Research Article Simultaneous determination of five anthraquinones ...

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Research Article

Simultaneous determination of five anthraquinones in a Chinese traditional preparation by RP-HPLC using an improved extraction procedure Yan-bin Shi1, Hui-li Li1, Hai-qin Wang1, Yan-biao Yang2, Xiao-yun Zhang1, Hui Wang1, Zong-jie Zhu2, Zhi-ye Zhang2, Cheng-an Zhang2 1. School of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu Province, China 2. Department of Pharmaceutics, the First People’s Hospital of Lanzhou, Lanzhou 730000, Gansu Province, China

OBJECTIVE: The stable quality of Chinese herbal medicines is a critical factor for their reliable clinical efficiency. An improved liquid-liquid extraction procedure and a liquid chromatographic method were developed to simultaneously analyze five anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) in a Chinese traditional hospital preparation, Fuyankang mixture, in order to quantitatively control its quality in a more effective way. METHODS: A more economical and repeatable extraction procedure based on conventional liquid-liquid extraction technique was developed and used to extract five marker components in Fuyankang mixture. These anthraquinones were separated in less than 20 min on a C18 column with methanol and 0.1% phosphoric acid (88:12, v/v) as mobile phase. The method was validated for specificity, precision, spiked recovery and stability. RESULTS: Compared to conventional liquid-liquid extraction, the improved liquid-liquid extraction was found to be more effective for simultaneous extraction of anthraquinones from an aqueous Chinese herbal preparation, especially for hydrophobic compounds. The improved extraction method was successfully applied to determine the content of five marker components in Fuyankang mixture by the means of reverse phase high-performance liquid chromatography. CONCLUSION: The improved extraction procedure may be suitable for routine quality control of Fuyankang mixture and other traditional preparations at city-level hospitals in China. KEYWORDS: improved liquid-liquid extraction; Fuyankang mixture; drugs, chinese herbal; chromatography, high-performance liquid; anthraquinones; quality control http://dx.doi.org/10.1016/S2095-4964(14)60037-6 Shi YB, Li HL, Wang HQ, Yang YB, Zhang XY, Wang H, Zhu ZJ, Zhang ZY, Zhang CA. Simultaneous determination of five anthraquinones in a Chinese traditional preparation by RP-HPLC using an improved extraction procedure. J Integr Med. 2014; 12(5): 455–462.  Received February 17, 2014; accepted May 7, 2014. Correspondence: Prof. Yan-biao Yang; Tel: +86-931-2352711; E-mail: [email protected]

1 Introduction Chinese herbal preparations are composed of several herbs containing complex chemical components. Unlike Journal of Integrative Medicine

pharmaceutical drugs, absolute quantitative standards have not yet been made for most Chinese herbal preparations, especially for traditional hospital preparations. Commonly hospital preparations are evaluated by techniques like visual inspection, relative density and thin-layer chro-

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matography (TLC). In fact, these evaluation methods do not describe the amount of active components. Hence, they cannot effectively control the quality of traditional hospital medicines. Fortunately, with the rapid development of China’s medical system, the quality of traditional medicine is more valued than before. Fuyankang mixture (batch No. 20120207, the First People’s Hospital of Lanzhou City, China) is an herbal preparation used for treatment of pelvic inflammation, vaginitis and appendagitis in women. In recent years, it has attracted more and more patients’ attention due to its safety and efficacy [1]. Fuyankang mixture consists of eight medicinal plant materials: radix et rhizoma of Rheum palmatum L. (Dahuang), cortex of Phellodendron chinense Schneid (Huangbai), caulis of Lonicera japonica Thunb (Rendongteng), pollen of Typha angustifolia L. (Puhuang), fructus of Lycium barbarum L. (Gouqi), flower of Carthamus tinctorious L. (Honghua), rhizoma of Dryopteris crassirhizomae Nakai (Guanzhong), and fructus of Morus alba L (Sangshen). The bioactive components in Fuyankang mixture are quite complicated, and include anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) from R. palmatum, berberine from P. chinense, chlorogenic acid from L. japonica, quercetin from T. angustifolia as well as other substances. The antimicrobial and anti-inflammatory activities of rhein-related anthraquinones[2,3], berberine[4,5], and chlorogenic acid[6,7] have been studied extensively, and may be responsible for some of the success of Fuyankang mixture in treating pelvic inflammation, vaginitis and appendagitis. However, strong interference from unknown water-soluble substances made it extremely difficult to separate and quantify these active polar components such as berberine and chlorogenic acid. In the present study, the five anthraquinone compounds considered to be the marker bio-active substances in Fuyankang mixture (aloe-emodin, rhein, emodin, chrysophanol and physcion; Figure 1) were effectively extracted with an improved liquid-liquid extraction procedure, identified and quantified using a reverse phase-high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array (PDA) detector. Purification and identification methods for all or part of the five target anthraquinones have been described well in the literature. These techniques included HPLC [8,9],

