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Licensed Unlicensed Requires Authentication Published by De Gruyter December 4, 2009

Determination of globotriaosylceramide in plasma and urine by mass spectrometry

  • Ralf Krüger , Kai Bruns , Silke Grünhage , Heidi Rossmann , Jörg Reinke , Michael Beck and Karl J. Lackner


Background: Fabry disease is an X-chromosomally inherited lysosomal storage disorder leading to accumulation of glycosphingolipids, mainly globotriaosylceramide (ceramide-trihexoside, Gb3). Concentrations of Gb3 in plasma and urine have been used to diagnose Fabry disease and to monitor enzyme replacement therapy with recombinant α-galactosidase.

Methods: Gb3 was purified from plasma or urine by combined liquid extraction/protein precipitation and solid-phase extraction, and was detected by flow-injection analysis electrospray mass spectrometry (MS) using multi-reaction-monitoring. Calibration was performed via standard addition using C17-Gb3 as internal standard. The most abundant isoforms were monitored for calculation of total Gb3.

Results: A MS-based assay for quantification of Gb3 in plasma and urine was established and validated. Intra- and inter-assay coefficient of variation (CV) of the method were ≤12%. However, at low concentrations the CV was 16%. The linear range covers roughly two orders of magnitude, down to 0.54 mg/L in plasma and 0.07 mg/L in urine. Careful adjustment of tuning parameters was necessary to obtain identical isoform intensities and quantitative results on different mass spectrometers. Gb3 concentrations in healthy controls were <4 mg/L in EDTA-plasma and <10 μg/mmol creatinine in urine. Significantly increased Gb3 concentrations were found in plasma and urine from male and female patients with Fabry disease.

Conclusions: An improved MS protocol for Gb3 quantification has been developed, validated, and shown to be suitable for diagnosis and monitoring of Fabry patients.

Clin Chem Lab Med 2010;48:189–98.

Corresponding author: Dr. Ralf Krüger, Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, Langenbeckstr. 1, 55131 Mainz, Germany

Received: 2009-7-10
Accepted: 2009-10-7
Published Online: 2009-12-04
Published in Print: 2010-02-01

©2010 by Walter de Gruyter Berlin New York

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