Fast Diagnosis of Neonatal Phenylketonuria by Gas Chromatography-Mass Spectrometry Following Microwave-Assisted Silylation

Abstract

Phenylketonuria (PKU) is a fairly common autosomal recessive disease. Phenylalanine (Phe) and tyrosine (Tyr) are the biomarkers of PKU, and it can be diagnosed by the measurement of Phe and Tyr in neonatal blood samples. A fast diagnostic procedure for neonatal PKU has been developed using microwave-assisted silylation followed by gas chromatography-mass spectrometry. Amino acids extracted from neonatal blood samples are rapidly derivatized with N, O-bis(trimethylsilyl)-trifluoroacetamide under microwave irradiation; the derivatives are then analyzed by GC-MS. The silylation conditions have been optimized, and the method validated for linear range, detection limit, precision and recovery. The proposed method is linear from 20 to 800 µM, detection limit is less than 0.35 µM, relative standard deviation (RSD) is less than 9.0%, and recoveries of 87% and 83% were obtained for Phe and Tyr. The proposed method was tested by the determination of Phe and Tyr in blood spots from eight PKU-positive neonates and twelve control neonates. Microwave irradiation considerably accelerates the derivatization reaction of amino acid with BSFTA, and shortens the whole analysis time. Microwave-assisted silylation coupled to GC-MS is a powerful tool for fast screening of neonatal PKU.