Development, validation, and application of bioanalytical methods for phenylketonuria and tyrosinemia diagnosis and monitoring

Abstract

The aim of this study is to develop and validate fast, accurate, and precise, high-performance liquid-with UV detection (HPLC-UV) and liquid chromatography tandem mass spectrometry (LC-MSMS) methods for phenylketonuria (PKU) and tyrosinemia (TRS) diagnosis and monitoring. Three methods were developed, HPLC-UV on blood serum, LC-MSMS on blood serum and LC-MSMS on dry blood spots (DBS). Chromatographic conditions in the HPLC-UV technique were optimized using central composite experimental design (CCD). Validation was accomplished using β-expectation tolerance intervals (β-ETI) for total error measurement that did not exceed 15%. The method applicability was determined using human serum from 442 volunteers and statistical study was conducted to investigate the effect of different factors in phenylalanine (Phe) and tyrosine (Tyr) levels in addition to comparative study with reference method. Optimal settings for HPLC-UV method were determined. The accuracy profiles were established. Mean analytical bias in spiking levels was acceptable, with relative standards deviation (RSD) below 5% in almost instances. The limit of (LOD) and limit of quantification (LOQ) were satisfactory in all methods. The epidemiologic statistical study reveals the most influencing factors on Phe and Tyr concentrations in the blood serum. The suggested approach analyzed Phe and Tyr in human blood samples either using HPLC-UV or LC-MSMS and presented no significant difference with the reference method. The developed methods offer valuable diagnosis and monitoring tool for PKU and TRS.