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Clinical Chemistry 53: 742-747, 2007. First published March 1, 2007; 10.1373/clinchem.2006.079012
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(Clinical Chemistry. 2007;53:742-747.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

Johanna E.M. Groener1,a, Ben J.H.M. Poorthuis1, Sijmen Kuiper1, Mariette T.J. Helmond1, Carla E.M. Hollak2 and Johannes M.F.G. Aerts1

Departments of1 Medical Biochemistry and 2 Internal Medicine, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.

aAddress correspondence to this author at: Department of Medical Biochemistry, University of Amsterdam, Academic Medical Center, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Fax 31-20-6915519; e-mail j.e.groener{at}amc.uva.nl.

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 µL 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 µL plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) µmol/L for Cer, 6.3 (1.9) µmol/L for GlcCer, and 1.7 (0.5) µmol/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.




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