Validation and application of volumetric absorptive microsampling (VAMS) dried blood method for phenylalanine measurement in patients with phenylketonuria.

[Display omitted] • Volumetric absorptive microsampling (VAMS) dried blood method for blood phenylalanine measurement. • Validation of bioanalytical method in compliance with regulatory guidance. • Correlation analysis of VAMS dried blood method with plasma method. • Application in clinical study to support primary efficacy endpoint. Frequent blood phenylalanine (Phe) measurement is required for phenylketonuria (PKU) patients for diagnosis and disease status monitoring. Though various methods are available for blood Phe measurement, there is a lack of validated quantitative methods for measuring Phe with less than 15% variability. A method to allow at home blood sample collection for the PKU community is in high demand. A volumetric absorptive microsampling (VAMS) dried blood collection high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method was developed and fully validated for blood Phe measurement in compliance with regulatory guidances. The method accuracy, precision, stability, selectivity, matrix and hematocrit effects were assessed. A venous plasma collection HPLC-MS/MS method was developed and validated as a reference method. 311 matching VAMS and plasma samples were collected from 24 PKU subjects in a Phase 2 clinical study. Phe measurements using the two methods were compared. Both VAMS and the plasma sample collection methods met the acceptance criteria for Good Laboratory Practice (GLP) bioanalytical analysis. Comparisons showed a high Pearson's correlation of 0.9813. The Passing-Bablok analysis showed that the difference was estimated to be less than 5% and Bland Altman analysis indicated that the difference was proportional with Phe concentration and for the majority of samples (88.85%) the measurement was within ±20% difference. Following 7 days treatment with 60 or 20 mg/kg/day PTC923 (Sepiapterin) or 20 mg/kg/day sapropterin, PKU patients exhibited respectively −206.4, −146.9, and −91.5 µmol/L reductions of blood Phe as measured by the VAMS method. Concordant results were obtained using VAMS and plasma methods, which demonstrated that VAMS is a reliable method for clinical applications to monitor blood Phe for PKU patients. [ABSTRACT FROM AUTHOR]