Prominence uflc hplc system

Serum (100 μL) was collected and isolated from mice 16 weeks (STCH and STKD) and 88 weeks (LTCH and LTKD) of age. Serum samples were extracted at dry ice temperatures using a 1:4 ratio of serum:methanol resulting in a total volume of 80% methanol. Samples were centrifuged and resulting supernatant was dried under vacuum. Samples were resuspended using 20 μL high-performance liquid chromatography (HPLC) grade water for mass spectrometry and 10 μL was injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/Sciex) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 249 endogenous water soluble metabolites as previously described [13 ]. All mass spectrometry data were collected and processed as previous reported [13 ]. Data are presented as mean ± SEM. Comparisons between two groups were performed using an unpaired t test.

To determine the relative levels of intracellular metabolites, extracts were prepared and analyzed by LC/MS-MS. Quadruplicate 10-cm plates (~80% confluent) were incubated in serum-free medium for 15 hours. Metabolites were extracted on dry ice with 4 mL 80% methanol (−80°C), as described previously (16 (link)). Insoluble material was pelleted by centrifugation at 3,000 × g for 5 minutes, followed by two subsequent extractions of the insoluble pellet with 0.5 mL80% methanol, with centrifugation at 16,000 × g for 5 minutes at 4°C. The 5-mL metabolite extract from the pooled supernatants was dried down under nitrogen gas using an N-EVAP (Organomation Associates, Inc). Dried pellets were resuspended using 20 μL HPLC-grade water for mass spectrometry. A 7-μL sample was injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC System (Shimadzu) via selected reaction monitoring of a total of 300 endogenous water-soluble metabolites for steady-state analyses of samples (17 (link)). The normalized areas were used as variables for the univariate statistical data analysis. All univariate analyses and modeling on the normalized data were carried out using Metaboanalyst 4.0 (http://www.metaboanalyst.ca). Univariate statistical differences of the metabolites between two groups were analyzed using two-tailed Student t test.

The purity and identity of the oligosaccharide fractions were evaluated via High Performance Liquid Chromatography (HPLC) equipped with evaporative light scattering detection (ELSD). Lyophilized samples were initially dissolved in DI water and then acetonitrile was added to make an oligosaccharide in 60% acetonitrile/40% water solution. Analyses was performed using Prominence UFLC-HPLC system (Shimadzu, Columbia, MD) equipped with a system controller (CMB-20A), degasser (DGU-20A), solvent delivery module (LC-20AD), autosampler (SIL-10A), column oven (CT20-A), and evaporative light scattering detector (ELSD-LT II; kept at 60 °C with nitrogen gas pressure of 350 kPa) on a HILICpak VN-50 4D analytical column and a HILICpak VN-50G 4A guard column (Shodex, New York, NY) for analysis of all samples. The column oven was set to 30°C for the analysis of FOS; and 60°C for analysis of GOS and XOS. Standard curves were prepared using commercially available xylose, xylobiose, xylotriose, and xylotetraose for XOS DP 1–4; fructose, sucrose, 1-kestose, and nystose for FOS DP 1–4, and galactose, lactose, and 6’-galactosyllactose for GOS DP 1–3. Peak integrations were done using the manufacturer’s LC-solution software (Shimadzu, Kyoto, Japan).

Polar metabolites were extracted from 100 mg flash-frozen atrial tissue samples with 1ml of ice-cold 80% (v/v) methanol and 0.6 ml acetonitrile and analyzed using a 5500 QTRAP hybrid triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) with SRM.^{26 (link)} Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/SCIEX). LC/MS-MS was run independently for samples from n=4/group. Data analysis was performed using Metabo Analyst 3.0²⁷

Cells were harvested in triplicate, and intracellular metabolites were extracted using 80% (v/v) aqueous methanol. Targeted liquid chromatography - tandem mass spectrometry (LC-MS/MS) was performed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) with Amide HILIC chromatography (Waters). Data were acquired in selected reaction monitoring (SRM) mode using positive/negative ion polarity switching for steady-state polar profiling of more than 260 molecules. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/SCIEX). Informatics analysis was carried out using MetaboAnyst.ca free online software.