Compound high-performance liquid chromatography injection Ginkgo Ginkgo flavonol glycoside content of
Authors: Yu Lin, Zhong Wei Fang, Liu Juan, HU Sheng-wide
【Abstract】 Objective To establish a compound injection of
Ginkgo flavonol glycosides in Ginkgo method for the determination. Methods HPLC, Zorbax-Extend-C18 column (5 μm,
4.6 mm × 150 mm); methanol 0.4% phosphoric acid (45:55) as mobile phase, flow rate of 1.0 ml * min-1, column temperature
30 ?, detection wavelength 360 nm. Results quercetin concentration 2.07 ~ 69.00 μg * ml-1, kaempferol
concentration of 1.91 ~ 63.60 μg * ml-1, isorhamnetin
concentration of 0.73 ~ 24.20 μg * ml-1 showed good linear
relationship within the framework of . Conclusion This method
is a short analysis time, specific and a good, stable, and can be used as quality control of compound ginkgo injection method.
Key words compound Ginkgo injection; flavonoids; high-
performance liquid chromatography
Abstract: ObjectiveTo establish a method for determining the flavonoids in Compound Ginkgo biloba Injection by HPLC. MethodsHPLC, Zorbax-Extend-C18 column (5 μm, 4. 6 mm × 150
mm) was used. The mobile phase consisted of methanol 0.4%
phosphoric acid (45:55) with flow rate of 1.0 ml * min-1.The
detection wavelength was set at 360 nm. The column temperature was set at 30 ?. ResultsThe linear range of quercetin, kaempferol and isorhamnetin were at the range of 2.07 ~ 69.00 μg * ml-1, 1.91 ~ 63.60 μg * ml-1 and 0.73 ~ 24.20 μg * ml-
1. ConclusionThe method is rapid, exclusive and steady, and can be used for the quality control for the injection.
Key words: Compound Ginkgo biloba Injection; Flavonoids; HPLC
Ginkgo injection compound is based on clinical experience and the development of traditional Chinese medicine compound injection from the Ginkgo biloba extract, Danshen and medicinal composition, with increases vascular flow, improve blood circulation, inhibit platelet aggregation, prevent thrombosis and so on. Ginkgo flavonoids as the main component
of which, at present, at home and abroad Ginkgo flavonoids quantitative methods are: complexation spectrophotometry, coulometric titration, TLC, high performance liquid chromatography, gas chromatography, capillary electrophoresis,
etc. [1], refer to the literature [2,3], comparing the advantages and disadvantages of various methods, this study opted for a RP-HPLC Determination of Compound Ginkgo
flavonoids injection method, injection of the compound ginkgo fluid provide the basis for the establishment of quality standards.
An apparatus and materials
1.1 Instrument
Agilent 1100 Series: quaternary pump, vacuum-line
Degasser, autosampler, column temperature box, a variable wavelength scanning UV detector, Agilent ChemStation.
1.2 Drugs and reagents
Reference substance quercetin, kaempferol reference substance, isorhamnetin reference substances were purchased in China pharmaceutical and biological products (Lot No. :10081-
9905 ,110861-200405 ,110860-200406), methanol (HPLC pure,
Tianjin City Kermel Chemical Reagents Development Center), phosphoric acid (AR, Tianjin Chemical Reagent Plant), water for drinking Wahaha purified water (Hangzhou Wahaha Group), compound ginkgo injection (Jiamusi University, College of
Chemistry and Pharmaceutical provided batch
060414,060415,060416).
2 methods and results
2.1 Chromatographic conditions and system suitability column Zorbax SB-C18 column (5 μm, 4. 6 mm × 150 mm), mobile
phase was methanol 0.4% phosphoric acid (45:55), detection wavelength of 360 nm a flow rate of 1.0 ml * min-1, column
temperature 30 ?, injection volume 10 μl. It was confirmed
that a peak in the order: quercetin, kaempferol and isorhamnetin. Theoretical plate number calculated by quercetin
peak of not less than 2500, isorhamnetin separation is greater than 1.5.
2.2 The reference substance solution and preparation of standard curve
Precision Weigh dry phosphorus pentoxide quercetin, kaempferol, isorhamnetin, methanol dissolves and is scheduled
as a reserve capacity of liquid, the concentration of 0.345,0.318,0.121 mg * ml-1, respectively, draw on three kinds of sophisticated control reserve liquid products of all 0.06,0.1,0.3,0.7,1.0,2.0 ml, in 10 ml Liang Ping, the constant
volume of methanol, 0.45 μm microporous membrane filter, as a reference substance mixture, in the above chromatographic conditions were determined to record peak area values, respectively, the concentration of reference substance for the horizontal axis, peak area value of the vertical axis, the regression calculation results show that: Quercetin concentration of 2.07 ~ 69.00 μg * ml-1 showed good linear
relationship within the framework of the regression equation: A = 57.375C-14.757, the correlation coefficient r = 0.999 7;
kaempferol concentration of 1.91 ~ 63.60 μg * ml-1 showed
good linear relationship within the regression equation: A = 50.194C 15.311, the correlation coefficient r = 0.999 6; isorhamnetin concentration of 0.73 ~ 24.20 μg * ml-1 showed
good linear relationship within the regression equation: A = 54.857 0C 1.230 9, the correlation coefficient r = 0.999 4.
