Determination of paeoniflorin from radix paeoniae rubrae by high - Performance liquid chromatography

Southeast-Asian J. of Sciences, Vol. 6, No. 1 (2018) pp. 87-95 DETERMINATION OF PAEONIFLORIN FROM RADIX PAEONIAE RUBRAE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Tran Khanh Duy∗, Trinh Duy Tai†, Nguyen Thi Minh Thuan‡, Vo Thi Bach Hue‡ ∗,†Faculty of Pharmacy, Lac Hong University, Dong Nai, Vietnam e-mail: duytk018@gmail.com ‡Faculty of Pharmacy Ho Chi Minh University of Medicine and Pharmacy Ho Chi Minh city , Vietnam e-mail: vothibachhue@gmail.com Abstract Radix Paeoniae rubrae is a medicinal herb used in traditional medicine as well as in the manufacture of medicinal products for the treatment of chest pain, anti-inflammatory, anti-coagulant, vasodilatation However, the quality of Radix Paeoniae rubrae is suspected because of not tight controlled. The main chemical component of this medicinal herb is paeoniflorin, a monoterpen glycoside. We conducted this study with the aim of developing the quantitative procedures of paeoniflorin in Radix Paeoniae rubrae by HPLC, which can detect cases of counterfeit or ex- hausted pharmaceuticals, this will help improve the quality control of this medicinal plant. The optimized conditions included isocratic mobile phase of 18% of acetonitrile and 82% of phosphate buffer 0.05M pH 4.5. Chromatographic system used Phenomenex Lunar C18 column (250 x 4.6 mm; 5 µm) at 25 0C, injection volume was 20 µL, flow rate at 1.0 mL/minute, as detector a spectrophotometer set at 230 nm. The procedures enables determina- tion of paeoniflorin in Radix Paeoniae rubrae within 22 minutes. The Key words: Paeoniflorin, Radix Paeoniae rubrae, determination, high-performance liquid chromatography. ∗ Corresponding author. 87 88 Determination of paeoniflorin from... analytical procedures was validated the suitability of the system, speci- ficity, linearity (yˆ = 26.5702x, R = 0.9999), repeatability with RSD = 0.8%, intermediate precision, accuracy with recovery rate 96.6 - 103.7% and range 6.4 - 25.6 µg/mL. 1. Introduction Radix Paeoniae rubrae is a medicinal herb used in traditional medicine as well as in the manufacture of medicinal products for the treatment of chest pain, anti-inflammatory, anti-coagulant, vasodilatation However, the quality of Radix Paeoniae rubrae is suspected because of not tight controlled. The main chemical component of this medicinal herb is paeoniflorin, a monoterpen glycoside. The Chinese Pharmacopoeia 2005 evaluated the quantitative criteria of Radix Paeoniae rubrae based on the content of paeoniflorin, but using the test method of Chinese Pharmacopoeia to quantify, we found that paeoniflorin peak was not separated from other peaks. Vietnamese Pharmacopoeia IV also has Radix Paeoniae rubrae monographs but not included the assay, so it is impossible to acurately assess the quality when this medicinal herb has been extracted active ingredients. From that fact, we conducted this study with the aim of developing the quantitative procedures of paeoniflorin in Radix Paeoniae rubrae by HPLC method, which can detect cases of counterfeit or exhausted pharmaceuticals, help improve the quality control of this medicinal plant. 2. Materials and Methods 2.1. Instrumentation All experiments were performed on a HPLC system of Agilent 1260 (USA) equipped with an auto sampler and Phenomenex Lunar C18 column (250 x 4.6 mm, 5μm) coupled with temperature control system for column (25 0C) in the Laboratory of analytical chemistry and drug quality control, pharmacy faculty, Lac Hong University. 2.2. Chemical and materials Acetonitrile (Merck), methanol (Merck), KH2PO4 (Merck). Paeoniflorin (Sigma Aldrich), purity of 99,0% was used as chemical reference substance (CRS). Radix Paeoniae rubrae was purchased in Ho Chi Minh city, August 2017. 