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J Chromatogr Sci. 2016 April; 54(4): 536–546.
Published online 2015 December 27. doi:  10.1093/chromsci/bmv182
PMCID: PMC4885386

Development of UPLC Fingerprint with Multi-Component Quantitative Analysis for Quality Consistency Evaluation of Herbal Medicine “Hyangsapyeongwisan”


Hyangsapyeongwisan (HSPWS), known as traditional herbal medicine, has been used in the treatment of gastric disease. Standardization of HSPWS is a necessary step for the establishment of a consistent biological activity for the production and manufacturing of HSPWS herbal preparations. A simple, sensitive and accurate method using ultra-performance liquid chromatography (UPLC) fingerprinting with a diode array detector (DAD) was developed and validated for systematic quality evaluation of HSPWS. Separation conditions were optimized using a Halo C18 2.7 µm, 4.6 × 100 mm column with a mobile phase of 0.1% phosphoric acid and acetonitrile, and detection wavelengths of 215, 250 and 350 nm. Validation of the analytical method was evaluated by tests of linearity, precision, accuracy and robustness. All calibration curves of components showed good linearity (R2 > 0.9996). The limit of detection (LOD) and limit of quantification (LOQ) were within the ranges of 0.004–0.134 and 0.012–0.406 µg/mL, respectively. The relative standard deviation (RSD) values of intra- and inter-day testing were within the range of 0.01–3.84%. The result of the recovery test was 96.82–104.62% with an RSD value of 0.14–3.84%. Robustness values of all parameters as well as the stability test of analytical solutions were within the standard limit. It showed that the developed method was simple, specific, sensitive, accurate, precise, reproducible and robust for the quantification of active components of HSPWS. Chromatographic fingerprinting with quantitative analysis of marker compounds in HSPWS prepared by different methods and commercial formulation was also evaluated successfully.


Oriental herbal medicines have been extensively used to prevent and cure different types of human diseases for thousands of years. Herbal remedies or medicines consist of portions of plant or unpurified plant extracts containing several constituents that work together synergistically (1). With the ever-increasing use of herbal medicines and the global expansion of the herbal medicines market, safety has become a major concern for both health authorities and the public sector in many countries (2). Consistency in composition and biological activity are essential requirements for the safe and effective use of therapeutic agents. In general, quantification of single marker compounds is simple and convenient, but it does not afford sufficient quantitative information for the other constituents in herbal medicine. Therefore, chromatographic fingerprinting is considered to be one of most important and acceptable approaches for quality assessment of herbal medicines during the last decade. Different parts of the same plant contain different concentrations of chemical compounds due to seasonal variation as well as stages of ripeness. The method of collection, drying, packing, storage, transportation and methods of extraction also affect herbal quality. Extraction method, temperature, solvent and time period are major parameters influencing the qualitative as well as quantitative evaluation of multi-components in herbal drugs. Therefore, the extraction condition needs to be optimized to obtain the highest yield of marker compounds (3).

For the establishment of consistent biological activity, ultra-performance liquid chromatography (UPLC) improves the quality of the data, and also increases productivity by giving more data per unit of work, with increased resolution compared with high-performance liquid chromatography (HPLC) (4).

Hyangsapyeongwisan (HSPWS) mix extract powder is a combinational oriental herbal formulation that consists of Atractylodis Rhizoma (Compositae), Citri Pericarpium (Rutaceae), Cyperi Rhizoma (Cyperaceae), Ponciri Fructus (Rutaceae), Agastachis Herba (Labiatae), Magnoliae Cortex (Magnoliaceae), Amomi Fructus (Zingiberaceae), Aucklandiae Radix (Compositae), Glycyrrhizae Radix (Leguminosae) and Zingiberis Rhizome (Zingiberaceae) (5). HSPWS was commonly used in East Asian countries (China, Korea and Japan) for the treatment of gastrointestinal disease such as functional dyspepsia, gastroenteritis and gastric dysmotility disease (5). It revealed potent antiradical activities on 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radicals, superoxide anions, nitric oxide and peroxynitrite radicals (6).

