High Performance Liquid Chromatography of Ganoderma lucidum spore powder capsule polysaccharide content of
【Abstract】 Objective To establish the Ganoderma spore powder capsule polysaccharide content in HPLC determination. Methods ShodexSH1011 (styrene-divinylbenzene copolymer
bonded sulfonic) sugar column, mobile phase of 0.6% sulfuric acid solution, detection wavelength 200 nm. The results of Ganoderma lucidum polysaccharides in the 0.266 ~ 8.51 mg / ml range a good linear relationship, r = 0.999 8, the average recovery was 100.4% (n = 9), RSD = 1.57%. Conclusion The method is simple and quick, non-interference, good
reproducibility and can be used for Ganoderma spore powder capsule quality control.
Key words Ganoderma lucidum spore powder of Ganoderma lucidum polysaccharides by high performance liquid chromatography capsule
Content Determination of Polysaccharides of Ganderma lucidum in Spore Powder Capsule by HPLC
Abstract: ObjectiveTo establish an HPLC method for determination of Ganoderma lucidum
Polysaccharide in spore powder capsule. MethodsA Shodex SH1011 column was used with 0.6% sulphuric acid solution. The detection wavelength was set at 200nm.ResultsThe linear relationship of the concentrations and peak areas was good in range of 0.266 ~ 8.51 mg /
ml (r = 0.999 8). The average recovery was 100.4% (n = 9), RSD = 1.57%. ConclusionThe method is simple, rapid and accurate and can be used for quality control of the capsule.
Key words: Spore powder of Ganderma lucidum ganderma capsule; Polysaccharides; HPLC
Ganoderma lucidum spore powder capsule from the Ganoderma spore powder capsule [WS
5026 (B 0026) 2004] to change formulations made with spleen Qi, Yang Anshen effect. For the Heart and deficiency, sickness and infirmity, as well as cancer patients after adjuvant treatment. The effective ingredients of Ganoderma lucidum polysaccharides, triterpenoids, with its polysaccharide content of about 0.75% [1]. Determination of polysaccharide used sulfuric acid - anthrone [2] and sulfuric acid phenol
spectrophotometric method [3,4]. The method error larger, more complicated operations. Determination of polysaccharide content using HPLC [5,6] have reported that Ganoderma
lucidum spore powder formulations using high performance liquid chromatography
polysaccharide not been reported. This selection of high-performance liquid chromatography
method for the fungus Ganoderma lucidum spore powder soft capsules in the Determination of polysaccharide in the establishment of a high separation efficiency, good stability, rapid analysis method for controlling quality of preparation provided a reliable method. The study methodology, in line with requirements determination.
An instrument and reagent
Shimadzu LC 10A high-performance liquid chromatograph, SPD 10Avp UV - visible
detector, N2000 chromatography data workstations, KQ 500DB CNC ultrasonic cleaner,
7725i six-way injection valve.
D anhydrous glucose reference substance (China Pharmaceutical and Biological
Products Institution, batch number 110833 200302, for the determination of use);
Ganoderma lucidum spore powder soft capsule (Nanjing Pharmaceutical Co., Ltd., batch number 20050301,20050302,20050303) sulfuric acid AR, water re-distilled water, other
reagents were analytical pure.
2 Methods and Results
2.1 Chromatographic conditions
Column: ShodexSH1011 (styrene-divinylbenzene copolymer bonded sulfonic) (8.0 mm ×
300 mm, 6 μm), mobile phase: sulfuric acid water (6:1 000), detection wavelength 200
nm, flow rate 0.6 ml / min, injection volume of 20 μl, column temperature 30 ?. In the
above-mentioned chromatographic conditions, D anhydrous glucose retention time of 4.987
min.
2.2 The product solution and reference substance for the test preparation solution
This product is 10 to take the contents mixed evenly, taking about 3.0 g, precision that will placed in 10 ml glass centrifuge tube, add n-hexane 8 ml, shock 2 min after
centrifugation (3000 r / min) 10 min, discard to n-hexane layer, and then repeat the above
steps once, water bath to shake off residual n-hexane, it will transfer them to the 10 ml
flask, add the appropriate amount of water, 100 ? water bath 1 h, lack of water, mixing,
filtration, take filtrate 2.0 ml by adding ethanol 8.0 ml, shake and place 2 min after centrifugation (3 000 r / min) 10 min, supernatant and sediment dissolved with 2.0 ml mobile phase, 1 h after 100 ? water bath that is too . Separate examination of the hydrolysis time. The results in Table 1. Examination of hydrolysis time in Table 1 (omitted)
The results can be completely hydrolyzed more than 1h, so choose 1h as the hydrolysis point in time.