2.3 Preparation of solution for the test items
Precision drawing compound Ginkgo Injection 5 ml in the Round-bottom flask, add methanol 10 mol / L hydrochloric
acid solution (4:1) mixture of 25 ml, constant reflux 1.5 h, cooled, transferred to 50 ml Liangping in and add methanol to the scale, shake, 0.45 μm microporous membrane filter, which was tested product solution.
2.4 Specificity test
Taking Ginkgo biloba extract does not contain the negative control injection, according to test products for the solution preparation method, preparation of negative control solution, were taken for the test product solution, reference
substance solution and the negative control solution, the same chromatographic conditions, sample measured determination results shown in Figure 1. Test results confirmed: samples of quercetin, kaempferol, isorhamnetin well separated from other
components, and the negative control in the corresponding retention time of no interference. Reposted elsewhere in the paper for free download http://
2.5 Precision Experiment
Take quercetin, kaempferol and isorhamnetin
concentrations were 10.35,9.54,3.63 μg * ml-1 of the
reference substance mixture in the above chromatographic conditions, a continuous sample six times. Quercetin, kaempferol and isorhamnetin peak area RSD were 0.98%, 0.90%, 1.00%.
2.6 Repeatability test
According to test products for the preparation methods, take samples of the same batch injection 6 Preparation of 6 copies of compound ginkgo products tested injection solution, measured under the above chromatographic conditions, quercetin, kaempferol, isorhamnetin Peak area RSD were 2.24%, 2.99%, 2.87%.
2.7 Stability for new injection preparation of compound ginkgo products tested solution, according to the above chromatographic conditions were measured, respectively, 0,1.5,4,6,24 h, quercetin, kaempferol and isorhamnetin peak
area RSD were 1.67% and 1.18%, 6.08%, concluded that the tested products of quercetin and kaempferol in 24 h within the stability of the well, and isorhamnetin in the 24 h has been unstable, the proposed test Samples should be hydrolyzed 6 h
after treatment completed within.
2.8 The average recovery
Experimental Determination of content of the compound have been taking ginkgo injection and the concentration of hydrolyzed samples of 6 were equivalent reference substance
solution (quercetin concentrations of 12.72 μg * ml-1,
kaempferol concentration of 9.54 μg * ml-1, isorhamnetin
concentration of 1.21 μg * ml-1), set up procedures for
injection, 10 μl sample plus 10 μl reference substance,
twice the volume of mixing into the sample, the same
chromatographic conditions were measured to calculate the total ginkgo flavonol glycosides average yield of 101.23%, RSD for the 1.43%.
2.9 Determination of Sample
According to test products for the preparation method,
three batches of samples taken in the same chromatographic conditions were determined and calculated into the sample. The results in Table 1. Table 1 compound injection of Ginkgo flavonol glycosides in Ginkgo assay results (omitted)
3 Discussion
"China Pharmacopoeia" chromatographic conditions [4] as a reference for the test materials and reference standards for high-performance liquid chromatographic conditions for screening experiments confirmed that, "Chinese Pharmacopoeia" basic conditions apply to this compound preparation, compound in the other components In the chromatographic conditions, no interference, and appropriate to increase the column temperature is conducive to shorten the analysis time and improve kaempferol and isorhamnetin separation effect.
Experiments also test products for the hydrolysis conditions were detailed exploration to finalize hydrolysis conditions and the chromatographic conditions can be used as injection of the compound of Ginkgo flavonol glycosides in Ginkgo method
for the determination.
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[2] Shu-Jun Wang, Hou Wei, Zhang Tian G, et al. HPLC
Determination of sustained-release capsules of Ginkgo biloba flavonoids in [J]. Chinese herbal medicine, 2004,35 (2): 163.
[3] Du safety, Wang Xianrong, Zheng-Hua Zhou, et al.
Ginkgo biloba flavonoid glycosides in HPLC Analysis of hydrolysis conditions [J]. Anhui Medicine, 2001,5 (3): 164.
[4] National Pharmacopoeia Committee. Chinese Pharmacopoeia [S]. Beijing: Chemical Industry Press, 2005:220. Reposted elsewhere in the paper for free download http://