Tan Shengjian, Peng Qingtao, Hu Wenxiang,^* Chen Peirang
(Institute of Military Medicine, Headquarters of General Equipment, Beijing 100101)

Received July.1, 2001

Abstract A RP-HPLC method for the determination of imperatorin in Yuanhu analgesic capsule was established. The sample was dissolved in ethanol and extracted by refluxing in water bath , then chromatographed on LUNA-C₁₈ column with mobile phase of methanol-0.5% phosphoric acid (69:31) . The flow rate was set at 0.8mL/min and the detection wavelength was of 300nm. The resolution between imperatorin and its neighboring peak was 2.0, the number of theoretical plates was 15367 according to the peak of imperatorin. The linear range was 19.50-58.50mg/mL, r was 0.9999, and the average recovery was 99.8%. This HPLC method is rapid and accurate, it can be used to determine imperatorin quantitatively in Yuanhu analgesic capsule.
Keywords RP-HPLC, Yuanhu analgesic capsule, Imperatorin

Yuanhu analgesic capsule is made of Yuanhu and Radix Angelicae Dahuricae which is recorded in the eighth volume (1993) of Medicine Standards published by the Ministry of Health of the People^'s Republic of China. Its function is to regulate the flow of vital energy and remove obstruction to it, to invigorate the circulation of blood and to relieve pain. It can be used to treat stomach pain caused by stagnation of the circulation of vital energy and blood stasis, to treat rib pain, headache and menses pain and so on. Imperatorin is the main active component of Radix Angelicae Dahuricae ^[1]. Quality of Yuanhu analgesic capsule is identified by the characteristics and general rule of capsule examination in Medicine Standards. It was reported^[2,3] that the concentration of imperatorin in Radix Angelicae Dahuricae of some medicines was determined by HPLC, but the determination method of imperatorin in Yuanhu analgesic capsule has not been reported. In this study, a RP-HPLC method for the determination of imperatorin in Yuanhu analgesic capsule was established.

1  INSTRUMENT AND REAGENTS
The HPLC system consists of LC-6A liquid pump of Shimadzu Corp. of Japan, SIL-6B automatic sample injector, SPD-6AV ultraviolet-visible spectrophotometer detector and Chromatopac C-R 6A data processor. Medicine for experiment was purchased from Medicine Corp. of Beijing and was identified by us. The standard sample of imperatorin was purchased from the Medicine and Biological Products Identification Institute of China. Yuanhu analgesic capsules were made by the Medicine Identification Institute of the Second Artillery of PLA . Methanol was of HPLC grade and ethanol was analytical reagent. The stock solution of imperatorin standard sample was ethanol solution of 97.50m g/mL.

2  METHOD AND RESULTS
2.1 Chromatographic conditions         
LUNA C₁₈ column (4.6mm ID×250mm, particle diameter 5m m), ODS protective column(5mm ID×50mm, particle diameter 5mm) of Analytical Apparatus Factory of Beijing were used . The column temperature was at 35°C. The mobile phase was the mixture of methanol-0.5% phosphoric acid (69:31) and the flow rate was set at 0.8mL/min. The detection wavelength was of 300nm, the injected volume was 10mL and the recorder paper rate was 1mm/min.
2.2 Experiment operations           
The standard sample of imperatorin was diluted to suitable concentration by ethanol, then injected. The negative sample without imperatorin was prepared according to Yuanhu analgesic capsule prescription. The sample of Yuanhu analgesic capsules and the negative sample were taken and extracted according to the sample determination method, injected and the chromatograms were given in Figure 1. From the chromatograms, it was known that the other components of Yuanhu analgesic capsule did not affected the determination of imperatorin. The retention time of imperatorin was about 16 min, the resolution between imperatorin and its neighboring peak was 2.0, the number of theoretical plates was 15367 according to the peak of imperatorin. Under the chromatographic conditions to be examined, imperatorin in Yuanhu analgesic capsule can be determined quantitatively.

Fig.1 The chromatogram of (a) imperatorin (b) negative sample of Yuanhu analgesic capsule (c) Yuanhu  analgesic capsule, the peak of 1 in chromatogram (c) is imperatorin

2.3 Linear relation and the minimum detect limit 
The standard sample was taken precisely and diluted to different concentration(19.50～58.50mg/mL) with ethanol, then injected 10mL respectively and the peak area was recorded. The linear regression was made between the mean peak area of imperatorin X and the injected concentration Y(mg/mL). The regression equation:
Y=0.000070964X+3.1591; r=0.9999.
The minimum detect limit was 0.1mg/mL according to the three times noise of baseline.
2.4 Extraction method 
The extraction rates of imperatorin by ethanol were examined by using different ultrasound time and different reflux time in water bath. Our results shown that the extraction rate was 96.3% by ultrasounding for 60min and that of by refluxing in water bath for 20min was 100%. Therefore, the extraction method of refluxing in water bath was selected. If take the extraction rate as 100% by refluxing for 40min, then the extraction rate was 99.9%, 100.5% and 98.3% respectively by refluxing for 20, 60, 90min.
2.5 Apparatus precision and method repeatability 
A sample solution was taken into the automatic sample injector, and injections were made in 5h for n=5, interday RSD% was 0.3170. The same sample was determined for 3 consecutive days and five times per day (n=5), intraday RSD% was 3.864.
2.6 Adding standard recovery 
Six parts of the same batch Yuanhu analgesic capsules were weighed precisely, among them, imperatorin standard sample was added precisely into five parts, the other part was the blank. Every part was operated according to the sample determination method for determining the imperatorin concentration. The average recovery was 99.8%, RSD% was 0.63 (n=5).
2.7 Sample determination
Fifteen Yuanhu analgesic capsules were taken and ground into powder. A certain amount of the powder was precisely weighed (about equivalent to Radix Angelicae Dahuricae 0.9g) into a 100mL Erlenmeyer flask with ground cap and 50mL ethanol was added. The mixture was extracted by refluxing for 40min in water bath, then the mixture was cooled down to room temperature and was filtered. 25mL continuous filtrate was precisely measured into a 50mL volumetric bottle, ethanol was added to the scale and the bottle was shaked evenly. A little was taken from the volumetric bottle and filtered through a 0.45mm membrane. An aliquot of 10mL was injected into the HPLC, the peak of imperatorin was recorded and its amount was calculated by the external standard method. Three batches samples were determined, the results of imperatorin was 0.66, 0.71 and 0.72mg/capsule respectively.

3  DISCUSSION       
By the stability experiment, it can be concluded that the concentration of imperatorin in the sample solution was stable at least for 48h, which was placed in brown volumetric bottle at 18°C.
    Different proportions of mobile phase of methanol-water and absorption wavelength were experimented repeatedly, the result showed that it could not obtain baseline separation for imperatorin with other components in 25 min until methanol-0.5% phosphoric acid (69:31) was selected as mobile phase.

REFERENCES         
[1] Yin Jian, Guo Ligong. Modern Research Medica and Clinical Application of Chinese Materia, Volume 1, Beijing: Xue Yuan Publisher, 1995: 259.
[2] Chen Yianxiang, Shen Anhua. Research of Chinese Traditional Patent Medicine, 1991, 11 (6): 26.
[3] Huang Xinsheng. Chinese Traditional and Herbal Drugs, 1988, 6: 19.

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