HPLC PDA Method for Simultaneous Determination of Nine Marker Components in Banhasasim-Tang

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1 Journal of Chromatographic Science, 2016, Vol. 54, No. 3, doi: /chromsci/bmv141 Advance Access Publication Date: 8 September 2015 Article Article HPLC PDA Method for Simultaneous Determination of Nine Marker Components in Banhasasim-Tang Chang-Seob Seo and Hyeun-Kyoo Shin* K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Korea *Author to whom correspondence should be addressed. hkshin@kiom.re.kr Received 9 January 2015 Abstract A simple and accurate high-performance liquid chromatography photodiode array (HPLC PDA) detection method has been developed and validated for simultaneous determination of nine components liquiritin, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, glycyrrhizin and wogonin in the traditional Korean formula, Banhasasim-tang decoction. A Gemini C 18 analytical column was used to separate the nine constituents and kept at 40 C by gradient elution with 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B) as mobile phases. The flow rate was 1.0 ml/min and the injection volume was 10 µl. The PDA detection wavelengths were set at 254, 275 and 350 nm. Calibration curves of all compounds showed good linearity with coefficients of determination within the test ranges. The limits of detection and quantification of all compounds were in the range and µg/ml, respectively. All recoveries of the nine marker compounds ranged from to % with relative standard deviation (RSD) values <1.25%. The RSDs of intraday and interday precision were <1.13 and 1.83%, respectively. The concentrations of the nine marker constituents were mg/g. Introduction General interest in traditional herbal medicines and their preparations has increased significantly (1). They contain many herbs among their various constituents and have been used widely for thousands of years in East Asian countries such as Korea, China and Japan (2). Therefore, both efficacy and standardization for quality control is required. Quality control, including chemical fingerprinting of herbal medicines, has been performed to ensure their consistency, safety and efficacy (3). Banhasasim-tang (BST), also known as Banxia-xiexin-tang in Chinese and Hange-shashin-to in Japanese, is a traditional Korean herbal formula consisting of eight medicinal herbs, Pinelliae Tuber (Araceae), Scutellariae Radix (Labiatae), Ginseng Radix (Araliaceae), Glycyrrhizae Radix et Rhizoma (Leguminosae), Zingiberis Rhizoma (Zingiberaceae), Coptidis Rhizoma (Ranunculaceae), Zingiberis Rhizoma Crudus (Zingiberaceae) and Zizyphi Fructus (Rhamnaceae) in 4 : 3 : 3 : 3 : 2 : 1 : 2 : 2 proportions (4). BST has been used clinically for the treatment of gastric disease, including acute and chronic gastritis, diarrhea and gastric ulcers (5 7). Several analytical methods have reported the simultaneous determination of bioactive components in herbal medicines forming BST using ultra-high-performance liquid chromatography (UHPLC), capillary electrophoresis and HPLC coupled with various detectors, including ultraviolet (UV), mass spectrometry, diode array detector (DAD) and electrochemical detection (8 14). However, there are a few studies on simultaneous determination of the marker constituents for quality control of the traditional Korean formulation, BST, using HPLC coupled with photodiode array (PDA) detection. Therefore, we performed simultaneous quantitation for quality control of BST. HPLC is a popular analysis method for quality control of herbal medicines because it is easy to learn and use. Moreover, it is a convenient, rapid, simple, accurate and powerful technique for the determination of multiple components in herbal medicines or traditional herbal formulas (15, 16). In the present study, we conducted simultaneous determination of the nine compounds baicalin, wogonoside, baicalein and wogonin from Scutellariae Radix, glycyrrhizin and liquiritin The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com 299