In this study, establishment of a ultra-performance liquid chromatography—diode array detector (UPLC-DAD) method was undertaken for the quantification of 13 marker compounds: atractylodin from Atractylodis Rhizoma, hesperidin from Citri Pericarpium, α-cyperone from Cyperi Rhizoma, Poncirin and naringin from Ponciri Fructus, rosmarinic acid from Agastachis Herba, magnolol and honokiol from Magnoliae Cortex, nerolidol from Amomi Fructus, costunolide and dehydrocostus lactone from Aucklandiae Radix and glycyrrhizin from Glycyrrhizae Radix have been chosen for standardization. Major compounds that are included in each herb were selected as marker compounds and a validation of the verified method was performed. Development of a simple and effective analytical method for the quantification of these active compounds would be a valuable tool for the quality control of this herbal formulation. This developed method was also applied for the analysis of other commercially available HSPWS samples prepared by different methods.


Materials and reagents

All the organic solvents used for extraction and isolation were purchased from Dae-Jung Chemicals Co. Ltd. (Siheung, South Korea). Silica GF254 and silica gel (200–300 mesh) were used for thin-layer chromatography (TLC) and column chromatography (CC), respectively (Merck, Darmstadt, Germany). Medium Pressure Liquid Chromatography (MPLC) Yamazen Pump 54 with a UV-10 V detector (Osaka, Japan) was used for separation of bioactive compounds. For structure elucidation, a Bruker FT-NMR spectrometer (Billerica, MA, USA) was used. Gas chromatography–mass spectrometry (GC–MS, 2010) Shimadzu (Kyoto, Japan) was used for determination of the molecular weight of isolated compounds. Marker compounds: atractylodin was purchased from Natural Product Banks (Seoul, South Korea); hesperidin, honokiol, magnolol, naringin and poncirin were purchased from Korea Food and Drug Administration (KFDA) (Cheongwon, South Korea); costunolide and rosmarinic acid were purchased from Sigma-Aldrich Co. (St Louis, MO, USA); glycyrrhizin was purchased from Chromadex (Irvine, CA, USA) and 6-gingerol and dehydrocostus lactone were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The purity of marker compounds was above 98%. The HPLC-grade solvents were purchased from Burdick and Jackson (Muskegon, MI, USA). The analytical grade phosphoric acid was purchased from Samchun Pure Chemicals Co. Ltd. (Yeosu, South Korea). The commercial formulation was received from Hanpoong Pharm & Food Co. (Jeonju, South Korea). All the necessary herbs were purchased through local suppliers, Human herb (Gyeongsangbuk-do, South Korea) and Omniherb (Suseong-gu, Daegu, South Korea).

Extraction, isolation and identification of α-cyperone

The dried rhizome of Cyperi Rhizoma (1.5 kg) was extracted with methanol (50 ± 5°C, 1 L × three times) for 2 h under reflux. The crude dried methanol extract was suspended in 10% methanol and subjected to fractionation using hexane. This fraction was loaded on a normal-phase silica gel column. The residues having similar TLC patterns were collected and concentrated. The concentrated fraction was further loaded on an MPLC C8 40–75 μm column using 65% methanol and yielded an oily compound. The structure determination was done by proton nuclear magnetic resonance (1H-NMR) and carbon-13 nuclear magnetic resonance (13C-NMR). Molecular weight was determined by observing the molecular ion peak in gas chromatography–mass spectrometry (GC–MS).

Colorless oil; GC–MS m/z: 218.1; 1H-NMR (500 MHz, CDCl3): δ 4.73 (2H, s, H-13), 2.70 (1H, d, J = 10.0 Hz, H-7), 2.46 (1H, m, J = 15.0, 14.0, 6.1 Hz, H-2b), 2.39 (1H, m, J = 15.0, 5.0 Hz, H-2a), 2.04 (2H, d, J = 10.0 Hz, H-6a), 1.83 (1H, dd, J = 13.5, 4.6 Hz, H-6b), 1.77 (5H, s, H-12, 14), 1.75–1.58 (4H, m, H-1, 9), 1.42 (1H, m, H-8a), 1.36 (1H, m, H-8b), 1.17 (3H, s, H-15); 13C-NMR (CDCl3, 125 MHz): δ 199.0 (C-3), 162.1 (C-5), 149.0 (C-11), 128.7 (C-4), 109.2 (C-13), 45.9 (C-7), 41.9 (C-1), 37.4 (C-9), 35.8 (C-2), 33.8 (C-10), 32.9 (C-6), 26.8 (C-8), 22.4 (C-15), 20.6 (C-12), 10.9 (C-14). These data indicated that the isolated compound was α-cyperone; the values were confirmed by comparing with values in the literature (7).

