A fast high performance liquid chromatographic (HPLC) analysis of amino acid phenylketonuria disorder in dried blood spots and serum samples, employing C18 monolithic silica columns and photo diode array detection

Abstract

A gradient high-performance liquid chromatography-photo diode array detection (HPLC-PDA) method applying a monolithic RP-C18 column was developed to facilitate the detection of amino acid phenylketonuria disorder within 6 min. Phenylalanine (Phe) and tyrosine (Tyr) extracted from a dried blood spot (DBS) and serum protein precipitated by perchloric acid (5% v/v) were injected directly. The proposed method offers sufficient chromatographic resolution (>2), a wide range of linearity (0.1–3200 µM), good accuracy (98.3–103.5 for DBS samples and 88.2–104.2 for the serum samples) and an acceptable precision (relative standard deviation of <9.0%). The statistical comparison of results, i.e. the obtained Phe and Tyr level in the DBSs and serum samples, did not show any significant difference. The applicability of the proposed method for screening newborns and monitoring of a diet therapy in PKU patients was examined by analysing 127 DBSs and 22 serum samples and confirmation of the results was done using liquid chromatography-mass spectrometry. The use of a monolithic column with gradient elution proved to be more suitable for a simple and fast analysis in screening newborns and economical, green and patient friendly for the regular monitoring of Phe and Tyr in PKU patients.