Date:
At present, more and more attention has been paid to the determination of multiple dissolution curves of solid preparations, which are gradually playing a pivotal role in preparation process development, prescription screening, pre-evaluation of bioequivalence tests of generic drugs, and evaluation of internal quality differences between products of the same variety and different sources. Therefore, in the research stage and prescription screening stage, how to determine the mass dissolution samples efficiently, accurately and quickly began to confuse the analysts; Based on years of work experience, after sorting and summarizing, I put forward the following ideas and views for the reference of the experimentered.
Dissolution test
For the tablet/paddle method, in order to avoid manual sampling "confusion", a fixed interval of time (e.g. 30 s) can be used to sample, so that parallel operation can be achieved at ease. Filter membrane adsorption Z is troubled. It is recommended to extract 15-20 ml of leachate in advance 1 min before the first sampling time point, filter to saturate the filter membrane, and slowly inject the filtrate back along the wall of the leachate cup. After the sampling time point, sample according to law, and take further filtrate for determination.
Determination of dissolution sample
Due to the uneven quality of the excipients, especially the film coat, the enteric coat, the sugar coat and the capsule shell, the UV method may lead to high UV absorption, resulting in a positive deviation of the mean dissolution degree (even 10%~30%). At the same time, because the excipients of the original reference preparation are generally unavailable, the interference of excipients on the measurement results cannot be evaluated 6.
Therefore, HPLC method is recommended, because most excipients are inert, do not produce peaks on the reverse phase chromatography column, and even if the peak retention time is short, they will not interfere with the principal component determination. In addition, due to the wide linear range of HPLC method, samples can be directly injected without dilution, and the tedious steps of sample dilution can be omitted because the absorbance of UV method is 0.20 ~ 0.80, so as to improve work efficiency.
If UV method is used for measurement, in order to eliminate interference, it is recommended to use dual wavelength subtraction (Z large absorption wavelength and no absorption wavelength at the far end). There are commercially available optical fiber stripping instrument, using the "corrected ultraviolet method". If the interference of excipients can be ensured, the instrument is still recommended. After all, a complete curve can be measured quickly and conveniently, which has practical value for the study of the intrinsic quality of drugs.
How to improve the determination efficiency of HPLC method
3.1 Sample Treatment
Due to the small amount of samples required by HPLC method, the sampling amount at each time point is 1-2 ml. Therefore, for small-size quick-release preparations (no more than 10 mg), the experimenters can use sample solution without filtration according to the actual situation (such as the solubility of excipients) and by observing the clarification of the dissolution solution. A method in which a liquid vial is placed directly in the vial for a period of time and then the sample is taken for determination.
This method is especially suitable for the automatic sampling device, because the main component of the small gauge or insoluble preparation is micro-pulverized, the surface energy is larger, so it is easy to have adsorption samples in the pipeline and filter membrane.
3.2 Chromatographic conditions
Chromatographic columns: Commercially available short analytical columns with 2-5 cm length and 5-10 µm particle size can shorten the analysis time, save the amount of mobile phase and reduce the detection cost.
Mobile phase: In order to speed up the measurement, it is entirely possible to increase the proportion of organic phase in the verified and determined mobile phase. The retention time was shortened under the condition that each chromatographic peak was completely separated.
Column temperature: After investigating the stability of the tested sample, the column temperature can be appropriately increased to 40 ~ 50 ℃. Normal reversed-phase column Z has a high tolerance temperature of 80 ℃, so testing below 60 ℃ is no problem.
Flow rate: The flow rate can be increased to 1.5 ~ 2.0 ml/min according to the column pressure.
Injection volume: For small-size preparations, the injection volume can be flexibly adjusted according to the precision of the reference solution. As long as the function of the instrument permits, 100-500µl is completely acceptable. It should be emphasized that it is recommended to use a reference solution with 10% of the labeled amount for precision verification, so as to ensure the accuracy of the determination of low concentration samples. For large-size preparations, when nearly all the dissolution, due to excessive concentration, there will be column overload or detector overload, resulting in poor peak shape
At this time, the injection volume can be reduced to 5 µl, making the peak area smaller and meeting the requirements of chromatographic peak precision.
Due to the small peak area, the chromatographic peak can be narrowed and sharpened by shortening the retention time, or the detection precision can be improved by increasing the sample size and other appropriate changes, so as to adapt to the test solution and meet the requirements of the system adaptability test. The test methods should also be verified.
Others
4.1 Use ultra-high velocity liquid phase
If the ultra-high speed liquid phase is used, the particle size of the ultra-high speed liquid chromatography column is small, if the sample liquid is not filtered, it may block the column. Therefore, it is suggested that the test should be verified first.
4.2 Liquid phase software programming
For data processing of a large number of samples, we must make full use of the software function of the instrument. It is not recommended to input each peak area into Excel software for calculation. Now the mainstream brand of the market chromatograph, all have the data batch processing and printing function, if you can master these functions, can greatly improve the work efficiency.
