Background
Lysosphingolipids, the N-deacylated forms of
sphingolipids, have been identified as potential
biomarkers of several
sphingolipidoses, such as Gaucher, Fabry, Krabbe and
Niemann-Pick diseases and in GM1 and
GM2 gangliosidoses. To date, different methods have been developed to measure various
lysosphingolipids (LysoSLs) in plasma. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a simultaneous quantification of LysoSLs (HexSph,
LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in dried blood spot (DBS). This LC-MS/MS method was used to compare the levels of LysoSLs in DBS and plasma in both affected patients and healthy controls. Methods
Lysosphingolipids were extracted from a 3.2 mm diameter DBS with a mixture of
methanol:
acetonitrile:water (80:15:5, v/v) containing internal stable
isotope standards. Chromatographic separation was performed using a C18 column with a gradient of water and
acetonitrile both with 0.1%
formic acid in a total run time of 4 min. The compounds were detected in the positive ion mode electrospray ionization (ESI)-MS/MS by multiple reaction monitoring (MRM). Results The method was validated on DBS to demonstrate specificity, linearity, lowest limit of quantification, accuracy and precision. The reference ranges were determined in pediatric and adult populations. The elevated levels of LysoSLs were identified in
Gaucher disease (HexSph),
Fabry disease (
LysoGb3),
prosaposin deficiency (HexSph and
LysoGb3) and
Niemann-Pick disease types A/B and C (LysoSM and LysoSM509). The correlation in the levels between DBS and plasma was excellent for
LysoGb3 and HexSph but poor for LysoSM and LysoSM509. Conclusions Despite the fact that plasma LysoSLs determination remains the gold standard, our LC-MS/MS method allows a rapid and reliable quantification of
lysosphingolipids in DBS. The method is a useful tool for the diagnosis of different
sphingolipidoses except for Niemann-Pick type C.