Quantification of Very Long-Chain LPCs

Very long-chain LPCs (C22:0-, C24:0-, and C26:0-LPC) were analyzed in all patient and control individuals using tandem mass spectrometry (MS/MS) with a Xevo TQD analyzer (Waters Corporation, Milford, MA) and the NeoBase™ 2 Non-derivatized MSMS kit (PerkinElmer, Waltham, MA,), following the manufacturer’s protocol with minor adaptations for plasma analysis. This kit simultaneously quantifies C26:0-LPC, amino acids, acylcarnitines, and succinylacetone based on the method previously described by Haynes et al. 2016 (34 (link)). In brief, 3 μl of plasma were applied to a 3.2 mm spot of Whatman 903 filter paper (Whatman International Ltd, Kent, UK). Analytes were extracted by adding 125 μl of the extraction working solution containing ²H4-C26:0-LPC as internal standard and then shaking at 45°C for 45 min. Following extraction, 5 μl of the supernatant were directly injected into Xevo TQD analyzer using both positive ionization and multiple reaction monitoring modes.