Masshunter quantitative analysis for qqq

Lipids were analyzed based on characteristic SRM transitions and retention time using Agilent MassHunter Quantitative Analysis for QQQ (v. B.07.00). Further processing, including normalization to suitable internal standards, was performed in Microsoft Excel. High-resolution LC–MS data were processed using XCalibur QuanBrowser 3.0.63 (Thermo Fisher Scientific).

Lipids from the isolated “fat pads” were resuspended in butanol:methanol (1:1, v/v) to a final concentration of 1 mg mL⁻¹. Lipids were chromatographically separated using a Waters BEH C8 column (Waters, Milford, MA) and a binary gradient with a flow rate of 0.2 mL min⁻¹ on an Agilent 1290 liquid chromatography (Agilent Technologies). The mobile phases were: A. H₂O:acetonitrile (10:90, v:v) with 10 mM ammonium formate and 0.2% acetic acid; B. H₂O:acetonitrile:isopropanol (5:15:80, v:v:v) with 10 mM ammonium formate and 0.2% acetic acid. The gradient was initially held at 1% B for 2 min, then increased to 20% B over the next 3 min, then increased to 70% B over 7 min, followed by a final increase to 90% B over 2 min. The eluted lipids were analyzed on an Agilent 6490 (Agilent Technologies) based on previously published methods (Reynolds et al., 2015 (link)). Results were analyzed by integration using Mass Hunter Quantitative Analysis for QQQ, version B.07.01 (Agilent Technologies) followed by export of the data to csv format and analysis in R (R Core Team, 2016 ).