An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples

Abstract

Background: Branched chain amino acid (BCAA) analysis is needed for the diagnosis and management of patients with maple syrup urine disease (MSUD). We report an improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of BCAAs and Allo-Ile in dried blood spot (DBS) samples. Methods: BCAAswere extracted from a 3 mmblood spot intomethanol/water containing stable isotope internal standards. Eluents were dried and reconstituted in the mobile phase. Gradient elution was performed on an Acquity™ BEH C 18 (100 × 2.1 mm, 1.7 μm) column. BCAAs were detected and quantified in the multiple reaction monitoring mode in a five-minute analysis. Results: The assay was calibrated to give best possible alignment with plasma results. Retrospective analysis of newborn DBSs from six classic MSUD patients showed elevated alloisoleucine (Allo-Ile) in all cases. Two of four patients with mild disease had normal values; the other two had significant elevations in Allo-Ile. Conclusions: Analysis of BCAA in DBS by UPLC-MS/MS is a useful second tier newborn screening test to identify classical MSUD and for monitoring of remote patients.