compounds in YPS using HPLC–PDA.

Compound	Conc. (μg/mL)	Intra-Day (n = 5)			Inter-Day (n = 5)			Repeatability (n = 6)
		Observed Conc. (μg/mL)	Precision (%) a	Accuracy (%)	Observed Conc. (μg/mL)	Precision (%)	Accuracy (%)	RSD (%) of Retention Time	RSD (%) of Peak Area
3-CQA	25.00	24.68	1.03	98.73	24.95	0.98	99.79	0.23	0.35
	50.00	49.95	0.60	99.90	50.23	1.74	100.46
	100.00	99.00	1.04	99.00	98.21	1.05	98.21
CFG	12.50	12.23	1.19	97.83	12.14	1.53	97.12	0.06	0.33
	25.00	25.16	1.50	100.62	24.70	2.72	98.81
	50.00	49.04	1.48	98.08	47.92	2.41	95.85
LIQA	25.00	25.16	1.48	100.65	25.66	2.30	102.64	0.06	0.33
	50.00	49.97	1.58	99.94	50.88	2.06	101.75
	100.00	97.95	1.20	97.95	98.67	1.44	98.67
FA	12.50	12.46	2.52	99.71	12.25	2.28	97.99	0.06	0.36
	25.00	24.84	0.50	99.35	24.51	2.46	98.02
	50.00	48.86	1.30	97.71	47.89	2.36	95.78
NAR	12.50	12.24	1.28	97.95	12.25	2.60	98.00	0.04	0.34
	25.00	24.76	0.44	99.03	24.48	2.61	97.91
	50.00	48.73	1.39	97.45	47.62	2.36	95.24
5-MVS	12.50	12.29	1.31	98.28	12.22	1.74	97.76	0.08	0.33
	25.00	24.77	0.49	99.10	24.49	2.57	97.96
	50.00	48.69	1.35	97.38	47.72	2.32	95.43
NAG	37.50	36.84	1.31	98.23	36.56	1.45	97.50	0.08	0.35
	75.00	74.60	0.60	99.47	73.66	2.64	98.22
	150.00	146.12	1.45	97.41	144.12	2.93	96.08
NHES	25.00	24.59	1.22	98.36	24.38	1.47	97.52	0.06	0.35
	50.00	49.69	0.48	99.39	49.27	2.65	98.54
	100.00	99.25	4.79	99.25	96.49	4.16	96.49
OPDH	12.50	12.26	1.39	98.04	12.18	2.00	97.48	0.05	0.35
	25.00	24.75	0.51	99.02	24.48	2.60	97.94
	50.00	48.64	1.39	97.27	47.47	2.39	94.94
ARC	12.50	12.26	1.37	98.06	12.15	1.56	97.23	0.13	0.47
	25.00	24.71	0.43	98.83	24.40	2.61	97.59
	50.00	48.55	1.35	97.10	47.46	2.33	94.91
GA	25.00	24.32	1.43	97.30	24.12	1.52	96.49	0.10	0.33
	50.00	49.64	0.58	99.28	49.03	2.70	98.05
	100.00	97.98	1.41	97.98	95.86	2.34	95.86
6-GIN	12.50	11.85	1.42	94.81	11.74	1.51	93.90	0.07	0.07
	25.00	24.14	0.38	96.57	23.79	2.16	95.16
	50.00	47.81	1.36	95.62	46.71	2.39	93.42

^a Precision is expressed as RSD (%) = (SD/mean) × 100.

). These results demonstrated that the developed HPLC assay is suitable for the simultaneous analysis of the 12 marker components in YPS.

3.3. UPLC–MS/MS Confirmation

Table 3 shows that five compounds (3-CQA, LIQA, NAR, NAG, and GA) were detected in the negative ion mode ([M–H]^–) at m/z 353.2, 549.3, 579.3, 279.2, and 821.6, respectively, whereas seven compounds (CFG, FA, 5-MVS, NHES, OPDH, ARC, and 6-GIN) were detected in the positive ion mode ([M+H]⁺) at m/z 469.3, 195.0, 453.0, 611.1, 304.9, 373.2, and 295.3, respectively. The precursor ion (Q1) and product ion (Q3) for the LC–MS/MS MRM method are shown in Table S3. Briefly, the Q3 peak of 3-CQA was detected at m/z 191.0 [M–H–caffeoyl]^– by elimination of the caffeoyl group from the Q1 peak [27]. CFG and 5-MVW were set at the Q3 peaks at m/z 307.2 and 291.3 in the form of [M+H–Glc]⁺, and these components released one glucose group at the Q1 peak [28,29]. FA and GA were detected at m/z 177.0 [M+H–H₂O]⁺ and 351.2 [M–H–2Glc]^– by the loss of one H₂O and two glucose groups from the Q1 peak, respectively [30,31]. LIQA was detected at m/z 549.3 (Q1) as [M−H]⁻, formed by the loss of the apiosy-glucosyl function group from Q1 [32]. NAR, NAG, and NHES were apt to lose the rutinose to give aglycone ions of m/z 271.2 [M–H–rutinose]^–, 271.0 [M–H–rutinose]^–, and 303.2 [M+H–rutinose]⁺, respectively [33,34,35]. The characteristic MS fragmentations of OPDH, ARC, and 6-GIN were observed at m/z [M+H–C₅H₉O–OH]⁺, 137.0 [dimethoybenzyl group–H]⁺, and 177.0 [M+H–C₆H₁₂O₂]⁺; this was assigned to the loss of a C₅H₉O molecule and OH group, and C₁₃H₁₅O₄ and C₆H₁₂O₂ molecules from the Q1 peak, respectively [36,37,38]. Our analysis carried out using the MRM conditions gave an r² value of >0.99 for the calibration curve showing good linearity. Furthermore, the LOD and LOQ values of these components were calculated to be in the ranges of 0.002–1.135 μg/mL and 0.005–3.404 μg/mL, using signal-to-noise ratios (S/N) of 3.3 and 10, respectively. These results are presented in Table S5.

