Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.
Keywords: dried blood spots; newborn screening; phenylalanine; phenylketonuria; tyrosine.