Separation of nerolidol from Amomi Fructus

The dried fruit of Amomi Fructus (1 kg) was subjected to extraction with methanol (50 ± 5°C, 1 L × three times) for 2 h under reflux. The crude dried methanol extract was suspended in water and subjected to fractionation using methylene chloride. This fraction was loaded on a normal-phase silica gel column. The residues having similar TLC patterns were collected and concentrated. The concentrated fraction was further chromatographed by an MPLC C8 (40–75 μm) column and an oily compound was isolated. The structure determination was done by 1H-NMR and 13C-NMR. Molecular weight was determined by observing the molecular ion peak in GC–MS.

Colorless oil; GC–MS m/z: 222.1; 1H-NMR (500 MHz, CD3OD): δ 5.92 (1H, dd, J = 18.0, 11.0 Hz, H-2), 5.20 (1H, d, J = 16.45 Hz, H-10), 5.10 (2H, m, J = 17.4 Hz, H-4), 5.02 (1H, d, J = 10.55 Hz, H-6), 2.08–1.96 (6H, m, H-5, 8, 9), 1.65 (3H, brs, H-14), 1.58 (6H, brs, H-12, 15), 1.49(2H, m, H-1), 1.23 (3H, brs, H-13); 13C-NMR (CD3OD, 125 MHz): δ 145.0 (C-2), 134.5 (C-7), 130.8 (C-11), 124.4 (C-10), 124.1 (C-6), 110.6 (C-1), 72.5 (C-3), 42.1 (C-4), 39.5 (C-8), 26.4 (C-13), 26.3 (C-9), 24.6 (C-12), 22.3 (C-5), 16.4 (C-15), 14.7 (C-14). This compound was identified as (3S, E)-nerolidol because the chemical shift value is 14.7 of C-14, which suggested that this compound is (E)-form and its optical rotation value ([α]25 dextrorotatory : +11.2°) supported the fact that the absolute configuration of C-3 was S. The values were confirmed comparing with literature values (8).

Extraction methods for preparation of HSPWS

HSPWS mix extract powder was formulated by extraction of bioactive compounds from each plant (100 g) separately to get dry powder. The obtained powder was mixed together giving a mixture containing the following compounds: Atractylodis Rhizoma (5.3 g), Citri Pericarpium (2.7 g), Cyperi Rhizoma (2.7 g), Ponciri Fructus (2.1 g), Agastachis Herba (2.1 g), Magnoliae Cortex (1.9 g), Amomi Fructus (1.9 g), Aucklandiae Radix (1.3 g), Glycyrrhizae Radix (1.3 g) and Zingiberis Rhizoma (4.7 g). HSPWS extract granule was prepared by; Atractylodis Rhizome (2.00 g), Magnoliae Cortex (1.50 g), Citri Pericarpium (1.50 g), Glycyrrhizae Radix (0.50 g), Amomi Fructus (0.67 g), Cyperi Rhizoma (1.33 g), Zingiberis Rhizome (1.00 g), Zizyphi Fructus (1.00 g) and Agastachis Herba (0.33 g) were mixed together and extracted in order to get dry powder. Water was used as the extraction solvent. The herbs were extracted with a herb-to-solvent ratio of 1:8 for 2 h at a temperature of 80 ± 5°C, and the extract was prepared to yield powder.

Chromatographic condition

The chromatograph consisted of an Agilent Technologies 1290 Infinity (Gangnam-gu, Seoul, Korea) UPLC system with a DEBAA03145 binary pump and DEBAF02229 diode array detector; a DEBAP03582 autosampler was used for sample analysis. The output signal of the detector was recorded using Chemstation Software version 1.3. For this formulation, chromatographic separation was carried out using a Halo C18 2.7 µm, 4.6 × 100 mm column. The mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid (B) with gradient elution for better separation. The gradient solvent was optimized and performed as 15% A (0 min), 15–16% A (0–13 min), 16–32% A (13–25 min), 32–59% A (25–39 min) and 59–71% A (39–45 min) at a flow rate of 0.8 mL/min. Detection was made in three different UV wavelengths. The column temperature was maintained at 45°C, and the injection volume was 4 µL.

