ABSTRACT
Objective: To establish an LC-MS/MS method for the determination of 4-( 4-amino-3-fluorophenoxy )-N-methylpyri-dine-2-carboxamide ( AFP-PMA) as a genotoxic impurity in regorafenib. Methods: The content of AFP-PMA was determined by an LC-MS/MS method. A Waters XBridge Shield RP18 column was adopted to separate the samples and the column temperature was 50℃. The mobile phase consisted of 5 mmol·L-1ammonium acetate aqueous (A)-acetonitrile (B) with gradient elution (0~9 min, 5%B→90%B) at a flow rate of 1. 0 ml·min-1. An electrospray ionization source (ESI) was used in a positive-ion and multiple reactions monitoring mode. The ion channel was m/z 262. 2→244. 1. Results:The standard curve was linear within the range of 2. 41-980. 90 ng·ml-1(r=0. 9998) and the limit of quantification was 8. 02 ng·ml-1. The limit of detection was 2. 41 ng·ml-1, which was e-quivalent to 0.000241% for the concentration of regorafenib. The average recovery was 100.95% and RSD was 2.37% (n=9). Conclusion:The method has good specificity, promising accuracy and high sensitivity, which can be used for determining the trace genotoxic impurity AFP-PMA in regorafenib.
ABSTRACT
Objective To investigate the effects of volatile oil from artemisia dracunculus on myocardial injury caused by viral myocarditis in mice and explore its possible mechanism.Methods Totally 160 adult male BALB/c mice were randomly divided into normal control group (10) and viral myocarditis group (150).Viral myocarditis mice models were reproduced by intraperitoneal inoculation with a solution of coxsackievirus B3 (CVB3),a viral strain with affinity to myocardium,and then randomly divided into model,astragalus group,and low-,medium-,and high-dose volatile oil from artemisia dracunculus groups.After 1 hour of viral infection,normal control group and model group mice were given normal saline by intragastric administration,astragalus group mice were injected with astragalus 0.1 mL in each mouse by intraperitoneal injection,and the mice in other three groups were given low,medium and high dose (2％,5％,10％) 0.3 mL volatile oil from artemisia dracunculus in each mouse by intragastric administration,respectively,once a day for one week consecutively.The mortality,heart/body weight ratio,the activity of natural killer cells (NK cell),virus titer in myocardial homogenate,serum cardiac troponin Ⅰ (cTnI) level and myocardial pathological changes were observed.Results ① Mortality:the mortality of model group was higher than that of the normal control group,astragalus group,low and medium dose volatile oil from artemisia dracunculus groups (60.0％ vs.0％,23.3％,20.0％,28.7％),and the difference in the mortality being of no statistical significance between model group and that of high-dose volatile oil from artemisia dracunculus group (60.0％ vs.47.6％,P ＞ 0.05);the mortality of astragalus group was obviously lower than that of high-dose volatile oil from artemisia dracunculus group (P ＜ 0.01),and the differences in comparisons between the mortalities of astragalus intervention group,and medium-and low-dose volatile oil groups were not statistically significant (all P ＞ 0.05),and the comparison of mortality between low-and medium-dose volatile oil groups were also not statistically significant (P ＞ 0.05).② Immunization parameters:on the 8th day after modeling,the activity of NK cells in the model group was significantly lower than that in the normal control group [(15.91 ± 3.87)％ vs.(38.50 ± 2.32)％],the activities of NK cells in astragalus group,medium-and low-dose volatile oil from artemisia dracunculus groups were significantly higher than that in model group [(19.38 ± 3.27)％,(18.54 ± 3.09)％,(18.36 ± 2.64)％ vs.(15.91 ± 3.87)％,all P ＜ 0.05].None of virus was detected in the myocardial homogenate in the normal control group,and the virus titers in astragalus group,low and medium dose volatile oil from artemisia dracunculus groups were significantly lower than the titer of the model group (10-9/mL:1.96 ± 0.44,1.95 ± 0.46,1.95 ± 0.48 vs.2.41 ± 0.51,all P ＜0.01).③ Myocardial injury parameters:the level of cTnI in the normal control group was less than 0.1 μg/L,obviously lower than that in the model group [(15.84 ± 3.89) μg/L],as well as the ratio of heart/body weight in model group was also significantly higher than that in normal control group (× 10-4:8.3 ± 1.3 vs.4.6 ± 0.1),and the cTnI and the ratio of heart/body weight of astragalus intervention group,low and medium dose volatile oil from artemisia dracunculus groups were markedly lower than those of model group [cTnI (mg/L):10.03 ± 2.35,10.81 ± 2.56,11.10 ± 1.89 vs.15.84 ± 3.89,ratio of heart/body weight (× 10-4):7.2 ± 0.8,7.3 ± 1.0,7.3 ± 0.6 vs.8.3 ± 1.3].In the normal control group,there were no inflammatory cell infiltration and necrosis in myocardial tissue,the scores of myocardial pathological changes were 0.In the model group,the scores of inflammatory cell infiltration (3.25 ± 0.45) and of necrosis (2.91 ± 0.51) were markedly higher than those in the normal control group.And the above scores in astragalus group,low and medium dose volatile oil from artemisia dracunculus groups were significantly lower than those of the model group (infiltration score:2.92 ± 0.39,2.95 ± 0.35,2.95 ± 0.37 vs.3.25 ± 0.45,necrosis score:2.46 ± 0.50,2.50 ± 0.51,2.54 ± 0.50 vs.2.91 ± 0.51,all P ＜0.05).Conclusions Volatile oil from artemisia dracunculus can protect cardiomyocytes by removing the virus and regulating the immune function in the body.But the protective effects of volatile oil from artemisia dracunculus is related to the dosage,and the effects of low and medium dose are better.