Van guard beh c18 pre column

Lipidomic analysis was performed according to Simcox et al^{43 (link),44 (link)}; briefly, 40μl aliquots of sera were combined with 250μls PBS and 225μls ice-cold MeOH containing internal standards (Avanti Splash cat#3307–07), and homogenized. Samples were then mixed with 750μls of ice-cold MTBE (methyl tert-butyl ether), rehomogenized, and separated by centrifugation (17,000g for 5 mins/4°C). The upper phase was transferred to a new tube, lyophilized and resuspended in 150 μls of isopropanol. Lipids were analyzed by UHPLC/MS/MS in positive and negative ion modes, at a dilution suitable to eliminate saturation issues. Extracts were separated on an Agilent 1260 Infinity II UHPLC system using an Acquity BEH C18 column (Waters 186009453; 1.7 μm 2.1 × 100 mm) maintained at 50 C with VanGuard BEH C18 precolumn (Waters 18003975), using the chromatography gradients described by Jain et al. The UHPLC system was connected to an Agilent 6546 Q-TOF MS dual AJS ESI mass spectrometer and run in both positive and negative modes as described. Samples were injected in a random order and scanned between 100 and 1,500 m/z. Tandem MS was performed at a fixed collision energy of 25 V. The injection volume was 2 μl for positive mode and 5 μl for negative mode.

UHPLC-HRMS measurements for determination of exact mass, sum formula and retention time were carried out with a Dionex Ultimate 3000 SL system consisting of SWPS 3000 SL autosampler, P680 pump module, TCC100 column oven, and PDA100 UV-detector coupled to a maXis 4 G UHR-TOF platform. The ionization mode was ESI with the following MS settings: capillary voltage 4000 V, end plate off-set -500 V, nebulizer gas pressure 1 bar, dry gas flow rate 5 L/min, dry gas temperature 200 °C, mass scan range 150–2500 m/z. The mass spectrometer was externally calibrated to sodium formate cluster using subsequent lock mass calibration. Metabolite profiling was performed using Waters Acquity BEH C18 columns (50 × 2.1 mm, 1.7 µm and 100 × 2.1 mm, 1.7 µm) connected to a Waters VanGuard BEH C18 pre-column. The standard methods for both columns are linear gradients with 5–95% ACN + 0.1 % FA over 6, 9, or 18 min. The LC-MS results were analyzed by Bruker Compass DataAnalysis V4.4.