# New multiplex LC-MS/MS method for lipid biomarker analysis of inherited neurodegenerative metabolic diseases

## TL;DR

This paper introduces a new, efficient method for detecting lipid biomarkers in inherited neurodegenerative diseases using mass spectrometry, improving diagnosis and monitoring.

## Contribution

A high-throughput multiplex LC-MS/MS method for analyzing 13 lipid biomarkers in various neurodegenerative metabolic diseases.

## Key findings

- The method confirmed diagnoses in 89 patient samples across multiple NMDs.
- It distinguished X-linked adrenoleukodystrophy from peroxisomal biogenesis disorder.
- Elevated sulfatides were identified in Krabbe disease and MEDNIK syndrome.

## Abstract

A significant number of inherited neurodegenerative metabolic diseases (NMDs) arise from altered lipid metabolism, including impaired degradation of sphingolipids and dysfunction in organelle-related machineries involved in lipid processing and trafficking. These lipid dysregulations profoundly impact cellular membranes, signaling pathways, and myelin integrity, contributing to the complex and multisystemic clinical phenotypes characteristic of NMD, which often complicate diagnosis and delay treatment initiation. Here, we present a high-throughput, multiplex LC-MS/MS method for the analysis of an extended panel of NMD biomarkers in plasma and dried blood spots. One-step sample extraction and targeted LC-MS/MS acquisitions in positive and negative ionization allowed the simultaneous measurement of 13 diagnostic biomarkers associated with GM1 and GM2 gangliosidosis, Fabry, Gaucher, and Krabbe diseases, acid sphingomyelinase deficiency, Niemann-Pick disease type C, X-linked adrenoleukodystrophy, peroxisomal biogenesis disorders (Zellweger syndrome), metachromatic leukodystrophy, and mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratoderma (MEDNIK)/MEDNIK-like syndromes, a disorder of cellular trafficking. The method was analytically and clinically validated, confirming the diagnosis of all targeted NMDs in samples from 89 patients. Additionally, the method allowed the differentiation of X-linked adrenoleukodystrophy from peroxisomal biogenesis disorder and revealed the elevation of C18- and C16-sulfatides in Krabbe disease and MEDNIK syndrome, respectively. This multiplex assay enhances diagnostic efficiency and expands the discovery of novel biomarkers, enabling the quantification of diagnostic markers for a wide range of NMDs. The method is suitable for diagnosis of NMD, as a first- or second-tier test in neonatal screening, as confirmatory testing of variant of unknown significance in genetic panels and for longitudinal monitoring in treatable diseases.