Determination of Tanshinone IIA in two kinds of Chinese traditional patent medicines by HPLC
Tan Shengjian, Peng Qingtao, Hu Wenxiang,
Wang Wenming
(Institute of Military Medicine, Headquarters of General Equipment, Beijing 100101)
Received Aug.8, 2001.
Abstract A HPLC method for the
determination of Tanshinone IIA in the Dan Qi Pian and Tong Mai
Chongji was established. The sample was dissolved in methanol and extracted by ultrasound,
then chromatographed on LUNA-C18 column with mobile phase of methanol-water
(84:16). The flow rate was set at 0.8mL/min and the detection wavelenghth was of 270nm.
This HPLC method is simple, rapid and accurate, it can be used to determine Tanshinone IIA in the Dan Qi Pian and Tong Mai Chongji quantitatively .
Keywords Tanshinone IIA, Dan Qi Pian, Tong Mai Chongji,
HPLC
Dan Qi Pian and Tong Mai Chongji are general
Chinese traditional patent medicines. Although they are edited in the eighth volume(1993)
of Medicine Standards published by the Ministry of Health of the People's
Republic of China, there was not any determination method for their effective components.
Hence it was difficult to examine the quality of the drugs. It is showed that Tanshinone IIA is a kind of important effective component in Dan Qi Pian and Tong
Mai Chongji[1]. The determination of Tanshinone IIA
have already been reported [2,3]. As complicated components existing,
determination method for Tanshinone IIA in Dan Qi Pian and Tong
Mai Chongji has not been reported. In this paper, a HPLC method for the determination of
Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji was
established.
1 INSTRUMENT AND REAGENTS
LC-6A liquid pump of Shimadzu Corp. of Japan, SIL-6B automatic sample injector, SPD-6AV
ultravoilet-visible spectrophotometric detector and Chromatopac C-R 6A data processor.
Standard sample of Tanshinone IIA was purchased from the
Medicine and Biological Products Identification Institute of China. Dan Qi Pian and Tong
Mai Chongji were self-made.(according to the eighth volume(1993) of Medicine Standards
published by the Ministry of Health of the People's
Republic of China). Methanol was HPLC grade.
2 METHOD AND RESULTS
2.1 Chromatographic conditions
LUNA C18 column (4.6mm ID×250mm, particle diameter 5mm) was used. The column temperature was
at 35°C. The mobile phase was methanol-water (84:16) and the flow rate was set at
0.8mL/min. The detection wavelength was of 270nm, the recorder paper rate was at 1mm/min.
2.2 Experiment operation
The standard sample was diluted to suitable concentration by methanol, then injected. The
negative samples without Tanshinone IIA were prepared
respectively according to Dan Qi Pian and Tong Mai Chongji prescription. The samples of
Dan Qi Pian and Tong Mai Chongji and their negative samples were taken and extracted
according to the sample determination method, injected and the chromatograms were given in
Figure 1 and 2. From the chromatograms, it was known that the other components of Dan Qi
Pian and Tong Mai Chongji had not affected the determination of Tanshinone IIA. The number of
theoretical plates was 22339 with regard to the peak of Tanshinone IIA in Dan Qi
Pian, and the resolution between Tanshinone IIA and its neighbouring peak was 3.3.
The number of theoretical plates was 22000 with regard to the peak of Tanshinone IIA in Tong Mai
Chongji, and the resolution was 3.0. Under the chromatographic conditions we selected,
Tanshinone IIA in Dan Qi Pian and Tong Mai Chongji can be determined
quantitatively.
Fig.1 The chromatogram of (A) Tanshinone IIA (B) negative sample of Dan
Qipian (C) Dan Qipian the peak of 1 in chromatogram (C) is Tanshinone IIA
Fig.2 The chromatogram of (A) Tanshinone IIA (B) negative sample of
Tong Mai Chongji (C) Tong Mai Chongji the peak of 1 in chromatogram (C) is
Tanshinone IIA
2.3 Linear range
The standard sample was taken precisely and diluted to five grade concentration with
methanol, then injected 10 mL
respectively and the peak area was recorded. The linear regression was made between the
mean peak area of Tanshinone IIA X and the injected
concentration Y(mg/mL), and
r was 0.9999. The linear range was 4.14~62.10 mg/mL.
2.4 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% of Dan Qi Pian and Tong Mai Chongji was both 0.5. The
same sample was determined for three consecutive days and five times per day (n=5),
intraday RSD% of Dan Qi Pian and Tong Mai Chongji was 4.2 and 3.9 respectively.
2.5 Adding standard recovery
Six parts of the same batch Dan Qi Pian and Tong Mai Chongji were weighed precisely
respectively, Tanshinone IIA 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 the determination of the Tanshinone IIA
concentration. The average recovery of Dan Qi Pian and Tong Mai Chongji was 100.7% and
100.4% respectively, and RSD% was 1.8 and 2.2 respectively.
2.6 Sample determination
2.6.1 Dan Qi Pian
Twenty Dan Qi Pians were taken and weighed precisely, the average weight of the
tabelet was calculated, then ground into powder. A certain amount of the powder (about
weight of ten tabelets Dan Qi Pian ) was taken and weighed precisely to a 25mL volumetric
flask, then a certain amount of methanol was added. Extracted by ultrasound for 5 minutes,
and supplemented the lost methanol to the calibration tails, then filtered. An aliquot of
10mL was injected , the peaks of Tanshinone IIA was recorded and
its amount was calculated by the external standard method. The average concentration of
Tanshinone IIA in Dan Qi Pian was 0.018mg/g, RSD was 0.7%(n=5).
2.6.2 Tong Mai Chongji
A certain amount(about 5 g) of Tong Mai Chongji was precisely weighed and taken into a
25mL volumetric flask. A certain amount of methanol was added. Extracted by ultrasound for
5 minutes, and supplemented the lost mathanol to the calibration tails, then filtered. An
aliquot of 10mL was injected , the peaks of Tanshinone IIA was
recorded and its amount was calculated by the external standard method. The average
concentration of Tanshinone IIA in Tong Mai Chongji was
0.075mg/g, RSD was 0.7%(n=5).
3 CONCLUSION
Under the chromatographic conditions selected by us, the HPLC method we established
can be used to determine Tanshinone IIA in Dan Qi Pian and Tong
Mai Chongji quantitatively. The method was sensitive, rapid and accurate.
REFERENCES
[1] Li Chunzhe, Yang Shaochun, Zhao Fudong. Acta Pharmacologica Sinica, 1984, 5 (1): 39.
[2] Okamura Nobuyuki, Kobayashi Keiao, Yagi Akira et al. J Chromatogr, 1991, 542 (2): 317.
[3] Li Xiangyun, Tu Wenqing. Zhong Cao Yao, 1995, 26 (10): 520.
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