2 300 Seo and Shin Figure 1. Chemical structures of the nine marker compounds in BST. Table I. Composition of BST Latin name Scientific name Amount (g) Origin Pinelliae Tuber Pinellia ternata China Scutellariae Radix Scutellaria Gurye, Korea baicalensis Ginseng Radix Panax ginseng Yeongju, Korea Glycyrrhizae Radix Glycyrrhiza China et Rhizoma uralensis Zingiberis Rhizoma Zingiber Taean, Korea officinale Coptidis Rhizoma Coptis japonica China Zingiberis Rhizoma Zingiber Ulsan, Korea Crudus officinale Zizyphi Fructus Ziziphus jujuba Yeongcheon, Korea Total from Glycyrrhizae Radix et Rhizoma and berberine, coptisine and palmatine from Coptidis Rhizoma using HPLC PDA. Experimental Plant materials The eight herbal medicines forming BST were purchased from Kwangmyungdang (Ulsan, Korea). The origin of the eight herbal medicines was confirmed taxonomically by Prof. Je-Hyun Lee, Dongguk University, Gyeongju, Republic of Korea. Voucher specimens (2012 KE38-1 KE3888) have been deposited at the K-herb Research Center, Korea Institute of Oriental Medicine. Chemicals and reagents Reference standard, liquiritin, coptisine, baicalin, palmatine, berberine, glycyrrhizin and wogonin were purchased from Wako Chemicals (Osaka, Japan). Wogonoside and baicalein were purchased from Tauto Biotech (Shanghai, China) and Sigma-Aldrich (St Louis, MO, USA), respectively. The purity of each component was determined to be 98% by HPLC analysis. The chemical structures of the eight marker compounds are shown in Figure 1. HPLC-grade reagents methanol, acetonitrile and water were obtained from J.T. Baker (Phillipsburg, NJ, USA). Trifluoroacetic acid was obtained from Sigma-Aldrich. Apparatus and conditions The quantitative determination was conducted with a Shimadzu Prominence LC-20A series HPLC system (Shimadzu, Kyoto, Japan) consisting of a solvent delivery unit (LC-20AT), online degasser (DGU-20A 3 ), column oven (CTO-20A), auto sample injector (SIL-20AC) and PDA detector (SPD-M20A). Data were acquired and processed using LCsolution software (version 1.24, Shimadzu). A Phenomenex Gemini C 18 column ( mm; particle size 5 µm, Phenomenex, Torrance, CA, USA) maintained at 40 C was used to separate the nine compounds. Gradient elution with two mobile phase systems consisting of 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B) was as follows: 10 60% B for 0 30 min, % B for min, 100% B for min and % B for min, with a reequilibrium time of 10 min. The flow rate and injection volume were 1.0 ml/min and 10 µl, respectively. The PDA detector wavelength ranged from 190 to 400 nm and was monitored at 254 nm (glycyrrhizin), 275 nm (liquiritin, baicalin, wogonoside, baicalein and wogonin) and 350 nm (coptisine, palmatine and berberine). Preparation of standard solutions The nine compounds were accurately weighed and dissolved in methanol at a concentration of 1.0 mg/ml. Each standard stock solution was maintained at 4 C and used after serial dilution with methanol before HPLC analysis.

3 Simultaneous Determination of Nine Marker Components in Banhasasim-Tang 301 Preparation of BST decoction and sample solution BST is composed of eight herbs, as listed in Table I (total weight = 5.0 kg, times the amount of a single dose), and was extracted in distilled water at 100 C for 2 h under pressure (98 kpa) using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, Korea). The extract solution was filtered using a standard sieve (No. 270, 53 µm) and freeze-dried. The yield of extract was 14.99% (749.5 g). Table II. System Suitability of the Nine Marker Compounds Compound Capacity factor (k ) Separation factor (α) Number of theoretical plates (N) Resolution (Rs) Liquiritin , Coptisine , Baicalin , Palmatine , Berberine , Wogonoside , Baicalein , Glycyrrhizin , Wogonin , For quantitative analysis, lyophilized BST extract (200 mg) was dissolved in distilled water (20 ml) and mixed. Subsequently, the solution was diluted 10-fold with distilled water for quantitative analysis of baicalin and wogonoside. The solution was filtered through a SmartPor GHP syringe filter (0.2 µm pore size, Woongki Science, Seoul, Korea) before injection into the HPLC instrument. Calibration curves, limit of detection and limit of quantification The calibration curves of the nine compounds were calculated by plotting the peak areas (y) versus the corresponding concentrations (x, µg/ml) using standard solutions. The tested concentration ranges were as follows: liquiritin, baicalin and glycyrrhizin ( µg/ml), coptisine, palmatine and berberine ( µg/ml), wogonoside ( µg/ml) and baicalein and wogonin ( µg/ml). These