2.3. Solution preparation Phosphate buffer 0.05M pH 4.5: Dissolve 6.8 g of KH2PO4 into 1000 mL of distiled water. Reference solution: Dissolve a quantity of paeoniflorin CRS, T. K. Duy, T. D. Tai N. T. M. Thuan and V. T. Bach Hue 89 accurately weighed, in mobile phase to produce a solution containing 16 μg per mL. Test solution: Accurately weigh 80 mg of the Radix Paeoniae rubrae powder to a 50 mL volumetric flask, add 35 mL methanol and ultrasonicate for 30 minutes. Allow to cool, add methanol to 50 mL, filter, 10 mL of filter fluid is diluted to 25 mL with the mobile phase in a volumetric flask. This solution was then filtered through filter paper with 0.45 m before injected. Solution used for validation: Accurately weigh 8.0 mg paeoniflorin CRS, dissolve in mobile phase to produce a solution containing 80 μg per mL (solution A). Test solution add paeoniflorin: Accurately weigh 80 mg of the Radix Paeo- niae rubrae powder to a 50 mL volumetric flask, add 5 ml solution A, produce in the same manner as the test solution. 2.4 Optimization of chromatographic conditions Different compositions of mobile phases were investigated: Methanol - phosphate buffer 0.05M pH 4.5 (40:65) (mobile phases 1) Acetonitrile - phosphate buffer 0.05M pH 4.5 (18:82) (mobile phases 2) Phenomenex Lunar C18 column (250 x 4.6 mm, 5 m) at 25 0C. Injection volume: 20 μL. Wavelengh of detector: 230 nm. Flow rate: 1.0 mL/min. The optimization was obtained when chromatographic parameters meet re- quirement such as theoretical plate number (N ≥ 3000), resolution (Rs ≥ 1.5), symmetrical factor (0.8≤ As ≤ 1.5) and peak purity. 2.5. Validation of analytical procedures This analytical procedures was subsequently validated according to the ICH guideline Q2 (R1) (ICH 2005) with respect to the system suitability, specificity, linearity, repeatability, intermediate precision, accuracy and range. Results and Discussions 3.1 Optimization of the mobile phase conditions With the mobile phase 1 condition, which was written in the Chinese Phar- macopoeia 2005, paeoniflorin peak was not separated from other peaks. The second mobile phase by isocratic of Acetonitrile - phosphate buffer 0.05M pH 4.5 (18:82, v/v) gave good parameters for paeoniflorin peak in test solution, theoretical plate number N = 14333, Rs = 4.41, As = 0.88 and paeoniflorin peak reached purity. Thus, it was chosen as optimum chromatography condition. 90 Determination of paeoniflorin from... Figure 1. Chromatograms of the test solution when analyzed by mobile phase 1 (a) and mobile phase 2 (b). 3.2. Validation of analytical procedures 3.2.1. System suitability System suitability was tested by performing six replicate injections refer- ence solution and test solution, determining capacity factor (k’), symmetrical factor (As), theoretical plate number (N), resolution (Rs), selectivity factor (α) and repeatability (RSD of retention times and peak areas) for the analytes of interest. The %RSD values of peak area and retention time for all peaks were less than 2% indicating the precise analysis of paeoniflorin by this sys- tem. All the results showed that the proposed analytical procedures met the requirements. 3.2.2. Specificity The chromatogram of the test solution obtained the peak has same reten- tion time as the peak of paeoniflorin in chromatogram of reference solution. The chromatogram of the test solution add paeoniflorin obtained the peak of paeoniflorin has an increase in peak area compared to before the addition. The chromatogram of dissolving solvent wasn’t obtained the peak has same reten- tion time with peak of paeoniflorin. The UV-Vis spectra of the active substance in the chromatogram obtained with reference solution and test solution were similar. Peak of the paeoniflorin in the chromatogram obtained with test solu- tion reached peak purity. Thus, the analytical procedures met the specificity T. K. Duy, T. D. Tai N. T. M. Thuan and V. T. Bach Hue 91 requirements. Figure 2. Test results for the purity of Paeoniflorin peak in the chromatogram of the test solution. Figure 3. Chromatogram obtained with the reference solution. 