The prevalently used LC–MS/MS methods for the simultaneous determination of synthetic phenol antioxidants (SPAs) and relevant metabolites suffer from a serious drawback of low sensitivity for 2,6-di-tert-butyl-4-hydroxytoluene (BHT) and a common issue of background contamination, which hampers the simple and accurate detection of these substances at trace levels in environmental samples. In this study, we developed an improved LC–MS/MS method for the simultaneous determination of eight SPAs and four relevant metabolites, including BHT. By use of atmospheric pressure chemical ionization (APCI), the sensitivity of BHT in the LC–MS/MS was enhanced approximately 260 times versus results obtained by using electrospray ionization (ESI), which allowed the analysis of BHT up to 1.1 ng/g in indoor dust and 0.06 ng/g in human plasma. Similarly, the sensitivity of 2,4,6-tri-tert-butylphenol and 2,6-di-tert-butyl-4-sec-butylphenol was also enhanced with APCI, which avoided their separate analysis by GC–MS. By installation of a C18 column after the eluent mixer and before the injector as a trap column, the target analytes leached out from the LC system were eluted at a much later retention time than those in the samples, which resulted in complete elimination of the instrumental background contamination. These improvements enable the method to be well applied to the real samples.

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In order to evaluate the quality of Bufonis Venenum commercial herbs, a three-step qualitative and quantitative research study was performed. Firstly, we tried to identify small molecules and peptides in Bufonis Venenum using pre-fractionation chromatography and high-resolution mass spectrometry. The database search of the small molecules and peptides of Bufonis Venenum revealed that the dried venom consisted of free/conjugated-type bufadienolides and peptides with a mass range of 0.4–2 kDa. Secondly, we used partial least squares (PLS) multivariate statistical analysis to screen bufadienolides markers (VIP > 1.5) responsible for the anti-tumor cell activity of Bufonis Venenum, including 21 identified bufadienolides and 7 unknown compounds. It is noticeable that these bufadienolide markers could not be recognized by traditional HPLC-UV based spectrum-effect relationship analysis (correlation coefficient ranging from −0.24 to 0.40). Finally, we proposed a weight coefficient-based corrected total contents of 9 bufadienolides as a quality evaluation indicator, which had good correlation with inhibitory effects on tumor cells of commercial Bufonis Venenum. The correlation coefficient increased from 0.4 to 0.6. Thus, our pre-fractionation chromatography and mass spectrometry strategy had significant advancement over the traditional spectrum–effect relationship method for chemical marker identification. These results could be crucial and helpful in the development of a quality evaluation method that could reflect the pharmacological activity of Bufonis Venenum.