Simultaneous quantification of G M1 and G M2 gangliosides by isotope dilution tandem mass spectrometry

Abstract

Objectives Gangliosides (GGs) are considered as diagnostic biomarkers and therapeutic targets and agents. The goal of this study was to develop a tandem mass spectrometry (MS/MS) method for the simultaneous measurement of both G M1 and G M2 gangliosides in human cerebrospinal fluid (CSF) samples in order to be able to determine their concentrations in patients with Tay-Sachs and Sandhoff disease and assess whether drugs or transplantation affect their concentrations. Design and methods An API-4000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standards. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 40 μL of human CSF sample was added and mixed with 400 μL of internal standard solution for deproteinization. After centrifugation, 100 μL of supernatant was injected onto a C-18 column. After a 2.5 min wash, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode. Results The within-day coefficients of variation were < 3% for G M1 and < 2% for G M2 and the between-day coefficients of variation were < 5% for both G M1 and G M2 at all concentrations tested. Accuracy ranged between 98% and 102% for both analytes. Good linearity was also obtained within the concentration range of 10–200 ng/mL (6.5–129.3 nmol/L) for G M1 and 5–100 ng/mL (3.6–72.3 nmol/L) for G M2 ( r ≥ 0.995). Conclusions A new simple, accurate, and fast isotope dilution tandem mass spectrometry method was developed for the simultaneous quantification of G M1 and G M2 gangliosides in a small amount of human CSF. Concentrations were measured in “normal” CSF and in CSF from patients with Tay-Sachs disease.