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Objectives: By using high performance liquid chromatography (HPLC), develop and validate an analytical method for astilbin. Methods: Development of chromatographic conditions for astilbin quantification in Rhizoma Smilacis glabra. The method was validated according to ICH guidelines.
JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 QUANTITATIVE STUDY FOR DETERMINATION OF ASTILBIN IN SMILAX GLABRA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Le Viet Duc*; Vu Binh Duong**; Trinh Nam Trung**; Pham Thi Thanh Huong** SUMMARY Objectives: By using high performance liquid chromatography (HPLC), we develop and validate an analytical method for astilbin Methods: Development of chromatographic conditions for astilbin quantification in Rhizoma Smilacis glabra The method was validated according to ICH guidelines Results: We investigated HPLC chromatographic conditions, including Phenomenex Gemini RP C18 column (4.6 mm x 250 mm, µm) at a flow rate of 1.0 mL/min, detection wavelength of 291 nm, and an isocratic elution of 0.1% aqueous acetic acid - acetonitrile (70:30, v/v) The method displayed an acceptable precision (RSD ≤ 1.37%) and accuracy The detection and quantification limits were 0.5 μg/ml and 1.5 μg/ml respectively Conclusion: The HPLC analytical method of astilbin was validated to meet requirements of ICH guidelines * Key words: Smilax glabra; High performance liquid chromatography; Astilbin INTRODUCTION Smilax glabra is a wild herb grown in the northern mountainous provinces of Vietnam According to Vietnamese traditional medicine, Smilax glabra is often used in treatment of tendon diseases, worms, ulcers, diabetes, and detoxification [1] Concerning to compositions of Smilax glabra, astilbin, which is the main active constituent (figure 1) with high content, has been revealed to have hypouricemic, anti-inflammatory, analgesic anti-oxidation effects [2, 3] Thus, quantitation for studies on extraction as well as quality control is very important Figure 1: The chemical structure of astilbin Vietnam Military Medical University has formulated several pharmaceutical products that contains Smilax glabra, such as: “Thong Phong Khang”, “Kien khop tieu thong” * Minishy of Defense ** Vietnam Military Medical University Corresponding author: Vu Binh Duong (vbduong2978@gmail.com) JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 to serve public health care In order to standardize Smilax glabra as well as its products, development and validation of astilbin quantitative method is required In this study, we developed and validated a quantitative analysis of astilbin in Smilax glabra using high-performance liquid chromatography (HPLC) 10 mL volumetric flask, filtered through 0.22 µm membrane before HPLC analysis * Chromatographic conditions: There were several reports on HPLC analysis of astilbin [5, 6] In this study, we selected chromatographic conditions to develop an appropriate analytical method * Validation of analysis method: MATERIALS AND METHODS Materials and equipment Smilax glabra was obtained from Son Lam Pharmaceutical Company complying the forth Vietnamese Pharmacopoeia Astilbin standard was purchased from Sigma Aldrich, 99.9% HPLC grade methanol (MeOH), acetonitrile (ACN), distilled water were purchased from Merck All reagents used were of analytical grade (PA) High performance liquid chromatography system (Waters 1525 Binary HPLC pump; USA) was applied in this study Methods * Sample preparation: - Standard preparation: Powder of standard astilbin is dissolved in MeOH to make the stock solution of 1.000 µg/mL Dilute the stock solution to get working standard solutions - Sample preparation: Accurately weighed about 1.0 g of powdered Smilax glabra, performed ultrasonic extraction using MeOH solvent (8 ml x times) The extracts were centrifuged and poured into 200 mL volumetric flask and diluted up to the mark with methanol Then diluted ten-folded using Validation of analytical method was carried out in accordance with the guideline of ICH (International Conference on Harmonisation) [4] for raw materials and finished products with the following criteria: system suitability, selectivity specificity; linearity; accuracy; precision; limit of detection (LOD) and limit of quantification (LOQ) RESULTS Chromatographic conditions Based on published researches [5, 6], we developed chromatographic conditions for astilbin analysis which consisted of C18 column (4.6 x 250 mm, μm) using a mobile phase of 0.1 aqueous acetic acid ACN (70:30, v/v) at a flow rate of mL/min UV - VIS detector was employed at 291 nm Injection volume of 10 µL was subjected to HPLC system For the proposed conditions, the chromatograms on the figure showed well-resolved and symmetrical peaks of astilbin with retention time of approximately 19.7 minutes, which is suitable for analyzing astilbin in Smilax glabra So, we choose these conditions of chromatographic method for validation JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 Method validation * System suitability, specificity: a time (19.43 ± 1.77, RSD = 1.77) and peak area (11,0192 ± 797, RSD = 1.77) were not more