Preparation of standard solutions

The stock solutions of the concentration of 1 mg/mL of each of the compounds: naringin, hesperidin, rosmarinic acid, poncirin, glycyrrhizin, 6-gingerol, costunolide, honokiol, dehydrocostus lactone, magnolol, α-cyperone, atractylodin and nerolidol (Figure 1) were prepared in 70% methanol–water. A dilution of stock solution was done for the establishment of a calibration curve. All solutions were filtered through a 0.22-µm PTFE hydrophilic filter.

Figure 1.
Chemical structures of standard compounds.

Preparation of sample solutions

All the extracts of individual plants (15 mg/mL) and formulation (30 mg/mL) were prepared in 70% methanol with an ultra-sonication water bath at 50°C. All solutions were filtered through 0.22 µm PTFE hydrophilic filter before injection.

Method validation

The method validation for linearity, limit of detection (LOD) and limit of quantification (LOQ), specificity, precision (inter- and intra-day and repeatability), robustness and accuracy was performed following the International Conference on Harmonization (ICH) Guidelines and United States Pharmacopeia (9).

Linearity, LOD and LOQ

All calibration graphs were plotted on linear regression analysis of the integrated peak areas (y) versus concentration (x, μg/mL) of the diluted standard solution at five different concentrations. These solutions were analyzed individually in triplicate for the establishment of the calibration curve. The correlation coefficient (R2) was greater than 0.999 for all standard components in order to determine the establishment of linearity (10). The LOD and LOQ were determined from the calibration curve, using the values of standard deviation (SD) and slope (S) of the calibration curve: LOD = 3.3 × SD/S and LOQ=10 × SD/S (11).


The specificity of the chromatographic method was studied by assessment of peak purity of marker components in the extract with the UV spectrum of standards provided by the DAD detector (12). Peak purity was confirmed by comparing the spectra on the upslope and downslope curves in the optimized chromatogram of the HSPWS sample (13).


The intra- and inter-day variability as well as repeatability were checked to determine the method precision. The intra-day test was analyzed in three different concentrations of each marker component under the optimized conditions within the same day (14). Inter-day precision was carried out by using the same concentrations used for intra-day precision on three consecutive days. The repeatability test was assessed by sextuplicate injections from the same vial of extractive solution/standard mixture in the same day and was expressed in terms of the relative standard deviation (12).


Accuracy of the method was measured by the recovery assay. Three different concentration levels of the 13 marker components were added to the known concentration of the HSPWS mix extract powder sample. Recovery (%) was calculated by the equation (amount found − original amount)/amount spiked × 100% (13).


The robustness of the proposed UPLC-DAD method was assessed by altering the chromatographic conditions like mobile-phase flow rate (±0.2 mL/min), column temperature (±5°C) and apparent pH of the buffer (±0.2 units), and the results of the experiments were evaluated with four responses. They were resolution between the two adjacent peaks, separation factor, capacity factor between peaks of interest with respect to the void volume and theoretical plate number which measured the peak efficiencies (15, 16).

Quantification of extractive solution

The optimized chromatographic method was applied for the quantitative determination of marker compounds in individual herbs as well as simultaneous assessment of standard components in an HSPWS sample prepared by different methods and commercial formulation. The marker components were quantified by linear regression of standards. Each sample was analyzed in triplicate to determine the mean content (17).


Optimization of the chromatographic condition

The UPLC chromatographic condition was maintained on the basis of certain criteria like column type, column temperature, mobile phase, detection wavelength, etc. To obtain optimal separation, five different columns were tested. Various columns (Halo C18 2.7 µm, 4.6× 100 mm; Acquity C18 1.7 µm, 2.1 × 50 mm; Halo PFP 2.7 µm, 4.6 × 100 mm; Halo Rp-amide 2.7 µm, 4.6 mm × 100 mm; Halo Rp-amide 2.7 µm, 4.6 × 150 mm), mobile phases (methanol–water and acetonitrile–water with different modifiers including acetic acid, formic acid, phosphoric acid and trifluoroacetic acid), column temperature (30, 35, 40 and 45°C), mobile-phase flow rates (0.8, 1, 1.3 and 1.5 mL/min) and different times were examined.

