Q exactive exactive plus orbitrap mass spectrometers

The plasma metabolomic profiles of participants were measured from plasma samples using a combination of four LC–MS methods that measure complementary metabolites: two methods that measure polar metabolites, a method that measures metabolites of intermediate polarity (e.g., fatty acids and bile acids), and a lipid profiling method (see below for method‐specific details). For the analysis queue in each method, participants were randomized and longitudinal samples from each participant were randomized and analyzed as a group. As the aliquots for the LC–MS methods were prepared from each sample, a pooled sample was created by combining an additional aliquot from each sample into a 50 ml conical centrifuge tube. The pooled sample was mixed by vortexing and sub‐aliquoted to create pooled plasma QC samples, which were injected in pairs at intervals of approximately 20 samples for QC and data standardization.
Samples were prepared for each method using extraction procedures that are matched for use with the chromatography conditions. Data were acquired using LC–MS systems consisting of Nexera X2 U‐HPLC systems (Shimadzu Scientific Instruments) coupled to Q Exactive/Exactive Plus orbitrap mass spectrometers (Thermo Fisher Scientific).

A combination of four LC–MS methods was used to profile metabolites in the fecal homogenates, as previously published^{97 (link)}; two methods that measure polar metabolites, a method that measures metabolites of intermediate polarity (for example, fatty acids and bile acids), and a lipid profiling method. The 95% ethanol solution in which stool samples were preserved was aliquoted into two 10 µL and two 30 µL aliquots in 1.5 mL centrifuge tubes for LC-MS sample preparation. Reference pooled material was generated by combining aliquots of each sample in the study. For the analysis queue in each method, subjects were randomized, and pairs of pooled reference samples were inserted into the queue at intervals of approximately 20 samples for quality control and data standardization. Samples were prepared for each method using extraction procedures that are matched for use with the chromatography conditions. Data were acquired using LC–MS systems comprised of Nexera X2 U-HPLC systems (Shimadzu Scientific Instruments) coupled to Q Exactive/Exactive Plus orbitrap mass spectrometers (Thermo Fisher Scientific). The method details are summarized below.