ultra-pressure liquid chromatography (UPLC)[10], capillary zone electrophoresis (CZE), electrochromatography[11-13], microemulsion electrokinetic chromatography (MEC)[14], HPLC-MS and GC-MS[15,16]. Moreover, TLC was widely applied to qualitatively identify the main components in traditional Chinese herbal extracts. This technique has also been employed, in conjunction with an ultraviolet scanner, to quantify anthraquinones levels[17,18]. RP-HPLC analysis was one of the most popular analytical method in China’s city-level hospitals. The extraction procedure is a crucial step for the effective determination of target components in traditional herbal preparations. Different extraction methods have been reported to improve analysis efficiency for multi-component preparations. High-speed counter current technology (HSCCC)[19], supercritical CO2 fluid extraction (SFE)[9] and solid-phase extraction (SPE)[20] were used to develop environmentally friendly extraction processes. However, these instruments are expensive, limiting their availability to control the quality of city-level hospital preparations on a routine basis. In recent years, microextraction methods have gained attention due to their rapidity, low cost and high enrichment factors. Some of these techniques include dispersive liquid-liquid microextraction (DLLME) [21], cloud point extraction (CPE)[22], single drop microextraction (SDME) [23], solid-phase microextraction (SPME) and its coupled technologies[24]. However, all these methods require strict separation conditions including the volume of solvent or surfactant used, skill level of technician and calibration of the instrument. Other extraction methods, such as ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE), have also been employed to improve extraction efficiency [25]. These high-energy approaches may cause damage to the structure of target compounds, especially for easily oxidized or hydrolyzed substances. Liquid-liquid extraction (LLE) is a traditional technique for separation and pre-concentration of hydrophobic constituents from aqueous solutions or suspensions. Unfortunately, repeatability of LLE depends strongly on highly variable human factors; this uncertainty is unacceptable in a quantitative environment. Thus, the aim of the present investigation was to develop a convenient, effective, and reproducible extraction method based on conventional LLE technique for the analysis of anthraquinones in Fuyankang mixture using RP-HPLC. This method is necessary to establish routine quality control for this preparation or other TCM preparations at city-level hospitals in China. 2 Materials and methods