For this formulation, the results showed that the Halo C18 2.7 µm, 4.6 × 100 mm column had better resolution than others. The gradient solvent system consisted of acetonitrile (A) and 0.1% phosphoric acid (B) at a column temperature of 45°C with a flow rate of 0.8 mL/min, and the run time was optimized to 45 min. Both standards and sample extract were analyzed in the wavelength range of 200–400 nm. Figure 2 shows a typical chromatogram of HSPWS mix extract powder sample and the corresponding marker compounds.

Figure 2.
UPLC chromatograms of HSPWS mix extract powder at various wavelengths; (A) 215 nm, (B) 250 nm, (C) 350 nm and (D) standard mixture at 215 nm. This figure is available in black and white in print and in color at JCS online.

Method validation

For the determination of linearity, five different concentrations of each standard solution were injected individually: naringin (250–7.81 µg/mL), hesperidin (200–6.25 µg/mL), rosmarinic acid (40–2.5 µg/mL), poncirin (300–9.37 µg/mL), glycyrrhizin (200–6.25 µg/mL), 6-gingerol (30–1.87 µg/mL), costunolide (10–0.62 µg/mL), honokiol (40–2.5 µg/mL), dehydrocostus lactone (10–0.62 µg/mL), magnolol (40–2.5 µg/mL), α-cyperone (250–9.81 µg/mL), atractylodin (30–1.87 µg/mL) and nerolidol (150–4.67 µg/mL). The correlation coefficient (R2) values for each standard were found to be >0.9996, which indicates a high degree of correlation between selected concentrations and their respective peak areas. Therefore, linearity was verified. Table I lists the regression equation, correlation coefficients, LOD, LOQ and calibration ranges obtained for the regression lines. Specificity was determined by comparison of UV absorption between standard marker compounds and the extract samples. Biomarkers and samples were measured at the same UV absorption wavelength, and peaks were pure and interference due to impurities was not observed (Figure 3).

Table I.
Regression Data, LOD and LOQ of Marker Compound (113) by UPLC
Figure 3.
UV spectra of 13 (113) marker compounds (dark curve) and HSPWS mix extract powder sample (gray curve).

As listed in Table II, the RSD of intra- and inter-day tests were found to be within the ranges of 0.01–2.91% and 0.07–2.64%, respectively. It was also noted that the RSD of the repeatability test was in the range of 0.23–2.63% (Table III). These data revealed that the described method had an acceptable degree of precision.

Table II.
Analytical Results of Intra-day and Inter-day Variability
Table III.
Repeatability Data of (113) Compoundsa

The average recovery percentages of the 13 marker compounds were in the range 96.82–104.62% with the RSD value in the range 0.14–2.63% (Table IV). The results of the recovery test showed that the established method was reliable and accurate.

Table IV.
Determination of Recoveries of 13 Compounds (113) in HSPWS Mix Powder

The robustness test was performed in order to evaluate the consistency of the developed UPLC methods. The chromatographic conditions which may affect the performance of the method, such as column temperature (30, 35, 40 and 45°C), solvent flow rate (0.8, 1.0, 1.3 and 1.5 mL/min.) and pH of buffer (1.84, 2.06, 2.25 and 2.49 units), were deliberately changed. The results were evaluated using four different analytical factors such as theoretical plate number, separation factor, resolution between two peaks and capacity factor. The degree of the reproducibility obtained as a result of small deliberate variations in the method parameters has proved that the method is sufficiently robust (Figure 4).

Figure 4.
Influence of liquid flow rate, pH of buffer and column temperature on UPLC chromatographic performance. The column temperature of 30–45°C on figure (A–D); buffer pH (1.84–2.26) on figure (E–H); and solvent flow ...

Quantitative determination of marker compounds of each herb

The developed UPLC chromatographic method was successfully applied to the quantitative determination of 13 major active compounds from individual herbs. Nerolidol was found to be the most abundant bioactive component (157.434 mg/g) in the dried extract of Amomi Fructus. In addition, the content of poncirin, naringin and hesperidin was also found markedly high from their respective herbs (Table V). Hence the quantitative determination of marker components from their plants would be a valuable tool to improve quality control for each herb used for the production of HSPWS herbal products . Representative chromatograms of each herb are shown in Figure 5.

