Monitoring tyrosinaemia type I: Blood spot test for nitisinone (NTBC)

Quantification of nitisinone, 2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione (NTBC) has been repeatedly described. Nevertheless monitoring of NTBC has not yet become part of routine therapy surveillance in tyrosinaemia type I (OMIM 276700). We developed a blood spot test to facilitate collection and transport of samples. Furthermore, the test material can be used for determination of other parameters like tyrosine and succinylacetone.

For quantification of NTBC in blood spots filter paper discs of 3.2mm diameter were extracted with 150 μL methanol containing mesotrione as internal standard (IS). Analysis was done by UPLC-MS/MS on a Xevo mass spectrometer (ESI+), (MRM). Parent ions were 330.05 for NTBC and 340.05 for IS, daughter ions were m/z 217.95 and m/z 125.95 for NTBC, and m/z 227.95 and m/z 103.95 for IS.

The calibration curve for NTBC in blood spots was linear from 0.1 μmol/L to 100 μmol/L. Recovery exceeded 73.1%, CV intraday and interday were below 9.6%. Instrumental run time was 2.5 min. Sensitivity of the method was 0.1 μmol/L. NTBC concentrations in plasma were higher than in blood spots by a factor of 1.56 ± 0.13.

As demonstrated in patients with tyrosinaemia type I quantification of NTBC by UPLC-MS/MS in blood spots is feasible and gives valuable information for monitoring NTBC treatment.