Development and Validation of a HPLC–PDA Method for the Simultaneous Determination of Berberine, Palmatine, Geniposide, and Paeoniflorin in Haedoksamul-tang

Abstract

Haedoksamul-tang (HST) is a traditional medical prescription comprising eight medicinal herbs: Angelica gigas, Cnidium officinale, Coptis japonica, Gardenia jasminoides, Paeonia lactiflora, Phellodendron amurense, Rehmannia glutinosa, and Scutellaria baicalensis. HST is used to treat blood circulation disorders and has anti-inflammatory, hemostatic, and anticonvulsant effects. In this study, a high-performance liquid chromatography/photodiode array detector (HPLC–PDA) method was developed and validated for the simultaneous determination of four marker compounds in HST, namely, berberine, palmatine, geniposide, and paeoniflorin. Four standard solutions and HST sample solutions were analyzed using a reverse-phase SunFire^®C₁₈ column (4.6 × 250 mm, 5 μm) using a 0.05% aqueous formic acid/methanol gradient. The column temperature, flow rate, injection volume, and wavelengths used were 28 ± 2 ℃, 1.0 mL/min, 10.0 μL, and 230 nm and 240 nm, respectively. Calibration curves of the four marker compounds showed good linearity (r² ≥ 0.9994), and limits of detection (LODs) and quantification (LOQs) were in the ranges 0.131–0.296 μg/mL and 0.398–0.898 μg/mL, respectively. Ranges of intra- and inter-day precisions and accuracies values were 96.74–102.53% and 97.95–100.83%, respectively, and relative standard deviation (RSD) values were all <4%. Recoveries averaged 92.33–116.72% with RSD values <5%. Quantitative analysis for the four marker compounds showed geniposide (10.77 mg/g) was most abundant in HST.

1. Introduction

Haedoksamul-tang (HST), also known as Onchungeum in Korea, Wen-Qing-Yin in China, and Unsei-in in Japan, is a traditional medical prescription comprising Hwangryunhaedok-tang (HHT) and Samul-tang (SMT). According to the Donguibogam (principles and practice of eastern medicine), HST has been traditionally used to treat abnormal uterine bleeding and abdominal pain in women or to treat flooding accompanied by a yellowish complexion, stomach aches, and alternating chills [1]. According to several studies, HST exhibits diverse therapeutic effects on inflammation [2,3,4,5,6,7], atopic dermatitis [8,9,10,11], blood stasis [12,13], Behcet’s syndrome [14,15], hyperlipidemia [16], and oxidative stress [17,18,19,20].

HST consists of eight medicinal herbs: Angelica gigas (Apiaceae), Cnidium officinale (Umbelliferae), Coptis japonica (Ranunculaceae), Gardenia jasminoides (Rubiaceae), Paeonia lactiflora (Paeoniaceae), Phellodendron amurense (Rutaceae), Rehmannia glutinosa (Orobanchaceae), and Scutellaria baicalensis (Lamiaceae). The major components of the eight medicinal herbs in HST are: flavones (e.g., baicalin, wogonin, and baicalein) from Scutellaria baicalensis [21], alkaloids (e.g., berberine, palmatine, coptisine, jatrorrhizine, and epiberberine) from Coptis japonica [22], alkaloids (e.g., palmatine, phellodendrine, magnoflorine, and berberine) from Phellodendron amurense [23], iridoids (e.g., geniposide, genipin, genipin-1-β gentiobioside, and geniposidic acid) from Gardenia jasminoides [24], iridoids (e.g., catalpol) from Rehmannia glutinosa [25], coumarins (e.g., decursin, decursinol, decursinol angelate, and nodakenin) from Angelica gigas [26], monoterpenoids (e.g., paeoniflorin, albiflorin, benzoylpaeoniflorin, and oxypaeoniflorin) and phenols (e.g., paeonol and (+)-catechin) from Paeonia lactiflora [27], and phthalides (e.g., senkyunolideA, Z-ligustilide, and Z-butylidenephthalide) and ferulic acid from Cnidium officinale [28]. Qualitative and quantitative analyses of HHT and SMT by high-performance liquid chromatography (HPLC) using photodiode array (PDA) detectors have been previously reported [29,30,31,32]. Recently, a method for simultaneous quantification of six biomarkers (berberine, baicalin, ferulic acid, geniposide, hydorxymethoxylfurfural, and paeoniflorin) in HST by high-performance liquid chromatography/diode array detector (HPLC-DAD) was developed [33]. However, analysis results depended on geographic sources and cultivation environments, and the analysis data was inadequate for standardizing HST quality. In the present study, we devised and validated a HPLC-PDA method for the simultaneous determination of four biomarkers in HST (berberine from Coptis japonica, palmatine from Phellodendron amurense, geniposide from Gardenia jasminoides, paeoniflorin from Paeonia lactiflora).

