Determination of the Content of Gastrodin in Jingtianshenma Tablets by High Performance Liquid Chromatography

Kun TANG, Hongzhen GUO, Jie QIAO, Bing LI

1. College of Life Science, Langfang Normal University, Langfang 065000, China; 2. Technology Innovation Center for Utilization of Edible and Medicinal Fungi in Hebei Province, Langfang 065000, China; 3. Edible and Medicinal Fungi Research and Development Center of Hebei Universities, Langfang 065000, China; 4. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China

Abstract [Objectives] The research aimed to establish a high performance liquid chromatography method for the content determination of gastrodin in Jingtianshenma Tablets. [Methods] A phenomenex Luna C18 (250 mm×4.6 mm, 5 μm) column was used; the mobile phase was acetonitrile-0.05% phosphoric acid solution (3∶97); the detection wavelength was 220 nm, and the column temperature was set at 25 ℃. [Results] Gastrodin showed a good linear relationship in the range of 0.098 4-0.590 4 μg with the peak area, and regression equation was Y=2 000 000X-51 999 (r=0.999 9). The limits of detection and quantification for gastrodin were 2.50 and 4.20 ng respectively, and the average recovery rate was 95.95%. [Conclusions] This method is sensitive, accurate and reproducible, with good linearity, and it is suitable for the content determination of gastrodin in health food Jingtianshenma Tablets.

Key words Jingtianshenma Tablets, Gastrodin, HPLC, Content determination

1 Introduction

Jingtianshenma Tablet is a health food made of Gastrodiae Rhizoma, Ginseng Radix Et Rhizoma and other valuable medicinal materials. It has the function of alleviating fatigue and is mainly suitable for people who are prone to fatigue. Gastrodiae Rhizoma is main raw material of Jingtianshenma Tablets, and the research shows that gastrodin contained by Gastrodiae Rhizoma is an active ingredient for relieving fatigue[1-2]. In the experiment, the method of high performance liquid chromatography determining gastrodin content in Jingtianshenma Tablets was established, and methodological research was conducted, which could provide reference for content determination of gastrodin in similar products.

2 Instruments and reagents

Waters HPLC (2707 Autosampler; 600 controller; Delta600; 2420ELS Detector; 2996 Photodiode Array Detector), electronic balance (1/100 000，Mettler Toledo, Switzerland), ELGA/water purification system (PURELAB Classic United States), KQ-700DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). Gastrodin control (National Institutes for Food and Drug Control, number of lot: 110807-201507, content: 95.4%); acetonitrile (chromatographic grade); phosphoric acid (GR); Jingtianshenma Tablets (samples 1, 2, 3, Beijing Changwei Technology Co., Ltd.).

3 Methods and results

3.1 Chromatographic conditions and system suitability test

Chromatographic column: phenomenex Luna C18(250 mm×4.6 mm, 5 μm); mobile phase: acetonitrile-0.05% phosphoric acid solution (3∶97), and the mobile phase was filtered by microporous membrane and degassed by ultrasonic before use; flowing speed: 1.0 mL/min; detection wavelength: 220 nm; column temperature: 25 ℃; injection volume: 5 μL.

System suitability test: number of theoretical plates (calculated by peak of gastrodin control solution) should not be lower than 5 000.

3.2 Preparation of solution

3.2.1Preparation of control solution. 10 mg of gastrodin control was weighed precisely, and mobile phase was added to dissolve and dilute to 20 mL. After shaken evenly, a stock solution of control sample containing 0.5 mg of gastrodin per 1 mL was prepared. 1 mL of stock solution was taken in 10 mL of graduated flask, and mobile phase was added to dilute to the graduate, preparing control solution containing 50 μg of gastrodin per 1 mL.

3.2.2Preparation of test solution. Sample was ground into fine powder and sieved by No.3 screen. 2.00 g of sifted fine powder was weighed precisely and set in conical bottle with stopper. 50 mL of 50% diluted ethanol was added precisely, and its weight was weighed. After ultrasonic treatment (120 W of power and 40 kHz of frequency) for 30 min, it was set to room temperature. Then, the weight was weighed, and the lost weight was made up with dilute ethanol. After filtration, 10 mL of continuous filtrate was weighed precisely and concentrated to near dry. Acetonitrile-0.05% phosphoric acid mixed solution (3∶97) was added for solution, and it was transferred to a volumetric flask (25 mL). Acetonitrile-0.05% phosphoric acid mixed solution (3∶97)was used to dilute to the graduate. After shaken evenly and filtration, subsequent filtrate was obtained, namely test solution.

