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

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. We developed a rapid and precise blood-spot method for Phe and Tyr determination using stable isotope dilution reversed-phase LC-MS. The advantages of this method are the very short sample preparation without derivatization, a short chromatographic run, the use of selective detection to eliminate interference, and the use of ideal internal standardization (stable isotope). The method described herein is now used routinely in our PKU clinic. l-Phe ( M r 165.19) and l-Tyr ( M r 181.19) were purchased from Sigma Chemical Co. Stable isotopes [ring-d5]-l-phenylalanine ( M r 170.23) …