than 2% Therefore, the method is comply to system suitability criteria Analyze the standard, blank and test solutions of astilbin simultaneously The spectrum of the peak at retention time of 19.7 of standard and test solutions proved that it was surely of astilbin (figure 3) a b b c Figure 3: UV-Vis spectrum of peaks at 19.7 minute of standard solution (a) and test solution (b) Figure 2: Chromatogram of blank solution (a), standard solution (b), test solution (c) Six replicate injections of a working standard solution of µg/mL were injected Retention time and peak area were evaluated The results indicated that relative standard derivations of retention 10 In figure 2, peak of astilbin was completely separated from peaks of impurities, symmetric and narrow width The retention time of astilbin in the chromatogram of the test solution and standard solution were the same There weren’t any corresponding peak in the chromatogram of blank solution Therefore, the method showed high selectivity and specificity JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 * Linearity: Peaks areas (µ AU.s) The working standard solutions from to 100 µg/mL was injected to HPLC system Results showed that in the test range, there was a linear regression between peak area and the concentration of astilbin The linear equation was y = 22172 - 15877, with correlation coefficient R2 of 0.999 (figure 4) Concentration g/ml Concentration (µg/mL) Figure 4: Behavior of peak area vs.concentration of astilbin * LOD and LOQ: LOD was determined to be 0.5 µg/mL and LOQ was 1.5 mg/mL * Precision: The precision of the method was carried out at concentrations: LQC, MQC and HQC The results showed that the relative standard deviations at the concentrations during intra and interday were less than 2% (table 1) Thus, the accuracy of the method was satisfactory to analyze astilbin Table 1: Intraday and interday precision of standard solutions PRECISION Repeatability Intermediate precision LQC (10 µg/ml) MQC (50 µg/ml) HQC (100 µg/ml) X ± SD (µAU.s) 212607 ± 2183 1051348 ± 6891 2218232 ± 4823 RSD (%) 1.03 0.66 0.22 X ± SD (µAU.s) 211941 ± 3877 1051937 ± 12870 2231226 ± 15550 RSD (%) 1.37 0.88 0.92 11 JOURNAL OF MILITARY PHARMACO-MEDICINE No7-2015 * Accuracy: The accuracy of the method is performed by spiking of 16 mg, 20 mg and 24 mg to 1.0 g of powdered Smilax glabra Extraction and chromatography analysis are conducted using the proposed procedure The recovered percentages of astilbin were compared with the original and determined as accuracy The results indicated that the percentages of recovery were found in the acceptance criteria of 80 to 120% (table 2) Therefore, the quantitative methods completely meet the requirements of ICH guidelines Table 2: Accuracy of astilbin in herbal samples SPIKED AMOUNT Recovery ( X ± SD, %) 16 mg 20 mg 24 mg 98.3 ± 4.6 100.0 ± 2.30 102.5 ± 1.00 CONCLUSION The HPLC method has been developed to determine astilbin in Smilax glabra using Phenomenex Genimi RP - C18 column, mobile phase containing 0.1% acetic acid ACN (70:30, v/v), flow rate of mL/min, UV - VIS detector at a wavelength of 291 nm, and the injection volume of 10 µl The validated quantitative method has high precision (98.3 ± 4.6% to 102.5 ± 1.00%) and accuracy (RSD ≤ 1.37%), the correlation coefficient (R2) of 0,999 Thus, the proposed 12 method is suitable to quantify astilbin in Smilax glabra as well as pharmaceutical products containing this compound REFERENCES Loi Do Tat Vietnamese Medicinal Plants and Herbs Medical Publishing House 2004, pp.498-499 Qing-Feng Zhang, Zhong-Rong Zhang, Hon-Yeung Cheung Antioxidant activity of Rhizoma Smilacis Glabrae extracts and its key constituent-astilbin Food Chemistry st 2009, July, , Vol 115, Issue 1, pp.297-303 Wen-Ai Xu et al Study on the correlation between constituents detected in serum from Rhizoma Smilacis Glabrae and the reduction of uric acid levels in hyperuricemia Journal of Ethnopharmacology 2013, Vol 150, Issue 2, pp.747-754 ICH Validation of analytical procedures, Text and Methodology Q2R1 2005 Liang Chen, Ye Yin, Hongwei Yi, Qiang Xu, Ting chen Simultaneous quantification of five major bioactive flavonoids in Rhizoma Smilacis Glabrae by high-performance liquid chromatography Journal of Pharmaceutical and Biomedical Analysis 2007, 43, pp.1715-1720 Qing-Feng Zhang, Yu-Xian Guo, Xinchen Shangguan, Guodong Zheng and Wen-Jun Wang Identification and quantification of polyphenols in Rhizoma Smilacis chinae by HPLC/DAD/ESI-MS/MS Journal of Liquid Chromatogrhaphy & Related Technologies 2013, 36, pp.2251-2260 ... astilbin quantitative method is required In this study, we developed and validated a quantitative analysis of astilbin in Smilax glabra using high- performance liquid chromatography (HPLC) 10 mL volumetric... retention time of approximately 19.7 minutes, which is suitable for analyzing astilbin in Smilax glabra So, we choose these conditions of chromatographic method for validation JOURNAL OF MILITARY... determine astilbin in Smilax glabra using Phenomenex Genimi RP - C18 column, mobile phase containing 0.1% acetic acid ACN (70:30, v/v), flow rate of mL/min, UV - VIS detector at a wavelength of