Received on February 24, 2004
Accepted on May 5, 2004

Endocrinology and Metabolism

Measurement of Urinary D- and L-2-Hydroxyglutarate Enantiomers by Stable-Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry after Derivatization with Diacetyl-L-Tartaric Anhydride

¹ Metabolic Unit, Department of Clinical Chemistry, VU Medical Center, Amsterdam, The Netherlands

^* To whom correspondence should be addressed. E-mail: e.struys{at}vumc.nl.

Background: The differential diagnosis of D-2-hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) can be accomplished only by the measurement of the corresponding 2-hydroxyglutarate (2-HG). Available methods for the determination of D- and L-2-HG in urine are either time-consuming and expensive or have not been extensively validated. We aimed to develop a method for their rapid and sensitive measurement.

Methods: We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of D- and L-2-HG with stable-isotope-labeled internal standards. Urine samples of 20 µL were mixed with 250 µL of methanol containing the internal standards and subsequently dried under nitrogen. The analytes were derivatized by use of diacetyl-L-tartaric anhydride (DATAN) to obtain diastereomers, which were separated on an achiral C₁₈ HPLC column and detected by MS/MS in multiple-reaction-monitoring mode.

Results: The use of DATAN as chiral derivatization reagent provided very well separated peaks of the formed diastereomers of D- and L-2-HG, with a total runtime of 5 min. The inter- and intraassays CVs for D- and L-2-HG ranged from 3.4% to 6.2%. Mean recoveries of D- and L-2-HG, evaluated on two concentrations, were 94%. Detection limit of the presented method was 20 pmol for a sample volume of 20 µL. Method comparison of the LC-MS/MS method with a gas chromatography-mass spectrometry method, in which D- and L-2-HG were derivatized with R-(-)-butanol, showed good agreement between the two methods.

Conclusions: Urinary D- and L-2-HG can be analyzed by MS/MS after derivatization with DATAN. The presented method may be suitable for the differential diagnosis of 2-HGA.