Uhplc qtof ms system

The LC–MS analysis was performed as described previously by Hanhineva et al.^{43 (link)}. In brief, the samples were analyzed using liquid chromatography quadrupole time-of-flight mass spectrometry UHPLC–QTOF–MS system (Agilent Technologies), which consisted of a 1290 LC system, a Jetstream electrospray ionization (ESI) source, and a 6540 UHD QTOF mass spectrometer. The samples were separated using reversed-phase (RP) chromatography (Zorbax Eclipse XDB-C18, particle size 1.8 µm, 2.1 × 100 mm; Agilent Technologies). The elution solvents were water (A) and HPLC grade methanol (B), both containing formic acid 0.1% v/v. The gradient was as follows for the ratio of solvent B: 0–10 min: 2 → 100% B; 10–14.5 min: 100% B; 14.5–14.51 min: 100 → 2% B; 14.51–16.5 min: 2% B. Data was acquired with both positive and negative polarity. Quality controls were injected at the beginning and at the end of the MS run and after every ten injections. Automatic data-dependent MS/MS analysis was performed on one sample representing each sample type. The sample tray was kept at +4 °C during the analysis. Three replicates for malted grain and rootlet and three technical replicates for whole grains, water extract, spent grain, and wort, were analyzed in a completely randomized order.

The metabolomic profiling was carried out on a UHPLC-Q-TOF MS system (Agilent, CA, USA) with a HILIC column (Agilent, CA, USA). The column conditions were set to include a column temperature of 25°C, a flow rate of 0.5 mL/min, and an injection volume of 2 μL. The samples were separated by an Agilent 1290 Infinity LC ultrahigh performance liquid chromatography system (UHPLC) and analyzed by a Triple TOF 6600 mass spectrometer (AB SCIEX) using electrospray ionization (ESI) positive and negative ion modes, respectively. The raw data were converted into. mzXML format by ProteoWizard and then XCMS software was used for peak alignment, retention time correction, and peak area extraction. The data extracted by XCMS were firstly subjected to metabolite structure identification, data preprocessing, and then to experimental data quality evaluation, and finally to data analysis. Data analysis includes univariate statistical analysis, multidimensional statistical analysis, differential metabolite screening, differential metabolite correlation analysis, and KEGG pathway analysis.

The sample preparation, instrument parameters and pre-processing of raw data were performed in the LC–MS Metabolomics Center at Biocenter Kuopio (University of Eastern Finland), and they are previously presented in detail [17 (link)]. Briefly, methanol (300 µl) was used to precipitate the proteins and extract the metabolites from plasma (100 µl). The non-targeted metabolite profiling was carried out using the UHPLC-qTOF-MS system (Agilent Technologies, Waldbronn, Karlsruhe, Germany), which consisted of a 1290 LC system, a Jetstream electrospray ionization (ESI) source and a 6540 UHD accurate-mass quadrupole-time-of-flight (qTOF) mass spectrometry. All samples were analysed using two different chromatographic techniques; i.e., reversed phase (RP) and hydrophilic interaction chromatography (HILIC). In addition, data were acquired in both ionization polarities; i.e., ESI positive (ESI+) and ESI negative (ESI−).

The sample preparation, instrument parameters and preprocessing of raw data were performed in the LC-MS Metabolomics Center at Biocenter Kuopio (University of Eastern Finland), and they are previously presented in detail^{26 (link)}. Briefly, an aliquot (100 µl) of plasma samples was mixed with 300 µl of methanol and mixed in vortex at maximum speed 15 s, incubated on ice bath for 15 min, and centrifuged at 16 000 × g for 10 min to collect the supernatant. The supernatant was filtered through 0.2 µm PTFE filters into HPLC vials. From every sample, aliquots of 4 µl were taken and mixed together in one tube and used as the quality control sample in the analysis. The samples were analyzed by the UHPLC-qTOF-MS system (Agilent Technologies, Waldbronn, Karlsruhe, Germany), which consisted of a 1290 LC system, a Jetstream electrospray ionization (ESI) source, and a 6540 UHD accurate-mass quadrupole-time-of-flight (qTOF) mass spectrometry. All samples were analyzed using two different chromatographic techniques, i.e., reversed phase (RP) and hydrophilic interaction chromatography (HILIC). Data were acquired in both ionization polarities; i.e., ESI positive (ESI+) and ESI negative (ESI−). The data acquisition software was MassHunter Acquisition B.04.00 (Agilent Technologies). The quality control samples were injected in the beginning of the analysis and after every 10 samples.

Cord blood samples were analysed in randomised order as a single uninterrupted batch with a UHPLC-QTOF-MS system (Agilent Technologies), as previously described [25 ]. Further details of the acquisition and structural annotation of features are given in supporting information 1.

In both cohorts, nonfasted EDTA blood samples were collected and processed within 4 h, according to identical processing protocols, and stored at −80 °C. Samples at each respective study site were shipped on dry ice to the International Agency for Research on Cancer (IARC) in Lyon, France for analysis. Samples were analyzed with a ultrahigh performance liquid chromatography‐ quadrupole time‐of‐flight mass spectrometry (UHPLC‐QTOF‐MS) system (Agilent Technologies) consisting of a 1,290 Binary LC system, a Jet Stream electrospray ionization (ESI) source, and a 6,550 QTOF mass spectrometer. Samples from each study center were analyzed in cohort‐specific batches, which consisted of five and six 96‐well plates for CORSA and ColoCare, respectively.
A detailed overview of the sample preparation and a complete description of sample analysis by UHPLC‐QTOF‐MS, pre‐processing of metabolomics data can be found in Supporting Information File S1. A summary of the data processing workflow is shown in Supporting Information Figure S1.