Objectives: Establishing an HPLC method that is suited with current conditions of the laboratory to quantify 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) in dry extract of Radix Fallopiae multiflorae.
Journal of military pharmaco-medicine No7-2016 QUANTIFICATION OF 2,3,5,4’TETRAHYDROXYSTILBENE-2-O-β-D-GLUCOSIDE IN DRY EXTRACT OF Radix Fallopiae multiflorae BY HPLC Hoang Viet Dung*; Nguyen Van Bach*; Pham Duc Thinh* SUMMARY Objectives: Establishing an HPLC method that is suited with current conditions of the laboratory to quantify 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) in dry extract of Radix Fallopiae multiflorae Material and methods: Parameters of the method are validated including: linear range, precision, accuracy Results: Chromatographic conditions of the method: using Sunfire RP-C18 column, acetonitrile and 0.1% aqueous formic acid as mobile phase following a multistep gradient program, monitored at 320 nm The established method has good precision (RSD = 2.37%) and accuracy (recovery ratio = 97.08 - 103.76%, average = 100.38%, RSD = 2.84%) Conclusion: HPLC method for quantifying THSG in dry extract of Radix Fallopiae multiflorae has been developed and validated * Key words: Fallopia multiflora; 2,3,5,4’-tetrahydroxystilben-2-O-β-D-glucosid; HPLC method INTRODUCTION Fallopia multiflora (Thunb.) Haralds was traditionally used to treat several diseases including: attenuated body, white-haired, good for blood [4] Actually, this medicinal plant is the major component in some modern products that are more easily used Following this trend, we are studying on preparing dry extract of Radix Fallopiae multiflorae as a raw material to prepare some products such as capsule and powder drug THSG is a compound of stilbenoid group that was seen in Radix Fallopiae multiflorae This compound possesses some important bioactivities as reducing blood lipid, anti-oxidant, antiinflammation… So that, THSG was selected as a bioactive marker for quality control of this dry extract as well as end-products afterwards This research is carried out to establish HPLC method for determining THSG in dry extract of Radix Fallopiae multiflorae that is suitable for our laboratory conditions and research target MATERIALS AND METHODS Materials and equipments - Materials: Dry extract of Radix Fallopiae multiflorae is prepared by Centre of Pharmaceutical Research-Training, Vietnam Military Medical University, in 12 - 2015 - Solvents: Acetonitrile (ACN) (Merck, HPLC grade), 0.1% aqueous formic acid (Merck, analytical grade) - Chemical: THSG (content 97.3%, provided by National Institute of Medicinal Materials) - Equipment: Waters HPLC system (Sunfire RP-C18 column; Autosample 2695; Detector PDA 2998; Empower sofware), Elmasonic S ultrasonic (Elma), Mettler Toledo ML204… * Military Medical University Corresponding author: Hoang Viet Dung (vietdungk85@yahoo.com) Journal of military pharmaco-medicine No7-2016 Dissolving and diluting an accurate amount of THSG standard with methanol by using volumetric flasks and Eppendorf pipets to obtain working standard solutions of about 10 µg/mL RESULTS AND DISCUSION System suitability For evaluating system suitability, a standard solution was injected times repeatedly into the HPLC system [3] Results are showed in table Table 1: Results of evaluating system suitability Content Peak area (µV*s) Retention time (minute) Result S = 233758 RSD = 1.32% tR = 6.89 RSD = 0.67% Tailing factor 1.14 Plate count 9700 9 A U 0.000 -0.001 -0.002 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 Minutes Figure 1: Chromatogram of the blank sample 0.10 T H S G - 0.08 0.06 0.04 0.02 0.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 Figure 2: Chromatogram of a standard solution of THSG 0.040 0.030 - Standard solution preparation: 0.001 A U - Sample solution preparation: 20 mg of dry extract of Radix Fallopiae multiflorae was ultrasonically shaked with about 15 - 20 mL of methanol in a volumetric flask of 25 mL for 15 minutes Methanol was added to volumetric line After that, this solution was filtered through a 0.45 µm filter membrane and injected into the HPLC system for analysis 0.002 0.020 A U Methods - Chromatographic conditions [1, 2, 5]: + Detector UV: 320 nm + Flow rate: 1.0 ml/minute + Injection volume: 10 µL + Column temperature: 280C + A multistep gradient program of mobile phase: - mins, ACN - 0.1% formic acid (23:77, v/v); - 10 mins from 23% to 100% ACN; 10 - 18 mins, 100% ACN; 18 20 from 100% to 23% ACN; 20 - 30 mins, ACN - 0.1% formic acid (23:77, v/v) 0.010 0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes Figure 3: Chromatogram of a sample solution of dry extract The results in table show the repeatibility of peak areas and retention times are good (both RSD < 2%) Peak of THSG is sharp and proportional (tailing factor = 1.14) Theoretic plate