2. Materials and Methods

2.1. Plant Materials and Chemicals

Scutellaria baicalensis was purchased from Hyunjin pharmaceutical Corp., Coptis japonica from Miryung herb medicine Ltd., Gardenia jasminoides, Phellodendron amurense, and Rehmannia glutinosa from Taechang pharmaceutical Corp., Angelica gigas, Cnidium officinale, and Paeonia lactiflora from Jechen traditional herbal market. All herbal materials were authenticated by Professor Eun Ju Jeong of the Department of Agronomy and Medicinal Plant Resources at Gyeongnam National University of Science and Technology (Jinju, South Korea). Voucher specimens (PNU-0028~PNU-0035) were deposited at the College of Pharmacy, Pusan National University.

Geniposide was isolated from G. jasminoides by column chromatography in our laboratory and used as a reference standard. The structure of geniposide was determined by nuclear magnetic resonance (NMR) [34], and their purities by HPLC (≥98.0%). Other reference standards: berberine (≥98.0%), palmatine (≥98.0%), and paeoniflorin (≥98.0%) were purchased from ChemFaces Biochemical Co., Ltd. (Wuhan, China). Water, acetonitrile, methanol (Honeywell Burdick and Jackson, Muskegon, MI, USA), and formic acid (DAEJUNG Chemicals & Metals Co., Ltd., Siheung-si, Korea) were of HPLC grade.

2.2. Preparation of Haedoksamul-Tang (HST) Extract

The eight herbal materials (100 g of each) were mixed and crushed for HST extraction, and then extracted for 3 h in 8 L of distilled water using an ultrasonic extractor. The extract was passed through filter paper (Advantec, Tokyo, Japan), concentrated under vacuum at 50 °C, and freeze-dried. The lyophilized HST extract was sieved to a particle size of <0.425 mm with a standard sieve and 117.6 g of HST (hereafter referred to as HST) was obtained (yield, 14.7%).

2.3. Preparation of Samples and Standard Solutions

Sample solutions contained HST (40.0 mg) dissolved in distilled water at a concentration of 20.0 mg/mL, and then sonicated for 10 min at room temperature and filtered through a 0.5 μm polytetrafluoroethylene (PTFE) syringe filter (13JP050AN, Advantec, Tokyo, Japan) prior to injection.

Standard stock solutions of the four reference standards (Figure 1), berberine, palmatine, geniposide, and paeoniflorin, were prepared at concentrations of 500.0 μg/mL, 500.0 μg/mL, 1000.0 μg/mL, and 1000.0 μg/mL, respectively, in methanol. All solutions were stored in a refrigerator at 4 °C until required for analysis.

2.4. High-Performance Liquid Chromatography–Photodiode Array (HPLC–PDA) Equipment and Chromatographic Conditions

Simultaneous analysis of the berberine, palmatine, geniposide, and paeoniflorin in HST was performed using a Waters Alliance system (Waters Corporation, Milford, MA 01757, USA) consisting of an e 2695 separation module and a 2998 photodiode array (PDA) detector. All chromatographic data were recorded and analyzed using Empower three chromatography data software. The four components were separated using a reverse-phase SunFire^® C18 column (100 Å, 4.6 × 250 mm ID, 5 μm particle size, Waters), which was maintained at 28 ± 2 ℃. The mobile phase consisted of 0.05% (v/v) formic acid in water (A) and methanol (B); solvents were degassed prior to analysis. The optimum gradient elution system used was as follows: 0–30 min, 10–50% (B); 30–35 min, 50–90% (B); 35–45 min, 90–90% (B); and 45–46 min, 90–10% (B). The flow rate and injection volume used were 1.0 mL/min and 10.0 μL, and detection wavelengths for quantification and analysis were set at 230 nm and 240 nm.

2.5. Method Validation

The HPLC analytical method devised was evaluated by determining its specificity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and recovery performance as detailed by the International Conference on Harmonisation (ICH) guidelines [35].

2.5.1. Specificity

The specificity was determined by comparing chromatograms and PDA spectral patterns (λ = 200–400 nm) obtained from standard mixtures and HST samples.

2.5.2. Linearity, Limit of Detection (LOD), and Limit of Quantitation (LOQ)

Calibration curves of berberine, palmatine, geniposide, and paeoniflorin stock solutions were plotted over the concentration ranges 2.50–25.00 μg/mL, 2.50–25.00 μg/mL, 2.50–25.00 μg/mL, and 2.50–25.00 μg/mL, respectively. Solutions were analyzed in quintuplicate, and calibration curves were obtained by plotting peak areas (Y) versus compound concentrations (x, μg/mL). Correlation coefficients (r²) obtained from calibration curves were used to evaluate linearity, and all r² values exceeded 0.999. LOD and LOQ were calculated using the standard deviations of y-intercepts (σ) and calibration curve slopes (S), as follows:

$$\mathrm{LOD}=3.3\times \frac{\mathsf{\sigma }}{\mathrm{S}}$$

(1)

$$\mathrm{LOQ}=10\times \frac{\mathsf{\sigma }}{\mathrm{S}}$$

(2)

2.5.3. Precision

The precision of the developed method was determined by inter-day and intra-day testing at three different concentrations. Intra-day precision was measured by performing analyses three times per day, and inter-day precision was measured by repeating analyses three times per day over three consecutive days. Precisions were determined by calculating relative standard deviation (RSD) using:

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

2.5.4. Recovery

Recovery testing was performed to verify the accuracy of the optimized HPLC–PDA method. Recovery was evaluated by spiking standard solution and HST samples with different amounts of berberine, palmatine, geniposide, and paeoniflorin. All analyses were performed in triplicate. Recovery was calculated as follows:

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

3. Results and Discussion

3.1. Optimization of HPLC–PDA Conditions

Optimal HPLC–PDA conditions were obtained by optimizing column type, column temperature, and mobile phase variables. For efficient separation of peaks, a SunFire^® C₁₈ column (5 µm, 100 Å, 4.6 mm × 250 mm, Waters) was used at a column temperature of 28 ± 2 ℃ with a water (0.05% formic acid)/methanol gradient elution system. The maximum ultraviolet (UV) absorption wavelengths of the four components were confirmed using PDA UV spectra (λ = 200–400 nm), and wavelengths of 230, 230, 240, and 230 nm were selected for berberine, palmatine, geniposide, and paeoniflorin, respectively (Figure 2).

3.2. Method Validation

3.2.1. Specificity

Method specificity was evaluated by comparing retention time and UV spectra of standard mixtures and HST samples. Comparisons showed retention times and UV absorbances of HST samples and the four reference standards were well matched (Figure 3). The wavelengths used for analysis were as follows; berberine (λ_max = 228.3, 263.8, and 346.2 nm), palmatine (λ_max = 226.0, 273.3, and 343.8 nm), geniposide (λ_max = 239.0 nm), and paeoniflorin (λ_max = 231.9 and 274.5 nm).

3.2.2. Linearity, LOD and LOQ

HPLC–PDA linearity results for berberine, palmatine, geniposide and paeoniflorin showed excellent linearity (r² ≥ 0.9994) in the concentration range 2.50–25.00 μg/mL for berberine and palmatine and 5.00–50.00 μg/mL of geniposide and paeoniflorin. The LOD and LOQ values of the four compounds ranged from 0.131–0.296 μg/mL and 0.398–0.898 μg/mL, respectively (

Table 1. Retention times, linear range, calibration curves, limits of detection (LODs), and limits of quantitation (LOQs) of the four compounds (n = 5).

Compound	λa (nm)	tRb (min)	RSDc (%)	Linear Range (μg/mL)	Regression Equationd	r2 e	LOD (μg/mL)	LOQ (μg/mL)
berberine	230	20.20	0.26	2.50–25.00	Y = 59,041.58 x + 7174.76	0.9998	0.131	0.398
palmatine	230	21.07	0.25	2.50–25.00	Y = 44,562.64 x + 7204.29	0.9999	0.179	0.543
geniposide	240	22.26	0.39	5.00–50.00	Y = 23,807.44 x + 6159.4	0.9998	0.217	0.659
paeoniflorin	230	24.11	0.38	5.00–50.00	Y = 19,555.98 x − 2874.87	0.9994	0.296	0.898

^a Detection wavelength; ^b Retention times; ^c Relative standard deviation; ^d Y: peak area, x: concentration (μg/mL).; ^e Coefficient of determination.

).

3.2.3. Precision

Precision was determined by intra-day and inter-day analysis. The intra-day and inter-day RSDs of the four compounds ranged from 0.111% to 2.857% and from 0.946% to 3.593%, respectively. Accuracy values ranged from 96.74% to 102.53% and from 97.95% to 100.83%, respectively (

Table 2. Precision and accuracy of the HPLC–PDA method for the four compounds.

Compound	Conc. (μg/mL)	Intra-Day (n = 3)			Inter-Day (n = 3)
		Measured Conc. (μg/mL)	RSD (%)	Accuracy (%)	Measured Conc. (μg/mL)	RSD (%)	Accuracy (%)
berberine	3.91	3.92	2.348	102.53	3.85	2.281	100.45
	5.21	5.02	0.697	98.28	5.04	1.068	98.70
	7.81	7.68	0.111	100.36	7.68	0.946	100.38
palmatine	3.91	3.90	2.127	101.79	3.86	2.840	100.83
	5.21	4.99	0.826	97.71	5.01	1.774	98.22
	7.81	7.69	1.230	100.48	7.69	1.109	100.49
geniposide	7.81	7.78	1.135	101.63	7.68	1.467	100.34
	10.42	9.88	0.280	96.74	10.00	1.061	97.95
	15.63	15.50	1.526	101.22	15.41	1.118	100.64
paeoniflorin	7.81	7.81	1.693	101.96	7.64	3.593	99.76
	10.42	10.03	1.731	98.23	10.09	1.274	98.85
	15.63	15.30	2.857	99.91	15.38	2.444	100.42

). The intra-day and inter-day RSDs of berberine, palmatine, geniposide, and paeoniflorin were all <4%. Thus, the developed method was found to be both reliable and reproducible.

3.2.4. Recovery

Recovery testing was performed in triplicate using the method developed by spiking HST samples with reference standards. The average recoveries of berberine, palmatine, geniposide, and paeoniflorin ranged from 92.33% to 116.72% and RSD values from 0.731% to 4.705% (

Table 3. Recoveries of the four compounds in Haedoksamul-tang (HST) samples (n = 3).

Compound	Spiked Amount (μg/mL)	Found Amount (μg/mL)	Recovery (%)	SD	RSD (%)
Berberine	2.60	58.33	92.97	0.032	3.486
	5.21	59.88	90.52	0.013	1.404
	7.81	72.74	108.46	0.040	3.693
palmatine	2.60	31.06	98.97	0.033	3.365
	5.21	31.94	92.33	0.043	4.705
	7.81	40.25	113.13	0.033	2.881
geniposide	5.21	319.60	104.35	0.009	0.893
	10.42	326.28	104.71	0.012	1.099
	15.63	367.28	116.72	0.009	0.731
paeoniflorin	5.21	141.21	95.42	0.040	4.148
	10.42	151.04	98.05	0.015	1.571
	15.63	178.61	114.41	0.034	2.955

). Therefore, the developed assay method was found to be suitable for analyzing levels of the four compounds in HST samples.

3.3. Quantitative Analysis of Four Marker Compounds in HST

The HPLC–PDA based method showed that HST contained 2.11 mg/g of berberine, 1.54 mg/g of palmatine, 10.77 mg/g of geniposide, and 7.04 mg/g paeoniflorin, and all RSD values were <3% (

Table 4. Amounts of the four compounds in HST (n = 4).

Compound	Mean (mg/g)	SD	RSD (%)
berberine	2.11	0.018	0.854
palmatine	1.54	0.033	2.141
geniposide	10.77	0.249	2.313
paeoniflorin	7.04	0.117	1.667

). In other words, geniposide, the major compound in Gardenia jasminoides, was the most abundant, followed by paeoniflorin, the major compound in Paeonia lactiflora.

4. Conclusions

Summarizing, we developed and verified a HPLC–PDA-based analytical method for the simultaneous determination of berberine, palmatine, geniposide, and paeoniflorin in HST. The optimized method utilized a 0.05% formic acid in water/methanol gradient elution system and a reverse-phase C₁₈ column. Validation of the optimized analytical method was performed by determining specificities, linearities, limits of detection (LODs), and limits of quantification (LOQs) and using precision and recovery tests. In addition, we confirmed that geniposide (the marker compound of Gardenia jasminoides) is abundant in HST. We hope that the method developed and data acquired during the course of this study will be found useful for efficient quality control and standardization of HST.