Agilent 1290 infinity 2 series uhplc system

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis based on the 16S rDNA was performed. The OTU table derived from QIIME was compared using the Kyoto Encyclopedia of Genes and Genomes and MetaboAnalyst (http://www.metaboanalyst.ca/) databases, and the metabolic function of the gut microbiota was predicted based on the findings. The abundances of functional genes were visualized as heatmaps by the R package.
For metabolite extraction, a 10-μL aliquot of each sample was mixed with 990 μL of the extraction solvent (acetonitrile/methanol/water, 2:2:1), and the mixture was vortexed for 30 s, incubated at -20 °C for 1 h, and then centrifuged at 12000 rpm and 4 °C for 15 min. Finally, the supernatant was diluted 10 times for ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis[24 (link)]. UHPLC separation was performed on an Agilent 1290 Infinity II series UHPLC system (Agilent Technologies). Mobile phase A included both 10 mmol/L ammonium formate and 10 mmol/L ammonia, while mobile phase B was acetonitrile. The temperatures for the column and the autosampler were set at 35 °C and 4 °C, respectively[25 (link)].

After the addition of 900 μL of extraction solution (acetonitrile-methanol-water, 2:2:1), these samples were vortexed for 30 seconds, homogenized at 45 Hz for four minutes, and sonicated for five minutes in an ice-water bath. Then, the homogenate and sonicate circle was repeated for three times, followed by incubation at −40°C for one hour and centrifugation at 12,000 rpm for 15 mins at 4°C. A 100-μL aliquot of the clear supernatant was transferred to an auto-sampler vial for the UHPLC-MS/MS analysis. The UHPLC separation was carried out using the Agilent 1290 Infinity II series UHPLC System (Agilent Technologies), which was equipped with a Waters ACQUITY UPLC BEH Amide column (100×2.1 mm, 1.7 μm). The mobile phase A was 1% formic acid in water, and the mobile phase B was acetonitrile. An Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies), equipped with an AJS electrospray ionization (AJS-ESI) interface, was applied for assay development. The MRM parameters for each of the targeted analytes were optimized using flow injection analysis, and were performed by injecting the standard solutions of the individual analytes into the API source of the mass spectrometer. The Agilent MassHunter Work Station Software (B.08.00, Agilent Technologies) was employed for MRM data acquisition and processing.