Abstract Background Newborn screening (NBS) is a population-based healthcare initiative designed to detect selected disorders that would benefit from early intervention. Newborn Screening Ontario (NSO), Canada, will be adding X-linked adrenoleukodystrophy as an NBS target in 2025. The disorder manifests in several forms, with childhood cerebral ALD (CCALD) being the most severe and the target of NBS. CCALD presents with adrenal insufficiency and aggressive inflammatory demyelination in the brain, leading to rapid cognitive and neurological deterioration. Treatment is by bone marrow transplantation. Lysophosphatidylcholine containing a saturated 26-carbon fatty acid (C26:0-LPC) measured by flow-injection analysis tandem mass spectrometry (MS/MS) is used as a first-tier test, with a more specific C26:0-LPC assay by liquid chromatography (LC) MS/MS being used as a second-tier test. Here, we report the clinical validation of a LPC LC-MS/MS assay measuring C26:0-LPC, C24:0-LPC, C22:0-LPC, and C20:0-LPC to be implemented as a second-tier test for CCALD screening in Ontario. Methods Various mobile phases and gradients were evaluated to optimize the chromatographic peak separation. Analysis was conducted using a Waters Xevo-TQS micro MS/MS instrument coupled to a Waters ACQUITY H class plus UPLCTM binary solvent LC system. An ACQUITY Premier BEH C8 VanGuard FIT Column, 1.7 µm, 2.1 mm x 50 mm, with a matching 5 mm guard column was employed. Analytes and stable isotope internal standards were monitored in positive electrospray ionization (ESI+) mode by multiple reaction monitoring (MRM), using the same quantification or qualification product ions for all species (M/Z 104.1 and 184.1, respectively) with the following precursor ions: 636.5 (C26:0-LPC), 608.5 (C24:0- LPC), 580.4 (C22:0-LPC), 552.4 (C20:0-LPC), 642.5 (C26:0-13C6-LPC), 614.5 (C24:0-13C6-LPC), 586.4 (C22:0-13C6-LPC), and 556.4 (C20:0-d4-LPC). Dried blood spot (DBS) samples used as calibrators and quality controls and blank filter paper spots used as blanks during the validation process were sourced in-house and from the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. Results Sample elution of DBS shaking with 100% methanol showed good recovery. The mobile phases A1 and B1 comprising water/acetonitrile (50/50, v/v) and methanol/acetonitrile (50/50, v/v), respectively, with 5 mM ammonium acetate, at a flow rate of 0.4 mL/min showed the best chromatographic separation (Figure 1). MS parameters were optimized, including cone voltage of 26 V, collision energy of 30 V, and capillary voltage of 4.25 kV. Method validation demonstrated good linearity ranging from 0.1 to 8.0 uM with R-squared value greater than 0.99. The limit of detection for C26:0 LPC was 0.007 uM. The between-day precision of C26:0 LPC was 0.5%. All analytes in DBS remained stable at -80, -20, 4, 25 and 45 degrees Celsius for up to 5 days. Conclusion We developed and validated an LC-MS/MS method for quantitation of C20:0, C22:0, C24:0 and C26:0 LPCs for CCALD second-tier NBS testing, with implementation anticipated in 2025.