count is 9,700 Therefore, the chromatographic system is suitable for analyzing samples Journal of military pharmaco-medicine No7-2016 Specificity Retention times of the peak in the chromatogram of sample solution and in the chromatogram of standard solution are coincident At this retention time, there is no peak in the chromatogram of blank sample Match coefficients, determined at points of a sample peak by comparison between the UV-Vis geometries at these points and that of the THSG peak using PDA detector, are from 0.991 to 0.996 These results prove that the established method has good specificity Linear range For evaluating the linear range of the established method, concentrations of THSG were analyzed [3] Results are showed in table and figure Table 2: Correlation between peak areas and concentrations of THSG N0 Concentration (µg/mL) Peak area (µV*s) 2.46 52208 4.92 112374 9.83 231882 14.75 344807 19.66 451939 The results of figure show linear correlation between peak areas and concentrations of THSG following the regression equation y = 22,426x + 4,709.3 with correlation coefficient R2 = 0.9979 Precision Precision of the established method is evaluated based on the repeatibility of individual experiments quantifying THSG in the raw materials [3] Results are showed in table Table 3: Results of evaluating precision N0 Weight of Peak area Content of THSG materials (µV*s) in materials (mg/g) (mg) 20.6 239516 12.65 20.5 235716 12.51 20.7 247719 13.02 20.5 237035 12.58 19.8 220023 12.09 20.3 234162 12.55 Statistic Average content = 12.57 mg/g RSD = 2.37% The results in table show the established method has good repeatibility with RSD = 2.37% The content of THSG in Radix Fallopiae multiflorae is 12.57 mg/g Accuracy Accuracy of the established method is evaluated by spiking an exact amount of THSG into the dry extract sample of Radix Fallopiae multiflorae Then, determining recovery ratio by comparing and recovery amount to the spiked amount of THSG [3] Figure 4: Graph of linear correlation between peak areas and concentrations of THSG Samples were injected times repeatedly into the HPLC system Results are showed in table Journal of military pharmaco-medicine No7-2016 Table 4: Results of evaluating accuracy N0 Addition amount (µg) Recovery amount (µg) Recovery ratio (%) 98.27 95.40 97.08 98.27 97.05 98.76 98.27 101.09 102.87 98.27 96.10 97.79 98.27 101.97 103.76 98.27 100.25 102.01 Statistic Average recovery ratio = 100.38% RSD = 2.84% The results in table show the established method has good accuracy with recovery ratio = 97.08 - 103.76%, RSD = 2.84% LOD and LOQ By diluting standard solutions of THSG and analyzing with the HPLC system, LOD and LOQ of the established method were determined based on signal to noise [3] LOD is about 0.1 µg/mL and LOQ is about 0.4 µg/mL ACKNOWLEDGEMENT This research was supported by “National Program for Tay Bac, VNU Hanoi” under grant number KHCN-TB.04C/13-18 REFERENCES Bharathi Avula, Vaishali C Joshi et al Simultaneous identification and quantification of anthraquinones, polydatin and resveratrol in polygonum multiflorum, various Polygonum species and dietary supplements by liquid chromatography and microscopic study of Polygonum species Journal of AOAC International 2007, Vol 90, No DQ Hana, J Zhao, J Xu Quality evaluation of Polygonum multiflorum in China based on HPLC analysis of hydrophilic bioactive compounds and chemometrics Journal of Pharmaceutical and Biomedical Analysis 2013, 72, pp.223-230 ICH guidelines Q2 (R1) validation of analytical procedures: text and methodology 1996 National Institute of Medicinal Materials CONCLUSION A suitable method had been developed and validated for quantifying THSG in dry extract of Radix Fallopiae multiflorae The established method has good precision and accuracy This study also provided valuable information for the content of THSG in dry extract of Radix Fallopiae multiflorae being about 12.57 mg/g Medicinal plants and animals in Vietnam Publishing house of Science and Technology Hanoi 2004, pp.529-531 Yan-Li Xu, Qi Dong, Feng-Zu Hu Simultaneous quantitative determination of eight active components in Polygonum multiflorum Thunb by RP-HPLC Journal of Chinese Pharmaceutical Sciences 2009, 18, pp.358-361 ... evaluated by spiking an exact amount of THSG into the dry extract sample of Radix Fallopiae multiflorae Then, determining recovery ratio by comparing and recovery amount to the spiked amount of THSG... quantifying THSG in dry extract of Radix Fallopiae multiflorae The established method has good precision and accuracy This study also provided valuable information for the content of THSG in dry extract. .. repeatibility of individual experiments quantifying THSG in the raw materials [3] Results are showed in table Table 3: Results of evaluating precision N0 Weight of Peak area Content of THSG materials