3.2.3. Linearity From solution A, dilute to 8 solutions at a concentration of 1.6 - 51.2 μg/mL (equivalent to 10 - 320% of the quantitative concentration) as described in Table 2. Carry out analyzes according to chromatography conditions described, 92 Determination of paeoniflorin from... Figure 4. Chromatogram obtained with the test solution. record retention time, peak area of paeoniflorin. Establish a linear regression equation for the peak area according to the paeoniflorin concentration. Using the regression function ”Regression” in MS - Excel 2013 with the F-test to test the suitability of the regression equation yˆ = ax + b and the t-test to test the significance of the coefficients a, b. 3.2.4. Repeatability Prepare six different test solution, analyze according to chromatography T. K. Duy, T. D. Tai N. T. M. Thuan and V. T. Bach Hue 93 conditions described. Record retention time, peak area, calculate content (%) of paeoniflorin according to the following formula: C(%) = ST ×mc × Cc ×DT × 100 Sc ×DC ×mT With ST , SC is the peak area of paeoniflorin in chromatogram obtained with test solution and reference solution; mC : weight of paeoniflorin to make reference solution; CC : purity of paeoniflorin CRS; DC , DT : the dilution of the reference solution and test solution, mT : weight of the Radix Paeoniae rubrae powder to make test solution. According to the results in Table 3, the content (%) of paeoniflorin in Radix Paeoniae rubrae is 2.67, RSD of the content over the six tests is 0.8% not to exceed 2.0%. The procedures met repeatability requirements. 3.2.5. Intermediate precision Two testers, independently analyzed on two different days, each tested 6 samples. Carry out the analysis according to the described chromatography conditions. The quantitative RSD value of each tester and of both testers was less than 2.0%. Anova analysis showed that the quantitative results between the two testers were not statistically significant difference (P-value = 0.095 > α = 0.05). The analytical procedures met intermediate precision requirements. 3.2.6. Accuracy Applying the method of adding paeoniflorin to the test solution, the amount of added paeoniflorin corresponding to 5 levels of 40%, 70%, 100%, 130%, 160% of the quantitative concentration. Each level contains 3 samples. Carry 94 Determination of paeoniflorin from... out analyzes according to the described chromatography conditions, record retention time, peak area of paeoniflorin, calculate concentration of paeoniflorin was found, recovery rate at each concentration level. T. K. Duy, T. D. Tai N. T. M. Thuan and V. T. Bach Hue 95 Recovery rates at all concentration levels ranged 96.6 - 103.7%. The analytical procedures met the requirements of correctness 3.2.7. Range Based on the results of linearity, precision and accuracy, figure out the analytical procedures has a range 6.4 - 25.6 mug/mL (corresponding to 40 - 160% of the quantitative concentration). Conclusions By using high-performance liquid chromatography with diode array detector, we have developed the quantitative procedures of paeoniflorin in Radix Paeo- niae rubrae as follows: isocratic mobile phase acetonitrile - phosphate buffer 0.05M pH 4.5 (18:82), as detector a spectrophotometer set at 230 nm, Phenomenex Lunar C18 column (250 x 4.6 mm; 5 m) at 25 0C, injection volume was 20 μL, flow rate at 1.0 mL/minute. Procedures validated system suitability, specificity, linearity, repeatability, intermediate precision, accuracy and range. This is the basis for providing test methods for the assay of Radix Paeoniae rubrae monographs, which improve the quality control of this medic- inal plant. 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