Table V.
Content of Marker Compounds of Each Herb in HSPWS Mix Extract Powder
Figure 5.
UPLC chromatograms of individual plants: (A) Ponciri Fructus, (B) Citri Pericarpium, (C) Agastachis Herba, (D) Glycyrrhizae Radix, (E) Zingiberis Rhizoma, (F) Aucklandiae Radix, (G) Magnolia Cortex, (H) Cyperi Rhizoma, (I) Atractylodes Rhizoma and (J) ...

Sample analyses

Using the established verified method, marker compounds of HSPWS prepared by different methods were quantitatively analyzed. Each sample was analyzed in triplicate to determine the mean content. The HSPWS sample consists of two different preparations on the basis of herb composition and preparation method.

For HSPWS mix extract powder, the result was expressed as the content of marker compound per Atractylodis Rhizoma (5.3 g) + Citri Pericarpium (2.7 g) + Cyperi Rhizoma (2.7 g) + Ponciri Fructus (2.1 g) + Agastachis Herba (2.1 g) + Magnoliae Cortex (1.9 g) + Amomi Fructus (1.9 g) + Aucklandiae Radix (1.3 g) + Glycyrrhizae Radix (1.3 g) + Zingiberis Rhizoma (4.7 g) of the sample. The result showed that poncirin (10.543 ± 0.517 mg/g) was the major chemical constituent in this sample. It was also noted that nerolidol > naringin > hesperidin were quantitatively found in the given order, 6-gingerol, costunolide, dehydrocostus lactone and atractylodin were detected in very small quantities.

For HSPWS extract granule, the result was expressed as the content of marker compound per Atractylodis Rhizome (2.00 g) + Magnoliae Cortex (1.50 g) + Citri Pericarpium (1.50 g) + Glycyrrhizae Radix (0.50 g) + Amomi Fructus (0.67 g) + Cyperi Rhizome (1.33 g) + Zingiberis Rhizome (1.00 g) + Zizyphi Fructus (1.00 g) + Agastachis Herba (0.33 g) of the sample. Hesperidin was the major constituent in this sample. Atractylodin, α-cyperone, 6-gingerol, costunolide, dehydrocostus lactone, naringin, nerolidol and poncirin were not detected because Ponciri Fructus and Aucklandiae Radix were not included in this sample. Moreover, nerolidol and α-cyperone were also not found. It could be due to the oily, volatile nature of these compounds.

Quality control of commercially available HSPWS products

The quality of three different commercially available HSPWS products prepared by different methods was evaluated by the established method. Each sample was analyzed in triplicate to determine the mean content. The results were expressed as the content of marker compound per dose and per gram of the sample. According to KFDA, the content of glycyrrhizin in the extract granule should be more than 2.7 mg/dose; hesperidin and glycyrrhizin in mix extract powder should be more than 12.5 mg/dose and 6.4 mg/dose, respectively, and the glycyrrhizin content in soft dried extract should be more than 2.5 mg/g. In this validated UPLC method, the content of glycyrrhizin in the extract granule was found to be 3.51 ± 0.39 mg/dose. It was also noted that the content of hesperidin and glycyrrhizin in the mix extract powder was found to be 8.69 ± 0.64 and 19.56 ± 0.01 mg/dose, respectively. Moreover, the glycyrrhizin content in soft dried extract was 3.23 ± 0.06 mg/g. Two major constituents in each commercial product were within the limit of KFDA, suggesting that these commercial products of HSPWS were all qualified according to the current standards. This study revealed that hesperidin was the major constituent in these commercial products of HSPWS. Naringin and poncirin were detected from the mix extract powder. Similarly, magnolol and honokiol were also quantified from the HSPWS extract granule and the soft dried extract. However, most of the major constituents were not detected from these commercial products, and so overall quality control of commercial products should focus on multiple constituents and not just on glycyrrhizin and hesperidin. Therefore, marker components in HSPWS commercial products and their remedial mechanisms could be further understood based on the investigation of these compounds.


Standardization and analysis of the marker compounds in herbal medicine are necessary for the quality, efficacy and safety of the medicine. Simultaneous qualitative as well as quantitative determination of chemical markers of each ingredient in this polyherbal preparation is required for which an optimal separation technique with a reliable, fast and validated UPLC-DAD method has been developed (18). In HSPWS mix extract powder, 13 standard compounds were selected for standardization. Among the 13 standard compounds, nerolidol and α-cyperone were not available in our market and so these compounds were isolated from their respective herb.

