Neobase 2 non derivatized msms kit

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.

The determination of 14 AAs, 35 ACCs, C0, succinylacetone, 2 nucleosides, and 4 lysophospholipids was performed in tear samples by the addition of isotopically labelled internal standards (ISs) for each analyte of interest prior to the extraction, according to the principle of isotope dilution internal standardization.
Schirmer’s strips imbibed by tears were cut and transferred into microcentrifuge tube (Eppendorf®, Hamburg, Germany). After adding the extraction solution (300 µL) containing ISs, each tear samples were shaken in a thermo mixer (750 rpm, 45 °C, 30 min) [35 (link),36 (link)].
The internal standards, as well as the extraction solution, were obtained from the NeoBase 2 Non-derivatized MSMS Kit (Perkin Elmer Life and Analytical Sciences, Turku, Finland). 100 μL for each extracted sample was transferred to a vial and leave at room temperature for an hour for succinylacetone derivatization [37 (link)].