Development of a new UPLC-ESI-MS/MS method for the determination of biopterin and neopterin in dried blood spot.

Background: (6R)-5,6,7,8-tetrahydrobiopterin (BH4) deficiencies are rare inherited defects of synthesis or regeneration of BH4. Due to the resulting hyperphenylalaninemia (HPA), some of them are detected by newborn screening and require the assessment of the pattern of neopterin (Neo) and biopterin (Bio) excretion in urine to be confirmed. Aim of present study was to develop a method for the measurement of these diagnostic biomarkers in dried blood spot (DBS).

Methods: After DBS extraction, samples were filtered and injected into the UPLC column coupled with a tandem mass spectrometer working in positive electrospray ionization.

Results: The chromatographic separation was accomplished in 6min. The LoQ was 0.57 and 1.45nmol/l of blood for Neo and Bio respectively and the response was linear over the range 0-100nmol/l of blood. The within- and between-day imprecision was <6.4 and 10.8% respectively. Reference ranges for newborns, infants and children/adult were established. The method was tested in 11 patients affected by BH4 defects.

Conclusions: The assessment of Neo and Bio in DBS is reliable and sensitive and may be proposed as a second tier test for the newborns with hyperphenylalaninemia (HPA) as well as a new potential diagnostic tool for symptomatic subjects with BH4 deficiencies.