MSACL 

40. A New UPLC-MS/MS Method for the Determination of Urinary Globotriaosylceramide (Gb3, GL3 or CTH)
Poster: Tue 6:30-7:30PM
Sabrina Forni
Baylor Research Institute
Sabrina Forni, Brandon Durant, Raphael Schiffmann, Lawrence Sweetman

Institute of Metabolic Disease, Baylor Research Institute
3812 Elm St, Dallas TX 75226
Introduction:
Fabry disease is an X-linked lysosomal storage disorder characterized by the accumulation of globotriaosylceramide (Gb3, GL-3 or CTH) in various tissues and organs. Incidence is one in 40 000 to one in 117 000 (Lancet, 2008). A screening test has already been implemented in our laboratory, based upon the extraction of urine dried on filter paper (Auray-Blais et al.2007).

Objectives:
Develop a second new fast and simple UPLC-MS/MS method for (Gb3) in liquid human and murine urine and control the relative matrix effect that is a predominant limiting factor in ESI-MS/MS assay for Gb3.

Methods:
Fifty micro liters of urine and 5μL of 2.5 μg/mL C17-Gb3 as internal standard in methanol are dispensed into one mL polypropylene tubes. The urine is dried in a rotary vacuum evaporator for about one hour, and then washed with 200 μL of HPLC grade water and the water discarded. The samples are reconstituted in 400 μL of methanol. Five μL are injected into a BEH C18 1.0x10 mm, 1.7 μm column for UPLC-MS/MS analysis with ESI in MRM mode for a five minutes run time.

Results:
Various methods for liquid urine sample clean up have been tested and evaluated for ion suppression by a post column infusion experiment and S/N ratio. The clean up with water was chosen over others such as Hybrid SPE for its simplicity and comparable results. The liquid urine method and the cards method are linear and reproducible up to 25 μg/mL of Gb3. The liquid urine method has been used to determine Gb3 in urine of WT and Fabry KO male and female mice and in human urine of hemizygous male Fabry patients and heterozygous female Fabry patients.

Conclusion:
The liquid urine method better controls ion suppression and relative matrix effect because of small sample size, small injection volume, and better separation from the rest of the matrix and no suppression due to interaction with filter paper.
Furthermore some customers still prefer to submit liquid urine rather than dried urine cards, so the two methods will be compared.
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