Background
Phenylketonuria patients on protein-restricted diets require monitoring of their blood phenylalanine concentrations. Small sample volumes are essential for neonates and infants, with the need for results to be generated in a timely manner by robust methods. We present a tandem mass spectrometry assay for phenylalanine (and tyrosine) which fulfils these criteria.
Methods
Plasma samples were deproteinized using a methanolic solution of phenylalanine-d5 and tyrosine-d4 that served as the internal standards for the assay. Following centrifugation each liquid phase was dried in a well of a microtitre plate and the amino acids converted to butyl esters using butanolic hydrogen chloride. After drying again and reconstituting in 80% acetonitrile in water, the samples were analysed using electrospray ionization tandem mass spectrometry.
Results
For both phenylalanine and tyrosine the limit of detection was 1 μmol/L and the carry-over less than 1%. The assay was linear to 3000 μmol/L in both amino acids (phenylalanine: r = 0·999; tyrosine: r = 0·992). Inter-sample precision varied with concentration but was 3·4-5·2% for phenylalanine and 4·3-7·6% for tyrosine. Between-batch precision was more variable, being 4·1-12·9% for phenylalanine and 8·4%-10·5% for tyrosine.
Conclusions
Good agreement was found between the assay and results from existing ion-exchange chromatography and high performance liquid chromatographic assays in this department. Carry-over and precision were acceptable for monitoring work and the linearity allowed most specimens to be analysed without dilution. Where available, tandem mass spectrometry offers a convenient means of transferring the routine work for both neonatal screening and monitoring to one instrument.