solutions were measured in triplicate for the assessment of calibration curves, and linearity was evaluated by correlation coefficient values. The standard solutions of the nine constituents were diluted with methanol to determine limit of detection (LOD) and limit of quantification (LOQ) values. The LOD and LOQ data under the present chromatographic conditions were determined by signal-to-noise ratios of 3 and 10, respectively. Figure 2. HPLC chromatograms of standard mixtures (A) and BST samples (B) at 254 nm (I), 275 nm (II) and 350 nm (III). Liquiritin (1), coptisine (2), baicalin (3), palmatine (4), berberine (5), wogonoside (6), baicalein (7), glycyrrhizin (8) and wogonin (9). Table III. Regression Equations, Linearity, LODs and LOQs of the Nine Marker Compounds Compound Linear range Regression equation a Coefficient of determination LOD b LOQ c Liquiritin y = x Coptisine y = x Baicalin y = x Palmatine y = x Berberine y = x Wogonoside y = x Baicalein y = x Glycyrrhizin y = x Wogonin y = x a y: peak area (mau) of compounds; x: concentration of compounds. b LOD = 3 signal-to-noise ratio. c LOQ = 10 signal-to-noise ratio.

4 302 Seo and Shin Table IV. Recoveries for the Assay of the Nine Analytes in BST Analyte Original Spiked Found Recovery a ± SD RSD Liquiritin ± ± ± Coptisine ± ± ± Baicalin ± ± ± Palmatine ± ± ± Berberine ± ± ± Wogonoside ± ± ± Baicalein ± ± ± Glycyrrhizin ± ± ± Wogonin ± ± ± a Recovery = (found original )/spiked 100. Table VI. Precision Data for the Assay of the Nine Analytes in BST Compound Spiked Intraday (n = 5) Interday (n =5) Observed Precision a Observed Precision Liquiritin Coptisine Baicalin Palmatine Berberine Wogonoside Baicalein Glycyrrhizin Wogonin a Precision is expressed as RSD = (SD/mean) 100. Table V. Repeatability of Retention Times and Peak Responses for the Nine Analytes (n =6) Compound Retention time (min) Peak response (mau) Mean ± SD ( 10 1 ) Precision and recovery RSD Mean ± SD RSD Liquiritin ± ± Coptisine ± ± Baicalin ± ± Palmatine ± ± Berberine ± ± Wogonoside ± ± Baicalein ± ± Glycyrrhizin ± ± Wogonin ± ± To evaluate the precision of the developed HPLC assay, intra- and interday variations were determined using a standard addition method with the samples spiked with low, middle and high concentration levels of standard compounds. The relative standard deviation (RSD) was used as an assessment of precision. Six replicates were measured to confirm the reproducibility using the standard stock solutions. The RSDs of peak areas and retention times for each compound were used to evaluate the reproducibility of this method. A recovery test was used to evaluate the accuracy of the method. The recovery test was conducted by adding three different known amounts (low, middle and high) of standard solutions to BST samples. This test was assessed using the calibration curves and performed in triplicate. Results Optimization of chromatographic conditions The HPLC conditions were investigated regarding column types, column temperatures and mobile phases for better chromatographic conditions. To accomplish the efficient separation of the nine components, different columns, such as the Phenomenex Gemini C 18 ( mm, 5 µm), Waters SunFire C 18 ( mm, 5 µm) and Optima- Pak C 18 ( mm, 5 µm), column temperatures (30, 35 and 40 C) and various mobile phases including acetic acid, formic acid and phosphoric acid and organic solvents, including methanol and acetonitrile, were examined. The most efficient separation conditions were selected with a Phenomenex Gemini C 18 column ( mm, 5 µm) and a gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid at 40 C after comparing the baselines, resolution and peak shapes of the nine compounds under the various conditions. The UV wavelength of the PDA for the quantification of each compound was selected at 254 nm (glycyrrhizin), 275 nm (liquiritin, baicalin, wogonoside, baicalein and wogonin) and 350 nm (coptisine, palmatine and berberine) based on retention time and UV spectra compared with those of the standards. Using the optimized analysis method, the nine compounds were and showed the suitability of the separation system without

