Quantitative determination method for genotoxic impurities in calcium dobesilate

Abstract

The invention discloses a quantitative determination method of genotoxic impurities in calcium dobesilate, which comprises the following steps: firstly, preparing a derivatization reagent by adopting an acidic acetonitrile solution, so that stable existence of calcium dobesilate, 2-sulfonyl-1, 4-benzoquinone, hydroquinone and 1, 4-benzoquinone can be ensured, and meanwhile, complete reaction of benzoquinone impurities with the derivatization reagent can be ensured; and fully dissolving the sample and impurities by using an acetonitrile aqueous solution. Aiming at the derivatized sample solution, the separation and quantification of the benzoquinone impurities are realized under the high performance liquid chromatography conditions that a chromatographic column taking terminated octadecylsilane chemically bonded silica as a filling agent is adopted, and a mixed solution of a buffer solution and acetonitrile with the pH value of 4.5 + / -0.5 is adopted as a mobile phase. The method is convenient to operate, good in detection specificity of the benzoquinone impurities in the sample, high in sensitivity, good in precision and good in reproducibility, two benzoquinone impurities in calcium dobesilate can be accurately and quantitatively analyzed, and the safety and quality controllability of calcium dobesilate raw materials and preparations are guaranteed.

Description

technical field [0001] The invention belongs to the field of drug analysis, in particular to a method for separating and measuring impurities in calcium dobesilate by pre-column derivatization and high performance liquid chromatography. Background technique [0002] Calcium dobesilate is a capillary protective drug that has been used in clinics for a long time. Its structural formula contains a hydroxy structure of diphenol, which is easily oxidized. Once in contact with oxygen in water or air, it is easily oxidized to produce 2-sulfonic acid. Acid-1,4-benzoquinone impurity, 2-sulfo-1,4-benzoquinone in solution increased rapidly even if the sample was placed in a temperature-controlled injector at 4°C, resulting in sample results that were out of limits and could not be Determine whether the overrun is caused by the unqualified sample itself or the preparation process. This impurity has a genotoxic structure and needs to be controlled, and this impurity is a new compound, w...

AI Technical Summary

Problems solved by technology

However, two genotoxic impurities, 2-sulfonic acid-1,4-benzoquinone and 1,4-benzoquinone, belong to trace components and are not easy to be detected; in addition, calcium dobesilate and trace impurity hydroquinone, due to The nature of its own group is lively and easy to be oxidized, while 2-sulfonic acid-1,4-benzoquinone and 1,4-benzoquinone have strong oxidizing properties, so the four components cannot coexist in the solution

[0007] First of all, for the quantification of 1,4-benzoquinone, because 1,4-benzoquinone and calcium dobesilate cannot coexist due to the oxidation-reduction reaction in the solution state, after quantitatively adding 1,4-benzoquinone to the raw material test solution , 1,4-benzoquinone did not appear in the system suitability solution, while 2-sulfonic acid-1,4-benzoquinone and hydroquinone increased significantly (such as Figure 4 shown), it can be seen that 1,4-benzoquinone reacts rapidly and completely with calcium dobesilate in solution state to generate 2-sulfonic acid group-1,4-benzoquinone and hydroquinone, so not only cannot 1,4-benzoquinone Quantification of quinones, while also interfering with the quantification of 2-sulfo-1,4-benzoquinone

[0008] In addition, for the quantification of 2-sulfonic acid-1,4-benzoquinone, due to the existence of 1,4-benzoquinone, the 2-sulfonic acid-1,4-benzoquinone measured in calcium dobesilate solution cannot Determine whether the source of the component is calcium dobesilate itself or a reaction product during solution preparation

At the same time, after the calcium dobesilate solution is prepared into a solution, the 2-sulfonic acid-1,4-benzoquinone will continue to increase, and if it continues to be placed, the impurity will still increase, indicating that the main component calcium dobesilate is unstable in the solution state , will be continuously oxidized to produce 2-sulfonic acid-1,4-benzoquinone, which further increases the difficulty of quantifying this impurity

[0009] In addition, the four structural formulas of calcium dobesilate, 2-sulfonic acid-1,4-benzoquinone, hydroquinone and 1,4-benzoquinone are similar, and the polarity is relatively large, and the retention is relatively good in the reversed-phase liquid phase. Weak, the peak time is very short, and because the impurity limit is low, the sample solution needs to be prepared with a high concentration to meet the quantitative requirements, resulting in a large peak width of the calcium dobesilate peak in the liquid chromatogram. The measured impurities will be included in the main peak of calcium dobesilate, which is difficult to separate (such as Figure 5 shown)

Images