Multiquant 3

All chemical structures were drawn using ChemBioDraw Ultra 12.0 (PerkinElmer Informatics, Waltham, MA, USA). The data acquisition was carried out with MultiQuant 3.0.2 and PeakView 2.1 from (ABSciex, Darmstadt, Germany). EICs were obtained with the use of MultiQuant 3.0.2 (ABSciex, Darmstadt, Germany), which created the base peak chromatograms for the masses that achieve a 0.01 Da mass accuracy width. The relative tolerance of the retention time was set within a margin of ±2.5%. Regarding the statistical analysis, the level of significance was estimated using Excel t-Test: two-sample assuming unequal variances.

MultiQuant 3.0.3 Software (AB Sciex) was used to integrate the data and calculate the concentration. Isotopically labelled standards were used for SCFA quantitation. For bile acid quantitation, we used Dehydrocholic acid as internal standard to correct for losses during sample preparation and isotopically labelled references to correct for ionization effects during measurement, according to the paper of Sinah et al. 2021^{90 (link)}. For comparison between two groups, Mann-Whitney U test (two-sided) was used to test for statistical significance. Metabolite-microbiota correlation analyses were performed on relative abundance zOTU level within the rhythmic in male Bmal1^fl/fl, but not Bmal1^IEC-/- samples with at least a 30% prevalence. Spearman correlation and adjusted p-values between targeted metabolomics and zOTUs were calculated using the rcor() function in R. Correlation matrixes were visualized within the R package “corrplot” v0.92 (Wei et al., 2017). Only correlations were plotted with a p value of <0.05 and coefficient values R ≤ − 0.5 and ≥0.5. Furthermore, M2IA online platform^{92 (link)} was used for the global similarity analyses (PA plot) between metabolome and microbiota data.

Mass spectrometry was performed in an AB Sciex Triple Quad™ QTRAP® 5500 Mass Spectrometer equipped with an Atmospheric-Pressure Chemical Ionization (APCI) source on a positive mode for carotenoid analysis. Also, 5 mmol/L of ammonium acetate was added to the mobile phases for improving compound ionization, as well as the column temperature (25°C). The conditions for mass spectrometry were adapted from Etzbach et al. (2018) with the following modifications: entrance potential (EP), 10 V; collision energy (CE), 30 V; collision cell exit potential (CXP), 8 V; time, 50 ms; curtain gas, 10 (API); medium collision gas (CAD); ion spray voltage, 5500 V; temperature, 450°C; and arbitrary units for ion source gas 1 (GS1), 30.0.
A selective reaction monitoring (SRM) experiment was performed to identify the analytes, the first mass transition following two or three mass transitions was used to confirm the compound profile, and these transitions were determined by the literature [16 (link)] (see Table 1).
The major carotenoids (see Figure S1 in Supplementary Materials) constituent from HPLC-DAD-UV were focused on these evaluations, and the SRM identification transition selected was 569.00/551.00. The Analyst® 1.5.1 software (AB Sciex®) and MultiQuant™ 3.0.3 software (AB Sciex®) were used for data analysis.
See Figure S1 in Supplementary Materials for comprehensive carotenoid image analysis.