[8] Figure 1 Chemical structure of analytes


2.1 Chemicals and reagents The reference standards for aloe-emodin (serial number

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(SN) 110795), emodin (SN 110757), rhein (SN 110756), chrysophanol (SN 110796) and physcion (SN 110758) were purchased from the Chinese National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Fuyankang mixture was made and donated by the First People’s Hospital of Lanzhou City (batch No. 20120207, Gansu, China). HPLC grade methanol (Shandong Yuwang Industry Co., Ltd., China), ethyl acetate, n-butanol and analytical grade acetone (Tianjin Fuyu Chemical Industry Co., Ltd., China) were purchased from local commercial company. Wahaha Purified Water (Hangzhou Wahaha Group Co., Ltd., China) was used as deionized water. 2.2 Apparatus HPLC analysis was performed on a Waters liquid chromatographic system equipped with a 1525 binary HPLC pump, an on-line degasser, a model 1500 column heater, a 2998 PDA detector (wavelength range from 210 to 400 nm) and a manual injector (Rheodyne 7725i, CA, USA). A personal computer with Empower Pro software (Waters, USA) was used to record the chromatograms and analyze the data. The separation was performed on a C18 column (250 mm × 4.6 mm, 5 μm, Diamonsil® , Dikma, China) with a SecurityGuardTM guard cartridge (Phenomenex, USA). 2.3 Chromatographic conditions In this system, the mobile phase was a mixture of methanol and 0.1% phosphoric acid (88:12, v/v), and was filtered through a 0.45-µm organic membrane prior to use. The flow rate was 0.8 mL/min. The column temperature was set at 40 ℃. The injection volume was 20 μL. The signals from the detector were monitored at 254 nm and analyzed with the Empower Pro software. The chromatography system was equilibrated by the mobile phase before the separation of samples was conducted. 2.4 Preparation of standard solutions Reference standards for the five anthraquinones were accurately weighed and dissolved each in 10 mL methanol to give a stock solution of 0.1 mg/mL for each standard. One mililitre of each stock solution was transferred into a round-bottom flask containing 25 mL deionized water with pH 3.2 (the pH value equals to that of Fuyankang mixture) and mixed well. Subsequently, the methanol was removed using a rotary evaporator under reduced pressure, yielding aqueous solutions of each of the five reference standards. The reference solution was transferred into a separating funnel, while a round-bottom flask with a magnetic stir bar, containing extraction solvent of 25 mL ethyl acetate-butanol (1:2, v/v), was placed on a magnetic stir plate. The solution in the separating funnel was dripped into the stirred extraction solvent (Figure 2), thereby rapidly emulsifying the aqueous sample in the organic phase. The organic phase was allowed to separate and the Journal of Integrative Medicine

solvent was recovered under reduced pressure leaving a yellow residue. If necessary, the aqueous phase was again extracted with an equal volume of organic solvent. This residue was completely dissolved into 10 mL acetone and filtered through Whatman filter paper. The filtrate was evaporated to dryness and the residue was reconstituted in 10 mL methanol, filtered through 0.22 μm millipore filters and used as the mixed standard solution, further diluted into a serial concentrations in the range of 0.05-5 µg/mL with methanol, and stored at around 4 ℃ until HPLC analysis.

Figure 2 The equipment of an improved liquid-liquid extraction

2.5 Preparation of sample Fuyankang mixture was extracted with the following two extraction methods. The extraction time was kept constant (about 2 min) in the case of liquid-liquid extraction by both methods to allow direct comparison. 2.5.1 Method I: conventional liquid-liquid extraction Fuyankang mixture (25 mL) was transferred into a separatory funnel and extracted with an equal volume of ethyl acetate-butanol (1:2, v/v), by manually agitating the funnel twice. The organic phase was collected and the solvent was evaporated under reduced pressure. The residue was suspended into 10 mL acetone and filtered through qualitative filter paper. The filtrate was collected and acetone was evaporated. The yellowish brown residue was dissolved in 10 mL methanol, filtered with organic millipore filters to obtain the final Fuyankang mixture extracts for HPLC analysis.

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2.5.2 Method II: improved liquid-liquid extraction Fuyankang mixture (25 mL) was transferred into a separatory funnel, and a round-bottom flask containing 25 mL ethyl acetate-butanol (1:2, v/v) with a magnetic stirrer bar was placed on a magnetic stirrer beneath the funnel. Then the mixture was extracted following the same procedure described in the Preparation of Standard Solutions (section 2.4). The final filtrate was used as the Fuyankang mixture sample for HPLC analysis. 2.6 Validation of HPLC method The standard solution of the five compounds, Fuyankang mixture sample and Fuyankang mixture sample spiked with the reference compounds were analyzed under the same chromatographic conditions. The retention times, peak height/area and ultraviolet characteristics were compared to evaluate the effectiveness of each extraction protocol. Additionally, theoretical plate, resolution, asymmetry and 3-point purity of analytes were estimated for both standard solutions and Fuyankang mixture samples. A calibration curve was made from six concentrations of the mixed standard solutions and linearity was evaluated in terms of the correlation coefficients (r). The calibration curve was made to span the potential range of analyte concentrations in the Fuyankang mixture. Limit of detection (LOD) and limit of quantitation (LOQ) were evaluated according to the following formula:

where 3 and 10 are the values of signal-to-noise (S/N) ratio for LOD and LOQ, respectively; SD is standard deviation obtained from ten analysis results of a low concentration of analyte; C is the concentration of the analyte; is the average HPLC response of ten analyses. We used a concentration of 0.1 μg/mL of the mixed standard solution to estimate LOD and LOQ. Standard samples at 0.1, 0.5 and 2.5 μg/mL were used to test the extraction methods for intraday and interday variability. For intraday variability, five replicates of mixed standard solutions at three concentrations were analyzed within one day. For interday variations, the solutions were analyzed in triplicate on three consecutive days. The replicates on the same day were repeat runs for the same extraction. The results of the analyses were expressed as relative standard deviation (RSD, %). Stability was evaluated by calculating the RSD values of peak area of corresponding analytes in the same Fuyankang mixture sample, which was stored at ambient temperature for different time, i.e., at 0, 2, 4 and 8 h. Nine samples of the Fuyankang mixture (Lot No: 20120207) were spiked with reference substances at three concentrations (30, 15, and 5 μg), meanwhile, three of the unspiked Fuyankang mixture samples were used as September 2014, Vol.12, No.5

control. All tested mixtures were extracted with the same procedure described in chapter 2.5.2. The results were expressed as the percentage recovery of each of the five analytes. 2.7 Statistical analysis The concentration of anthraquinones in the extracts was calculated from the respective peak area using calibration curves and expressed as mean ± standard deviation. Statistical significance of the differences obtained for the two extraction methods was evaluated by student t test for pairwise comparisons using SPSS 13.0 statistical software. P < 0.05 was considered to be statistically significant. 3 Results 3.1 Chromatographic specificity As seen in Figure 3, the retention times of aloe-emodin, rhein, emodin, chrysophanol and physcion in Fuyankang mixture sample were around 5.2, 6.4, 8.7, 12.6 and 16.0 min, respectively. These time intervals were consistent with corresponding reference substances in standard solution. The peak heights of the five compounds in the spiked Fuyankang mixture sample increased concomitantly with those of the spiking compounds. Ultraviolet characteristic of each compound was used to monitor the analytes throughout analysis. Performance parameters for both Fuyankang mixture and the standard solution were: peak resolution was more than 2.0; asymmetry factor was between 0.80 and 1.15; the mean value of theoretical plate number was higher than 1 500; the three-point peak purity value for all the analytes was above 0.998 0. These results indicated that the proposed method was sufficient for quantitative determination of the five compounds in Fuyankang mixture. 3.2 Calibration, linearity, LOD and LOQ The 6-point concentration curves (ranging from 0.1 to 5.0 µg/mL) were calculated as the linear least squares regression of the peak areas (AU, x-axis) of analytes and the corresponding concentrations (µg/mL, y-axis) in triplicate assays. As shown in Table 1, the calibrations were highly linear, with the value of correlation coefficient r ≥ 0.999 0. The LODs were in the range 0.006 7-0.017 9 µg/mL, and the LOQs were 0.022 5-0.059 6 µg/mL. 3.3 Precision and stability As shown in Table 2, the RSD values of intraday and interday variability were between 1.40%-3.06% and 1.93%-5.81%, respectively. For all analytes, the variability at the lowest concentration (0.1 µg/mL) was greater than at the higher concentrations. RSD values of the peak area for the tests of sample stability were less than 5.97%, indicating that the sample solution was stable when stored at room temperature for 8 h. 3.4 Spiked recovery The recovery of analyte spikes (30, 15 or 5 µg) for each

