| Genotoxic impurities have the characteristics of low concentration and high risk,which brings about the strong genotoxic effect on human body and affect the drug quality seriously.With the stringent regulatory requirements,genotoxic impurities have become a dominating negative factor that affecting the cost of drug research and development,as well as the time to market.Therefore,it is imperative to study the basic materials used in API and their possible side effects.Detecting in advance to determine possible or potential genotoxic impurities in API is also very important.This research originates from the sub-project of the API project of a pharmaceutical company in Shanghai,the title is "Development and validation of API analysis methods".Based on the requirements and practical factors of company’s production,it is necessary to develop a series of cost-effective methods for analyzing genetic toxicity impurities of API in order to meet the validation schemes approved by both sides.In this paper,high performance liquid chromatography and ultra-high performance liquid chromatography-mass spectrometry were used to detect formaldehyde and sodium p-aminobenzene sulfonate in API respectively.The method was also optimized.On the premise of matrix effect and methodological verification,the system suitability,precision,linearity,accuracy and durability of the two methods were investigated.The results show that both methods reveal lower detection limits and better recovery efficiency and precision,which means better accuracy and reliability,that meeting the testing requirements.Using these two methods to detect the target residues in API,the conclusions are as follows:(1)As for the genotoxic impurity formaldehyde: on the basis of sample pretreatment and optimization of liquid chromatography conditions,the methodological validation of high performance liquid chromatography for formaldehyde detection was carried out.The results show that the system possesses good adaptability and specificity,and the peak shape of high performance liquid chromatography was good,namely no interference between adjacent peaks.The retention time of standard-added samples was the same as that of target in standard solution.The minimum detection limit was 0.02 ug/mL and the quantitative limit was 0.04 ug/mL.The linear range of the method was 0.04-0.30 ug/mL(linear correlation coefficient r was 0.9999).The average recovery rate was up to 95.0% no matter in the high,medium and low concentration standard addition recovery test.Meanwhile,the relative standard deviation(RSD)was 1.8% and 1.2% in the repeatability and intermediate precision test,respectively.The method exhibits reliable in the durability and solution stability test.(2)As for potential genotoxic impurity sodium p-aminobenzene sulfonate: On the basis of optimizing the conditions of mass spectrometry and liquid chromatography,the method of detection of sodium p-aminobenzene sulfonate by ultra-high performance liquid chromatography-mass spectrometry was validated.The results show that the method takes on good adaptability,namely no interference with adjacent peaks.Meanwhile,the retention time of the added standard sample was the same as that of the target in the standard solution.The minimum detection limit was 9.8680 ng/mL and the quantitative limit was 29.6039 ng/mL.The method shows good linearity,ranging from 0.03 to 0.05 ug/mL(linear correlation coefficient r was 0.9986).In the spiked recovery test of no matter high,medium and low concentrations,the average recovery was up to 84.1%.During the repeatability and intermediate precision experiments,the relative standard deviation(RSD)was 7.3% and 2.9%,respectively.The method exhibits reliable in the stability and solution stability tests.(3)After four consecutive batches of samples,it was found that the residual amount of formaldehyde in the drug substance was 4-18 mg/kg,while there was no any residue of sodium p-aminobenzene sulfonate in API. |
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