FIA-MS/MS Method Validated for Dried Blood Spot Amino Acid Monitoring in Phenylketonuria Management
A flow injection analysis tandem mass spectrometry method was validated for simultaneous monitoring of phenylalanine, tyrosine, leucine, and valine in dried blood spots from PKU patients, replacing the enzymatic method limited to phenylalanine alone. The assay achieved precision and accuracy below 15% CV with excellent correlation to the enzymatic reference. Notably, 50% of PKU patients showed low tyrosine, leucine, or valine concentrations that would have gone undetected by the previous single-analyte method, improving dietary management.
The original study
Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management.
- Authors
- Bruno C, Dufour-Rainfray D, Patin F, Vourc'h P, Guilloteau D, Maillot F, et al.
- Journal
- Clinical biochemistry
- Type
- Journal Article, Validation Study
- PMID
- 27450222
Original abstract
OBJECTIVES: Phenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method. DESIGN AND METHODS: We ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method. RESULTS: We developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method. CONCLUSION: Based on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method.