Development, Validation, and Clinical Application of an HPLC-Based Method for Determining Adenosine Deaminase...

Adenosine deaminase (ADA) deficiency is a rare genetic condition leading to severe combined immunodeficiency. Timely and accurate quantification of ADA activity is crucial for both diagnosis and treatment monitoring. This study describes the development and analytical validation of a high-performance liquid chromatography (HPLC)-based method for measuring ADA activity in dried blood spot (DBS) samples.

ADA activity was quantified by measuring the enzymatic conversion of adenosine to inosine and hypoxanthine, followed by chromatographic separation and detection. Method validation was conducted according to current bioanalytical guidelines and included assessment of selectivity, linearity, sensitivity, precision, trueness, extraction recovery, and stability. The study involved 3 groups of samples taken from healthy pediatric donors (n = 116), carriers of pathogenic mutations (n = 13), and patients with genetically confirmed ADA deficiency (n = 11).

The assay demonstrated high selectivity and linearity over the tested concentration ranges (coefficient of determination > 0.99 for both analytes). The lower limit of quantification was 0.312 μg/mL (2.29 μmol/L) for hypoxanthine and 0.5 μg/mL (1.86 μmol/L) for inosine. A reference interval for ADA activity in DBS was established at 14.94 to 43.28 nmol/h/mg. The mean ADA activity was 25.06 nmol/h/mg of protein in the control group, 14.08 nmol/h/mg in mutation carriers, and 2.18 nmol/h/mg in patients with ADA deficiency.

The developed method allows precise and reliable determination of ADA activity in DBS. It can be applied for the diagnosis and monitoring of patients with ADA deficiency, as well as in scientific studies related to purine metabolism.