Magnolia bark might be an important traditional medicine for treating a variety of conditions such as cancer, neuronal disease, inflammatory disease and cardiovascular disease. Most studies reported that Magnolol (2–11%) and honokiol (0.3–4.6%) were found to be major active chemical compounds in Magnoliae Cortex. Citri Pericarpium contains three flavone glycosides: hesperidin, narirutin and naringin, among which hesperidin was predominant (19, 20). The HPLC profile of the water extract of Glycyrrhizae Radix showed that liquiritin and glycyrrhizin were major chemical constituents. A literature survey showed that costunolide and dehydrocostus lactone were isolated from Aucklandiae Radix. Costunolide (35.7 mg) and dehydrocostus lactone (43.6 mg) were obtained from the dried rhizome of this plant (21). The amount of poncirin (2.15%) and naringin (1.65%) were quantitatively determined from Ponciri Fructus. These two are the major compounds obtained from this plant (22). α-cyperone, a well-known major chemical constituent of Cyperi Rhizoma, showed anti-inflammatory activity by inhibiting lipopolysaccharide-induced cyclooxygenase-2 and interleukin-6 in RAW 264.7 cells (23). 6-Gingerol is the major pungent principle of Zingiberis Rhizoma with numerous pharmacological properties including antioxidant, anti-inflammatory and antitumor activities (24).

One of the herbs (Zizyphi Fructus) that are included in the HSPWS extract granule was not taken in this study due to the absence of a marker compound that should meet the linearity range in the current method validation.

The available literature indicates that the constituents of most of the herbs constituting HSPWS are well studied. However, the specific investigation on the major constituents of the herbal formula has not been reported yet. Full evaluation of HSPWS products depends on the analysis of the multiple active ingredients. Therefore, it is necessary to develop a sensitive and reliable analytical method to identify and characterize multiple active constituents in HSPWS products to ensure the safety, efficacy, and stability of the herbal prescription.

In addition, chemical structures of the markers are different (Figure 1). As the maximum absorbance wavelengths of these components are also different, it is difficult to obtain a sensitive determination of all markers in a single wavelength. Therefore, multiple wavelength detection was selected in order to obtain a sensitive determination of all components. However, all standard compounds were clearly detected in the standard mixture using only one wavelength. Thus, for the standard mixture sample a single wavelength was selected.

Various parameters used in method validation are specificity, linearity, precision, robustness and accuracy, which were to be within the range of regulations of ICH, KFDA and USP guidelines. Different kinds of herbs are mixed together and boiled with water to get a decoction, which is usually done by patients themselves. This traditional prescription method has been in practice for at least 2000 years (25). The major drawback of this traditional method is that the boiling temperature, time and amount of water used for the decoction are difficult to control; also the quality of the decoction may be inconsistent among different practices and thus the efficacy and safety of the decoction could not be assured (26). Therefore, the extraction procedure, solvent and herb-to-solvent ratio have been evaluated. Different procedures showed a variation in the content of marker compounds.

From the result, all marker components were detected in the mix extract powder sample. Among the components, glycyrrhizin and hesperidin were found to be at a relatively high level in the three different commercial products of HSPWS. However, atractylodin, costunolide, dehydrocostus lactone, naringin, poncirin, nerolidol, 6-gingerol, α-cyperone and rosmarinic acid were not detected in the commercial formulation; this could be due to the absence of Ponciri Fructus and Aucklandiae Radix, which were not included in the extract granule and soft dried extract of the HSPWS commercial formulation, and also due to the thermolabile and volatile nature of the compounds. Nevertheless, the quantification and analysis of multiple components in HSPWS commercial products is necessary in order to optimize their quality for better therapeutic activity.


The proposed UPLC-DAD fingerprinting method combined with quantitative analysis of 13 marker compounds is an efficient tool for the quality consistency evaluation of HSPWS. The results demonstrated that the developed method is simple, sensitive, accurate, precise, reproducible, robust and specific and could be readily employed as a suitable quality control method for the HSPWS-derived extract. Variation in the content of active constituents in the HSPWS sample prepared by different methods was evaluated. Most of the compounds were not detected in commercial formulation. The method was also successfully used for quantitative determination of marker constituents in HSPWS prepared by different methods.


This paper was supported by Wonkwang university in 2013, Iksan 570-749, South Korea.


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