Precision said the scheduled 48 h vacuum drying of the D anhydrous glucose
reference substance 21.28 mg, buy 10 ml flask, add the mobile phase was dissolved and diluted to the scale, shake, made with D per ml anhydrous glucose 2.128 mg of the
solution, filtration, that is, too.
Adaptability of the system, an experimental study. The results in Table 2. Table 2 Experimental results of system suitability study (omitted)
2.3 The linear relationship of the study
Precision drawing reference substance solution of 5 ml in 10 ml volumetric flask in
the dilution constant volume, followed by two-fold dilution prepared with the reference
substance solution, according to the above-mentioned chromatographic conditions, precision drawing 20 μl injection of liquid chromatography, continuous sample 2 times measured. To
peak area of the vertical axis (Y), in order to sample the concentration of X-coordinate
(X), draw the standard curve. The regression equation was Y = 321 56X 4 999.2, the correlation coefficient r = 0.999 8, the linear range of 0.25 ~ 8.00 mg / ml. The results
in Table 3. Table 3 Standard curve drawing results (omitted) reposted elsewhere in the paper for free download http://
2.4 Precision Experiment
Precision drawing reference substance solution obtained, continuous sampling six
times, the measured RSD value of 0.6% (n = 6), consistent with the requirement of experiment. The results in table 5.
2.5 Stability test
Take the same sample (batch number 20,050,401), prepared according to 2.2, according to the above-mentioned chromatographic conditions, precision drawn into the liquid chromatograph 20 μl every 2.5 h sample 1 measured 12.5 h for the stability of the test solution . The RSD value of 0.2%, in line with the requirement of experiment. The results in Table 4. Table 4 Stability test results (a little) Table 5 accuracy test results (a little)
2.6 Repeatability test
Take the same sample (batch 20,050,401) 6 copies, prepared according to 2.2, respectively, precise drawing 20 μl, continuous nebulization two times, the measured RSD
value of 1.5%, in line with the requirement of experiment. The results in table 6. Table 6 Repeatability results (omitted)
2.7 The average recovery experiment
Take the same sample (batch 20,050,401) 9 copies, adding a certain amount of
reference substance, were prepared according to 2.2, that too. Precision drawing 20 μl
were injected into high-performance liquid chromatography, continuous sample injection three times measured. Calculation of the average recovery was 100.4%, the RSD value of
1.57%, in line with the requirement of experiment. The results in table 7. Table 7 The average recovery results (omitted)
2.8 Determination of medicinal herbs
Take different batch ingredients of Ganoderma lucidum spore powder is about 1.0 g,
precision that will, according to 2.2 method according to the preparation, 20 μl for each
sample measured. The results in Table 8. Table 8 Determination of medicines results (omitted)
4 different batches of Ganoderma lucidum spore powder medicine because of its content
from different sources and have some differences, because of Ganoderma lucidum spore powder polysaccharide content of medicinal herbs is not the existing statutory standards, the production of internal control standards should be established to carry out strict control, the average content of to 114.40 mg / g, rate to fall 20% as the internal control standards for 91.52 mg / g.
2.9 Determination of Sample
Were collected from 10 batches (batch
20050401,20050402,20050403,20050501,20050502,20050503,20050504,20060301,20060302,20060303) of 10 samples of the contents, taking about 3.0 g, precision that will, according to 2.2 were prepared, namely, too. Precision drawing for the test product solution 20 μl
injection, continuous injection two times, measured, that is too. The results in table 9. Table 910 batches of samples assay results (omitted)
From the above results can be seen that high degree of accuracy of the method can be used to assay samples. 10 batches of samples the average level of 40.18 mg / g, and the transfer rate of 74.72%. Based on the average content of 20.54 mg / tablets, medicine and technology may lead to consider the contents of the floating, the average content of the sample rate to fall 20%, as each and content limits. Finished content limits defined as follows: This product contains Ganoderma lucidum polysaccharides in each and anhydrous D-
glucose (C6H12O6) of dollars, not less than 16 mg.
3 Discussion
3.1 The choice of the mobile phase was considered the use of phosphoric acid, but it will be difficult when the hydrolysis of polysaccharides, so use sulfuric acid as mobile phase.
3.2 Preparation of test materials for the solution found in the sample by adding the
mobile phase 1h and 1.5h absorption peak area close to, consider saving time, so choose the optimum hydrolysis time 1h.
3.3 The preparation of the sulfuric acid used in accessories - anthrone and sulfuric
acid - phenol spectrophotometric determination of Ganoderma lucidum polysaccharide in
there is a certain interference results show that this method had no significant effect on the determination of accessories.
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