MSACL 2025 Abstract

Newborn Screening for X-linked Adrenoleukodystrophy: Method Development and Validation of LC-MS/MS assay for Lysophosphatidylcholine Quantification

Kandeepan Karthigesu (1), Nathan Mclntosh (2), Nathalie Lepage (2), Matthew Henderson (2), Murray Potter (1).
(1) Department of Pathology and Molecular Medicine, Health Sciences, McMaster University, Hamilton, Canada (2) Newborn Screening of Ontario (NSO), Children's Hospital of Eastern Ontario (CHEO), Ottawa, Canada

Kandeepan Karthigesu, PhD (Presenter) McMaster University >> POSTER (PDF)

Presenter Bio: Dr. Kandeepan Karthigesu is an experienced Research Scientist with a strong foundation in clinical laboratory research, mass spectrometry, and method development. Demonstrated expertise in newborn screening, metabolomics, and molecular research, with practical experience in LC-MS/MS, GC-MS, and other biochemical and immunological analyzers. Skilled in developing and validating analytical methods that support clinical decision-making, improve diagnostic accuracy, and enhance patient care. Deeply engaged in the clinical biochemistry field to meaningful health outcomes and committed to advancing healthcare through scientific innovation, analytical precision, and collaborative practice.

Relevant Financial Disclosures (within past 24 months, reported on Aug 07, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

BACKGROUND:
X-linked adrenoleukodystrophy (X-ALD), due to pathogenic variants of the ABCD1 gene, is the most common peroxisomal disorder and affects 1:17000 births. Among the several forms, the childhood cerebral ALD (CCALD) is the most severe and fatal at the age of 4-8 years in males. CCALD presents with adrenal insufficiency and aggressive inflammatory demyelination. Treatment by bone marrow transplantation is effective, but must be done before onset of clinical neurological symptoms. Hence, early diagnosis is crucial in a clinical setting. Inclusion of X-ALD for newborn screening (NBS) in Canada has been limited due to a lack of validated analytical methods. This study aimed to clinically validate C20:0 to C26:0 lysophosphatidylcholines (LPCs) using liquid chromatography (LC) MS/MS and to implement it as a second-tier test for CCALD screening in Ontario.

METHODS:
Screen positive samples from the first-tier method under flow injection analysis (FIA) MS/MS were subjected to C20:0-LPC, C22:0-LPC, C24:0-LPC, and C26:0-LPC measurements using LC-MS/MS. To achieve this, various mobile phases, gradients, columns, internal standards, and MS parameters were evaluated to optimize the method. Analysis was conducted using a Waters Xevo-TQS micro MS/MS instrument coupled to a Waters ACQUITY H class plus UPLC 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. Underivatized 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: 552.4 (C20:0-LPC), 580.4 (C22:0-LPC), 608.5 (C24:0- LPC), 636.5 (C26:0-LPC), 556.4 (C20:0-d4-LPC), 586.4 (C22:0-13C6-LPC), 614.5 (C24:0-13C6-LPC), and 642.5 (C26:0-13C6-LPC). Dried blood spot (DBS) samples used as calibrators, quality controls, and blank filter paper during validation were sourced in-house and from the U.S. Centers for Disease Control and Prevention (CDC).

RESULTS:
Sample elution of DBS shaking at 600 rpm and 28°C 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 with gradient elution starting at 85% A1, showed the best chromatographic separation. MS parameters were optimized, including cone voltage 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 μM with an R-squared value greater than 0.99 for all LPCs. The limit of detection (LOD) for C20:0 LPC, C22:0 LPC, C24:0 LPC, and C26:0 LPC was 0.035, 0.008, 0.012, and 0.018 μM, respectively. The between-day precision of C20:0, C22:0, C24:0, and C26:0-LPCs were 6.3%, 11.9%, 9.2%, and 7.0%, respectively. Two-way ANOVA followed by multiple comparisons using Tukey’s HSD (Day 0 vs. Day 5; storage temperatures: 80°C, -20°C, 4°C, 25°C, and 45°C) revealed no significant differences in the concentration of C26:0 LPC extracted from DBS.

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.