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Figure 3 Chromatograms of the five analytes

A: Mixed standard solution (all concentrations of compounds 1 to 5 were 2.5 µg/mL); B: Fuyankang mixture sample spiked with reference substances (30 µg); C: Fuyankang mixture sample (Lot No. 20120207). Peaks: 1=aloe-emodin, 2=rhein, 3=emodin, 4=chrysophanol, 5=physcion. Analytical conditions: column, Diomonsil® C18; mobile phase, methanol-0.1% phosphoric acid (88:12, v/v); flow-rate, 0.8 mL/min; column temperature, 40 ℃; detection wavelength, 254 nm.

Table 1 Calibration curves and sensitivity for determination of five analytes Compound Aloe-emodin Rhein Emodin Chrysophanol Physcion

Calibration curve y=0.055 0×10-4x-0.017 0 y=0.034 4×10-4x-0.013 7 y=0.066 0×10-4x+0.015 1 y=0.059 2×10-4x+0.027 3 y=0.039 6×10-4x+0.044 3

Correlation coefficient (r) 0.999 3 0.999 3 0.999 0 0.999 1 0.999 1

LOD (µg/mL) 0.017 9 0.007 8 0.008 6 0.006 9 0.006 7

LOQ (µg/mL) 0.059 6 0.026 0 0.028 8 0.023 1 0.022 5

y represents concentration of analyte; x represents peak area. LOD: limit of detection; LOQ: limit of quantification.

of the five compounds ranged from 91.47% to 107.81%. Table 3 shows the RSD for recovery at the medium spike level (15 μg). The average recoveries were between 91.69% and 105.86%. Journal of Integrative Medicine

3.5 Comparative analysis of two extraction methods The improved liquid-liquid extraction was applied to Fuyankang mixture from three batch numbers; we prepared three samples from each batch. In order to compare their

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www.jcimjournal.com/jim Table 2 Intraday and interday precisions of the method Intraday precision RSD (%)

Compound

0.1 µg/mL

0.5 µg/mL

Interday precision RSD (%)

2.5 µg/mL

0.1 µg/mL

0.5 µg/mL

2.5 µg/mL

Aloe-emodin

3.06

2.46

2.68

5.81

4.33

3.45

Rhein

2.97

1.83

1.56

2.49

2.40

2.29

Emodin

2.31

2.28

2.31

2.75

2.62

2.62

Chrysophanol

2.28

1.40

2.06

2.29

2.60

2.57

Physcion

2.40

1.47

1.97

2.15

2.04

1.93

Table 3 Spiked recoveries of five analytes in the Fuyankang mixture sample Compound

Initial amount (µg)

Aloe-emodin

Amount added (µg)

24.80

15

96.41±4.08

Rhein

39.85

15

91.69±3.21

Emodin

17.43

15

105.86±2.58

Chrysophanol

35.05

15

95.66±2.25

7.75

15

98.69±0.99

Physcion

extraction efficiency the conventional liquid-liquid extraction method was also used to prepare 3 samples from each batch of Fuyankang mixture. Analyte concentrations measured from the extracts are shown in Table 4. Student t test showed that the differences between the concentrations of corresponding analytes were statistically significant (P < 0.05) except for in the case of emodin. The higher yield and lower variability of the improved liquid-liquid extraction method makes it the preferred technique for the extraction of anthraquinones present in Fuyankang mixture, particularly for those more hydrophobic components, such as chrysophanol and physcion. Table 4 Contents of five analytes in Fuyankang mixture analyzed by two methods Contents of compounds (mean ± standard deviation, µg/mL)

*

Recovery (mean ± standard deviation, %)

Compound

Improved LLE method (Method Ⅱ)

Aloe-emodin

2.062±0.098

1.987±0.157*

0.034

Rhein

3.296±0.188

*

3.140±0.200

0.028

Emodin

1.539±0.118

1.423±0.313

0.261

Conventional LLE method P value (Method Ⅰ)

