Conclusion
We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.
Methods
Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays.
Results
The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean.