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New psychoactive substances (NPS) have been rapidly emerged as legal alternatives to controlled drugs, which raised severe public health issue. The detection and monitoring of its intake by complete metabolic profiling is an urgent and vital task. Untargeted metabolomics approach has been applied for several NPS metabolites studies. Although the number of such works is relatively limited but with a rapidly growing need. The present study aimed to propose a procedure that includes liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis and a signal selection software, MetaboFinder, programed as a web tool. The comprehensive metabolites profile of one kind of NPS, 4-methoxy-α-pyrrolidinovalerophenone (4-MeO-α-PVP), was studied by using this workflow. In this study, two different concentrations of 4-MeO-α-PVP along with as blank sample were incubated with human liver S9 fraction for the conversion to their metabolites and followed by LC-MS analysis. After retention time alignment and feature identification, 4640 features were obtained and submitted to statistical analysis for signal selection by using MetaboFinder. A total of 50 features were considered as 4-MeO-α-PVP metabolite candidates showing significant changes (p < 0.00001 and fold change >2) between the two investigated groups. Targeted LC-MS/MS analysis was conducted focusing on these significantly expressed features. Assisted by chemical formula determination according to high mass accuracy and in silico MS 2 fragmentation prediction, 19 chemical structure identifications were achieved. Among which, 8 metabolites have been reported derived from 4-MeO-α-PVP in a previous literature while 11 novel 4-MeO-α-PVP metabolites were identified by using our strategy. Further in vivo animal experiment confirmed that 18 compounds were 4-MeO-α-PVP metabolites, which demonstrated the feasibility of our strategy for screening the 4-MeO-α-PVP metabolites. We anticipate that this procedure may support and facilitate traditional metabolism studies and potentially being applied for routine NPS metabolites screening.

The current approaches remain insufficient for measuring chicken egg spoilage or present analytical limitations. This study aimed to complement the existing analyses and identify novel markers using liquid chromatography-high resolution mass spectrometry-based foodomics strategies. In the discovery set, comparative untargeted metabolomics was utilized to identify marker candidates in microbially inoculated chicken eggs. Markers were annotated by spectral matching with authentic standards, experimental libraries, or in silico fragmentation. In the validation set, targeted metabolomics was employed to verify the markers in stored chicken eggs from five farms. Statistical differences at a p -value < 0.001 revealed increases in lactic and 3-hydroxybutyric acids and decreases in phosphocholine, LPE(O-18:1), LPC(16:0), and LPC(18:0) in stored eggs. Receiver operating characteristic curve analysis of the six combined markers yielded an AUC of 0.956 and a sensitivity and specificity of ∼90%. Four phospholipids were highlighted as a novel class of spoilage markers. Our findings may contribute to further industrial implementation, benefiting the quality assurance and food safety of poultry egg production.

Analyzing and presenting data from multiple groups are much more informative than that from two groups. However, common tools such as S plot and volcano plot are only available for identifying the significant features between two groups and are restricted to multiple-group comparisons. This study proposed novel visualization plots which not only overcame the restrictions of the above methods but also utilized the p values of multiple tests as the x-axis. The novel visualization plots included a parametric method and a nonparametric method. The parametric method was a combination of an analysis of variance and Welch's analysis of variance; the nonparametric method used the Kruskal-Wallis test. During the selection of significant features, machine learning algorithms were used to determine the cutting points of the x-axis. As a proof of concept, the real data from the experiments of 4-MeO-α-PVP metabolites and fish spoilage metabolomics were illustrated via our visualization method. The results showed that the novel visualization plots were much efficiently presented to identify significant metabolites in multiple-group comparisons. Especially, the positive predicted values of the nonparametric method and the cutting points determined by logistic regression were higher than those of other machine learning algorithms in determining the cutting points for multiple groups. • New visualization plots outweigh volcano plot and S plot for multiple-group study. • Parametric method requires normality of data and Bonferroni's adjustment is suggested to utilize on cut point of x-axis. • Nonparametric method is flexible on data type and machine learning method is suggested to use on cut point of x-axis. • As proof-of-concept, two methods perform well for multiple-group comparisons. [ABSTRACT FROM AUTHOR]