5 Simultaneous Determination of Nine Marker Components in Banhasasim-Tang 303 Table VII. The Amount of Each Marker Compounds in the BST (n =3) Batch (#) Concentrations (mg/g) ± SD ( 10 1 ) Liquiritin Coptisine Baicalin Palmatine Berberine Wogonoside Baicalein Glycyrrhizin Wogonin ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 obstruction from other components (Table II). Typical chromatograms of standard solutions and the BST extracts are shown in Figure 2. Linearity, range, LOD and LOQ The regression equations of the calibration curves, coefficients of determination (r 2 ), range, LOD and LOQ are presented in Table III. The linearity of the established method was evaluated from the r 2 value of the calibration curves of each compound. Calibration curves of the nine compounds showed good linear regressions, with r 2 values of in the seven tested concentration ranges. The LOD and LOQ values for the nine components were and µg/ml, respectively. Recovery and precision For the recovery test, three different concentrations (low, middle and high) of standard solutions were added to the BST sample. The average recovery of the nine compounds was in the range % and the RSD values were below 1.25%. The recovery data are summarized in Table IV. The RSD of peak areas and retention times for each compound were used to evaluate the reproducibility of this method. As a result, the RSD values for reproducibility assessment of the nine compounds were from 0.42 to 0.56% for peak areas and from 0.02 to 0.03% for retention times (Table V). Therefore, this established method showed good reproducibility under optimized conditions. The precision of this method is summarized in Table VI. The RSD values of intra- and interday variations for each analyte were and %, respectively. These results suggest that the established method has satisfactory recovery, reproducibility and precision for simultaneous analysis. Quantitative analysis of BST samples The newly established HPLC PDA analysis method was applied to the simultaneous determination of the nine marker compounds in BST. The concentrations of the nine components were mg/g and the results are summarized in Table VII. Among these components, baicalin, which is a marker component of Scutellariae Radix, was the most abundant compound (41.09 mg/g) in this sample. and spinosin from Ziziphus jujuba (14). We tried to analyze 16 components of the above. However, the seven compounds excluded the nine compounds including liquiritin, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, glycyrrhizin and wogonin were not detected because of the measurement conditions in this analysis system. Consequently, we tried to analyze nine components, such as baicalin, wogonoside, baicalein and wogonin from S. baicalensis, glycyrrhizin and liquiritin from G. uralensis and berberine, coptisine and palmatine from C. japonica in BST using HPLC PDA. Using the optimized analysis method, the nine compounds were separated within 35 min and showed good linear regressions with r 2 values of The retention times of the liquiritin, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, glycyrrhizin and wogonin were observed at 15.14, 18.80, 19.45, 20.54, 20.84, 22.41, 26.10, and min, respectively. Previously, the analytical method was reported for the HPLC profile of the main compounds from the fractions of BST by HPLC DAD (19). Kase et al. (19) conducted the three-dimensional HPLC profile of chemical constituents, whereas we performed the simultaneous determination and method validation of marker components in BST. As a result, nine compounds were successfully separated and HPLC PDA analysis method was validated. Conclusion In this work, a rapid, accurate, convenient and reliable HPLC PDA detection method was established and successfully applied for the quantitative analysis of nine marker components in extracts of the traditional Korean herbal medicine BST. Validation of the method showed good linearity, reproducibility, intra- and interday precision and recovery. The established method may be useful for the quality control of BST samples and related botanical preparations. Funding This research was supported by a grant (no. K14030) from the Korea Institute of Oriental Medicine. Conflict of interest statement. None declared. Discussion For the simultaneous quantitation of nine marker components in BST, a traditional Korean medicine, HPLC PDA method was established and validated. 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