Evaluation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of Glucosylceramide and Galactosylceramide Isoforms in Cerebrospinal Fluid of Parkinson's Disease Patients

Abstract

Mutations in GBA1, encoding glucocerebrosidase beta 1 (GCase), are the most common genetic risk factor for Parkinson's disease (PD). GCase dysfunction leads to an accumulation of glucosylceramide (GluCer) substrates in different organs and fluids. Despite the challenges in quantifying GluCer isoforms in biological samples, their potential clinical interest as PD biomarkers justifies the development of robust assays. An extensively evaluated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying 14 GluCer and galactosylceramide (GalCer) isoforms in human cerebrospinal fluid (CSF) samples is presented. Sample pretreatment, HPLC, and MS/MS parameters were optimized. Evaluation was performed according to the recommendations of the Clinical and Laboratory Standards Institute and European Medicines Agency guidelines. Four 7-point calibration curves were generated, with a linearity interval from 2.5 to 200 nM (R² ≥ 0.995). The limit of quantification was set at 5 nM. Between-run precision and accuracy were up to 12.5 and 9%, respectively. After method validation, we measured the levels of GluCer and GalCer isoforms in CSF human samples, including 6 healthy controls (HC), 22 idiopathic GBA1 wild-type PD (iPD) patients, and 5 GBA1-associated PD (PD-GBA) patients. GluCer/GalCer median ratios were found to be higher in the CSF of PD-GBA patients, particularly in severe GBA1 mutations, than those in iPD and HC. The observed trends in GluCer/GalCer ratios among groups provide novel information for the comprehensive analysis of sphingolipids as potential biomarkers of PD.

Figure 1

Chromatogram of the 200 nM standard, showing the RTs of GluCer and GalCer isoforms.

Figure 2

Boxplots (median and interquartile range) for GluCer concentrations and GluCer/GalCer ratios. (A) Sum of the total GluCer isoform concentration and (B) normalization by the total GalCer ratio among the groups of patients. (C) GluCer normalized to the GalCer ratio for each isoform, with a focus on C22:0, C23:0, and C24:0.