**

Chrysophanol

2.948±0.117

2.519±0.199

0.001

Physcion

0.625±0.046

0.518±0.049**

0.001

**

P < 0.05, P < 0.01, vs method Ⅱ. Student t test was used to compare the differences between the two methods. LLE: liquid-liquid extraction. September 2014, Vol.12, No.5

4 Discussion Different organic solvents were used to improve extraction efficiency assessed by peak height of analytes and peak resolution. Those solvents included ethyl acetate, ethyl acetate-butanol, chloroform, dichloromethane and chloroform-butanol. Polar solvents such as acetone and methanol were used to exclude polar impurities in the later stage of the extraction procedure. Among them, ethyl acetate used alone showed low efficiency for rhein extraction; chloroform or dichloromethane demonstrated higher efficiency for physcion but poor efficiency for extraction of the other target compounds. A chloroform-butanol mixture was also tested, but failed to improve the extraction efficiency. A solution of ethyl acetate-butanol (1:2, v/v) improved the overall extraction efficiency, and was selected as extraction solvent for this application. Acetone was better at solubilizing the target anthraquinones than methanol, thus it was used to remove polar impurities. Additionally, we tried a self-made solid phase cartridge consisting of a syringe and silica G powder to remove polar impurities, but it did not give the expected result. An improved extraction procedure was finally developed and described as Preparation of Standard Solutions in section 2.5 (Method II). Chromatographic conditions were optimized to save analytical cost, simplify operation and generally increase the practical applicability of the technique. These optimizations included a shorter run time, cheaper mobile phase and isocratic elution mode. With reference to

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the literature[26-28], we explored gradient programs such as methanol and 0.01% phosphoric acid at different ratios, isocratic elutions including 0.1% acetic acid-19% acetonitrile in methanol, methanol-0.1% formic acid and acetonitrile-0.01 % phosphoric acid. Finally, the chromatographic conditions described in section 2.3 were finalized. Table 4 shows that the concentrations of the five analytes obtained by method II were higher than those obtained by method I, and the RSD% values of method II were lower than those of method I. These findings showed that the improved LLE method is more effective and more repeatable. The improvement can be explained with the Fick’s diffusion equation with certain factors. The Fick’s equation is described below[29]:

6 Acknowledgements This work was supported by the Science and Technology Project of Chinese Traditional Medicine of Gansu, China (No. GZK-2011-72) and Agricultural Biotechnology Research and Development Project of Gansu Province, China (No. GNSW-2013-14). 7 Conflict of interests The authors declare that they have no competing interests. REFERENCES 1

Where D is diffusion coefficient, A is diffusion surface area, K is oil-water partition coefficient, h is the thickness of diffusion layer, and Cs and Ce are concentrations of analyte in the aqueous sample and extraction solvents. Obviously, the improved LLE method (method II) can increase A, while decrease h efficiently. Thus it increases the mass transfer rate between two incompatible phases, particularly for those hydrophobic substances with higher affinity to extraction solvents. Compared with conventional LLE operated by shaking a separatory funnel (method I), the improved LLE can increase the repeatability of extraction owing to the quantifiable extraction parameters including sample dripping rate, stirring rate and extraction time, etc. Compared to conventional LLE, the improved LLE (method II) was found to be more effective for simultaneous extraction of anthraquinones from an aqueous Chinese herbal preparation. This improved extraction method was successfully applied to determine the content of five target components in Fuyankang mixture by the means of RPHPLC. 5 Conclusion An improved liquid-liquid extraction method has been developed and successfully applied to simultaneously quantify concentrations of five anthraquinones in Fuyankang mixture. The results demonstrated that the improved method was more effective than conventional liquid-liquid extraction. An important aspect of the proposed method is its high efficiency and reproducibility and also its low costs in comparison with other advanced chromatographic techniques. Thus, it can be much more easily transferred to other laboratories for routine analysis and may be a good alternative to the conventional liquid-liquid extraction techniques for the quality control of herbal mixture or decoction at city-level hospitals in China. Journal of Integrative Medicine

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