An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

In vitro measure is a suitable measure for inspecting the dissolution of solutions of solid Chinese medicine preparation which take into account the effects of drug disintegration, and the dissolution behavior, and the content of active ingredient. The detection of multi-index component can better reflect the neutral influence, and the dissolution difference of different components. Demonstrating the procedure will be Quinyun Du a extraordinary student from my laboratory.

To begin, prepare the reference substance stock solution by weighing 10.6 milligrams of salidrocide, 5.24 milligrams of gallic acid, and 5.21 milligrams of ethyl gallic acid separately on an electronic analytical balance. Then add them individually into a five milliliter volumetric flask. Then add HPLC grade methanol to dissolve, and dilute to five milliliters.

And finally, shake well to obtain the reference substance stock solution with mass concentrations of 2.120, 1.048, and 1.042 milligrams per milliliter, respectively. To prepare the test sample solution, extract 2.8 grams of rhodiola granules with 10 milliliters of HPLC grade methanol, using an ultrasonic cleansing machine for 30 minutes. Then filter it with a 0.22 micrometer filter for the system adaptability test.

Then prepare a mixed reference solution that contains 0.590 milligrams per milliliter salidrocide, 2.030 milligrams per milliliter gallic acid, and 1.930 milligrams per milliliter ethyl gallate. To obtain the theoretical content of each characteristic component of rhodiola granules, place 2.8 grams of rhodiola granules in 500 milliliter conical flask. Add 200 milliliters of ultrapure water, and ultrasonically extract for 60 minutes.

Then filter it with a 0.22 micrometer filter. After determining the content of the test solution, set the chromatographic conditions for high performance liquid chromatography as described in the manuscript. To investigate the linear relationship, dilute the reference stock solutions of gallic acid and ethyl gallate, and the reference stock solutions of salidrocide to obtain the gradient concentration solution for drawing a standard curve.

Then inject 10 microliters of the mixed reference solution into the HPLC system six times daily, and run the samples with the same HPLC conditions, while recording the peak area of each feature component. Next inject 10 microliters of the prepared sample solution, and determine the peak areas of the HPLC according to the chromatographic conditions at different time intervals. Take six samples of the same batch of rhodiola granules to prepare the test sample solution as demonstrated previously, and inject 10 microliters of each sample into the HPLC system.

Run the samples as demonstrated previously to determine reproducibility. After preparing six portions of the same batch of rhodiola granules for the test solution, add about 50%of the reference substrate of each index component. Run these samples in the HPLC system, as demonstrated previously, to calculate the recovery rate as described in the manuscript.

To carry out the dissolution test, add 100 milliliters of ultrapure water into the dissolution cup of the drug dissolution apparatus, and maintain the temperature at 37 degrees Celsius with a rotation speed of 100 rpm. Next add 2.8 grams of rhodiola granules into a dissolution cup, and start recording the duration of dissolution immediately. Collect a total of one milliliter of the sample with an injector at different time intervals, and make up the volume in the dissolution cup with a dissolution medium at the same temperature immediately.

Immediately fill the collected samples through a 0.22 micrometer microporous membrane, and take the subsequent filtrate. After determining the content of each component, at each time point by HPLC, calculate the dissolution of each time point, and cumulative dissolution as described in the manuscript. The chromatogram of the sample, and the reference solution were obtained.

Three different peaks corresponding to gallic acid, salidrocide, and ethyl gallate shows that the elution gradient had a good resolution for the three index components in rhodiola granules. The dissolution curves of each component show that the cumulative dissolution of gallic acid was over 80%after one minute. Whereas for salidrocide and ethyl gallic acid, it was over 65%after five minutes.

However, the cumulative dissolution of each index component decreased after 30 minutes. Adjusting the dilution issue of the standard curve, according to the preliminary experiment of the sample treatment, samples must be collected quickly to avoid them missing special collection time point.