Phenylalanine and Tyrosine Quantification by Stable Isotope Dilution Liquid Chromatography–Mass Spectrometry from Filter Paper Blood Spots

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Abstract
Phenylketonuria (PKU) is one of the more common inborn errors of metabolism, affecting 1 in 10 000–15 000 individuals ( 1). Newborn screening for PKU is performed in all 50 states and started with the development of the bacterial inhibition screening assay by Guthrie and Susi ( 2). Frequent monitoring of blood Phe concentrations is required to maintain the concentration as close as possible to the reference interval [60–120 μmol/L (1–2 mg/dL)] to prevent damage to the brain from increased Phe concentrations and to prevent a negative nitrogen balance from an overly Phe-restricted diet. Moreover, the high risk of birth defects for the fetus of a PKU mother requires extremely frequent monitoring of blood Phe concentrations and would greatly benefit from a rapid and accurate blood spot method. Phe determination from dried blood spots is feasible by various methods: bacterial ( 2), fluorometric ( 3), liquid chromatography (LC) ( 4), and tandem mass spectrometry (MS) ( 5). Of these methods, only tandem MS allows use of a stable isotope as internal standard. Although tandem MS is the most rapid method available, the extremely high equipment cost and the need for sample derivatization are disadvantages.