A validated analytical method was developed for the determination of free astragaloside Ⅳ in Astragali Radix by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Effects of each step of pretreatment were studied on the determination of astragaloside Ⅳ in Chinese pharmacopoeia(2015)(ChP).Furthermore,impacts of n-butanol extraction and ammonia impurity removal on the determination and content variation of astragaloside Ⅳ were investigated.The differences of components caused by pretreatment were analyzed by Fourier transform ion cyclotron resonance mass spectrometry(FT-ICR-MS).Results showed that there was a good linear relationship of the proposed method for astragaloside Ⅳ in the range of 0.387 0-2475 mg/L,with a correlation coefficient(r) of 0.999 8.The limit of detection(LOD) and limit of quantitation(LOQ) were 1.0 mg/kg and 3.0 mg/kg,respectively.The recoveries at three spiked levels ranged from 94.5% to 105%,with a relative standard deviation(RSD,n=6) of 1.4%.The relative content of astragaloside Ⅰ was the highest when Astragali Radix was directly extracted with methanol.The impurity removal process with ammonia after n-butanol extraction led to the homologues turned into astragaloside Ⅳ by hydrolysis,which significantly increased the content of astragaloside Ⅳ.Moreover,it was found that there exists a new isomer of astragaloside Ⅱ,named as neoastragaloside Ⅱ in Astragali Radix.