HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma.

BACKGROUND

Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (GlcCer), and ceramide trihexoside (CTH).

METHODS

After addition of sphinganine as internal calibrator, we extracted lipids from 50 microL plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at lambda(ex) 340 nm and lambda(em) 435 nm.

RESULTS

Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra- and interassay CVs were <4% and <14%, respectively, and recovery rates were 87%-113%. The limit of quantification was 2 pmol (0.1 pmol on column), and the method was linear over the interval of 2-200 microL plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) micromol/L for Cer, 6.3 (1.9) micromol/L for GlcCer, and 1.7 (0.5) micromol/L for CTH. Plasma concentrations of GlcCer were higher in Gaucher disease patient samples and of CTH in Fabry disease patient samples.

CONCLUSIONS

HPLC enables quantification of total Cer, GlcCer, and CTH in plasma and is useful for the follow-up of patients on therapy for Gaucher or Fabry disease.