3.4. Quantitative Analysis of the 12 Marker Components in YPS Samples Using HPLC–PDA

The 12 marker components (3-CQA, CFG, LIQA, FA, NAR, 5-MVS, NAG, NHES, OPDH, ARC, GA, and 6-GIN) were analyzed in the YPS samples using the developed and validated HPLC analytical method. Quantification of these components was carried out at 254 nm for GA; 275 nm for LIQA; 280 nm for NAR, NAG, NHES, ARC, and 6-GIN; 290 nm for 5-MVS; 300 nm for CFG; 310 nm for OPDH; 320 nm for FA; and 325 nm for 3-CQA, based on the λ_max of the ultra-violet (UV) spectra. The peak of each analyte was identified by comparing the UV spectrum and the retention time of the reference standard. The amounts of the 12 marker components in the three batches of the YPS samples were calculated using the calibration curves of each analyte. The amounts of these analytes in the samples were found to be in the range of 0.14–9.00 mg/g (

Table 4. Amount of the 12 marker components in YPS using HPLC–PDA (n = 3).

Compound	Batch No.
	1		2		3
	Mean (mg/g) ± SD (× 10–1)	RSD (%)	Mean (mg/g) ± SD (× 10–1)	RSD (%)	Mean (mg/g) ± SD (× 10–1)	RSD (%)
3-CQA	4.28 ± 0.66	1.53	4.22 ± 0.56	1.32	4.15 ± 0.83	1.99
CFG	0.62 ± 0.05	0.80	0.61 ± 0.07	1.19	0.60 ± 0.12	2.03
LIQA	3.05 ± 0.26	0.84	2.90 ± 0.65	2.24	2.96 ± 0.39	1.31
FA	0.32 ± 0.04	1.23	0.30 ± 0.07	2.27	0.30 ± 0.08	2.74
NAR	1.30 ± 0.15	1.19	1.27 ± 0.25	1.93	1.24 ± 0.24	1.95
5-MVS	0.59 ± 0.03	0.49	0.58 ± 0.10	1.71	0.50 ± 0.15	2.97
NAG	9.00 ± 0.74	0.82	8.93 ± 0.07	0.08	8.84 ± 0.65	0.74
NHES	6.48 ± 0.42	0.65	6.39 ± 0.59	0.92	6.28 ± 0.94	1.50
OPDH	0.69 ± 0.05	0.78	0.67 ± 0.10	1.53	0.64 ± 0.17	2.64
ARC	0.94 ± 0.09	0.96	0.92 ± 0.03	0.31	0.92 ± 0.12	1.28
GA	6.02 ± 0.51	0.85	5.95 ± 0.37	0.61	5.85 ± 0.95	1.62
6-GIN	0.15 ± 0.02	1.19	0.15 ± 0.04	2.65	0.14 ± 0.02	1.16

).

3.5. Simultaneous Determination of the 12 Marker Components in YPS Sample Using UPLC–MS/MS MRM Mode

For a quality evaluation of YPS, quantification of the 12 marker components was carried out using the optimized UPLC–MS/MS MRM assay. The retention times (min) of the 12 marker components were as follows: 3-CQA, 1.98; CFG, 4.30; FA, 4.65; LIQA, 4.66; NAR, 5.12; 5-MVS, 5.30; NAG, 5.30; NHES, 5.61; OPDH, 6.58; GA, 8.10; ARC, 8.20; and 6-GIN, 8.94 min (Figure 2). The amounts of these analytes ranged from 2.35 to 853.11 μg/g (

Table 5. Amount of the 12 marker components in YPS by LC–MS/MS MRM mode (n = 3).

Compound	Amount (μg/g)
	Mean	SD	RSD (%)
3-CQA	466.95	14.45	3.10
CFG	35.70	0.49	1.37
FA	26.91	0.78	2.89
LIQA	310.68	6.89	2.22
NAR	137.57	2.13	1.55
5-MVS	19.29	0.27	1.42
NAG	756.78	16.17	2.14
NHES	721.47	14.93	2.07
OPDH	56.13	1.48	2.64
GA	853.11	60.47	7.09
ARC	107.85	1.52	1.41
6-GIN	2.35	0.21	9.03

).

4. Conclusions

In this study, a simultaneous analysis for the quantification of the 12 marker components in YPS was carried out using an easy and convenient HPLC system, and a rapid and sensitive LC–MS/MS system, to construct basic data with which to assess the analytical quality of a commonly used, traditional Korean medicine. For the quality assessment of YPS, a simultaneous analysis was carried out using our established and optimized analytical method. These methods are expected to be very useful in quality assessments of YPS and other chemically related herbal prescriptions.