3.3 Methodological investigation

3.3.1Specificity. Referring to the formulation and process of Jingtianshenma Tablets, negative sample not containing Gastrodiae Rhizoma was prepared, and negative sample solution was prepared according to the method of Section3.2.2. Control solution, sample solution and negative sample solution were determined according to chromatographic conditions of Section3.1. The results showed that other components in the sample did not interfere with the determination, and the method had strong specificity.

3.3.2Stability. The same test solution was taken, and 5 μL of sample was injected according to chromatographic conditions of Section3.1at 0, 2, 4, 6, 8, 10 and 12 h, andRSDof peak area of gastrodin was 1.34% (n=6). The results showed that the sample solution was stable for at least 12 h.

3.3.3Linearity. 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0 and 12.0 μL of gastrodin control solution under Section3.2.1was measured precisely for injection, and peak area was determined according to chromatographic conditions of Section3.1. Injection volume of gastrodin (X, μg) was taken as horizontal ordinate, while peak area (Y) was taken as longitudinal coordinate, obtaining linear regression equationY=2 000 000X-51 999 (r=0.999 9). It showed that injection volume of gastrodin showed good linear relationship with peak area in the range of 0.098 4-0.590 4 μg.

3.3.4Precision. 5.0 μL of gastrodin control solution was taken, and continuous injection for 6 times was conducted under chromatographic conditions of Section3.1, and peak area was determined.RSDof peak area of 6 consecutive injections was 0.33%. The results showed that the precision of the instrument was good.

3.3.5Repeatability. Test sample was taken, and 6 test solutions were prepared in parallel by the method of Section3.2.2, and it was determined according to chromatographic conditions of Section3.1. The results showed that average content of gastrodin in the sample was 7.50 mg/g, andRSDwas 2.48% (n=6). It showed that the method had good repeatability.

3.3.6Recovery rate. 6 samples with known gastrodin content (7.60 mg/g) were taken, and each copy was 0.100 0 g. It was weighed precisely and set in conical bottle, and an appropriate amount of control sample was added respectively. It was processed according to preparation method of test solution under Section3.2.2, and injection volume was 10 μL, and recovery rate of gastrodin was determined. The results were determined via sample adding recovery test, and recovery rate of gastrodin in sample was between 93.32% and 98.37%. Mean was 95.95%, andRSDwas 1.78% (Table 1), with good recovery rate.

Table 1 Result of sample adding recovery experiment of gastrodin (n=9)

3.4 Sample determinationAccording to above chromatographic conditions, content of gastrodin in three batches of Jingtianshenma Tablets samples was detected. The results showed that mean gastrodin content in three batches of samples was 7.569 mg/g, andRSDwas 0.568% (Table 2). It illustrated that the determination method of gastrodin in this product was applicable, controllable and stable.

Table 2 Determination results of gastrodin content in three batches of samples

4 Discussion

Gastrodiae Rhizoma is main raw material in the formula of Jingtianshenma Tablets, and gastrodin is main chemical composition of Gastrodiae Rhizoma and monomer composition with the highest content. Modern pharmacological research shows that gastrodin could enhance anti fatigue ability of mice[3-4]. In theChinesePharmacopoeiaof 2015, gastrodin is taken as main efficacy component index of quality control of Gastrodiae Rhizoma[5]. Therefore, gastrodin is taken as main iconic component of Jingtianshenma Tablets.

Mobile phase of chromatographic conditions referred to that used by content determination of gastrodin in theChinesePharmacopoeiaof 2015, but the mobile phase used in theChinesePharmacopoeiaof 2015 was acetonitrile-0.05% phosphoric acid solution (3∶97), and there was ideal separation effect between gastrodin peak in the product and adjacent chromatographic peak. Methodological findings showed that HPLC method established in the test could accurately measure gastrodin content in Jingtianshenma Tablets. The method has simple operation, good reproducibility and strong specificity, so it could provide reference for methodological research of determining gastrodin content in Jingtianshenma Tablets and its similar products.