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  • Titill er á ensku Validation of an Ultraperformance Liquid Chromatography Method for Measuring Phenylalanine and Tyrosine in Dried Blood Spots
  • Bakkalár
  • Útdráttur er á ensku

    Introduction: Inborn errors of metabolism (IEM) are a collective group of rare genetic disorders that have a high combined incidence and are associated with considerable morbidity. In the United States, many of these conditions are screened for at birth. Phenylketonuria (PKU) is the most common IEM and is characterized by an inability to metabolize the essential amino acid (AA) phenylalanine. PKU patients require lifelong monitoring of blood phenylalanine levels, which is usually performed by chromatographic analysis of plasma from venous blood. However, this represents a moderately invasive approach and often presents logistical challenges for patients living in geographically-isolated areas. Alternatively, a more convenient approach for PKU patients may be offered by collection of capillary whole blood onto filter paper at home, allowing sample transport to the laboratory by regular mail for AA analysis. This study sought to validate a previously published method for quantitative analysis of AAs in dried blood spots (DBS) using ultraperformance liquid chromatography (UPLC).
    Methods: A previously published method for AA analysis in DBS using UPLC was validated in the clinical core laboratory at Boston Children’s Hospital (BCH). The assay was validated for precision by replicate analysis of DBS samples containing known concentrations of phenylalanine and tyrosine. Intra-assay precision was determined by analyzing samples, across two concentrations, in replicate (n = 10) in a single analytical run. Inter-assay precision was determined by replicate analysis (n = 5) of DBS samples for phenylalanine and tyrosine across two concentrations over five consecutive days. A method comparison study was performed by analysis of phenylalanine and tyrosine in DBS samples (n = 30) and their corresponding plasma samples using blood from PKU patients collected for routine blood phenylalanine monitoring.
    Results: Intra-assay precision, expressed as coefficient of variation (%CV), was 9.8% and 9.6% for phenylalanine and tyrosine, respectively. Inter-assay precision, also expressed as %CV was 15.6% for phenylalanine, and 13.8% for tyrosine. Linear regression analysis demonstrated excellent correlation between DBS and plasma for both phenylalanine (r2 =0.964) and tyrosine (r2 =0.918), with slope values of 0.15 and 0.115, respectively. Bland-Altman analysis revealed a clear negative proportional bias for both amino acids, with a mean bias of -197.9 for phenylalanine and -58.1 for tyrosine across the range of concentrations tested.
    Conclusions: The DBS method performs with acceptable precision for both phenylalanine and tyrosine across the range of clinically-relevant concentrations. Measurement of phenylalanine and tyrosine in DBS samples holds promise for clinical monitoring of PKU patients on treatment. This method has the potential for expansion to additional AA, increasing its application for other aminoacidopathies and genetic metabolic disorders. This method is now available for implementation in the clinical chemistry laboratory at Boston Children’s Hospital.

  • 20.5.2019

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