Quantitative determination of plasma phenylalanine and tyrosine by electrospray ionization tandem mass spectrometry.
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 micromol/L and the carry-over less than 1%. The assay was linear to 3000 micromol/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.
Hardy
D T
DT
Institute of Child Health, Division of Reproductive and Child Health, University of Birmingham, UK. David.Hardy@bhamchildrens.wmids.nhs.uk
Hall
S K
SK
Preece
M A
MA
Green
A
A
D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D015997:Neonatal Screening; D010649:Phenylalanine / Q000097:blood*; D010661:Phenylketonurias / Q000097:blood* / Q000175:diagnosis; D011786:Quality Control; D012016:Reference Values; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014443:Tyrosine / Q000097:blood*
D014443:Tyrosine; D010649:Phenylalanine