Towards newborn screening for ornithine transcarbamylase deficiency: fast non-chromatographic orotic acid quantification from dried blood spots by tandem mass spectrometry.

BACKGROUND

Orotic acid (OA) is the key parameter in the detection of ornithine transcarbamylase deficiency (OTC-D). Inclusion of OA into newborn screening compatibility with existing analytical procedures is necessary.

METHODS

OA was eluted from dried blood spots with methanol containing deuterated [1,3-(15)N2] OA as internal standard. Quantification by tandem mass spectrometry was accomplished without chromatographic separation. Samples were measured in MRM mode for the masses m/z 155.1 → 111 for OA and 157.1 → 113 for d2 OA.

RESULTS

OA was determined in a wide range of concentrations with high precision, LOD and LOQ being 0.21 and 0.65 μmol/L, respectively. Values correlated well with those obtained after chromatography. Pretreatment of samples with HCl-butanol regularly used for acylcarnitine measurement did not significantly affect quantitative results. Inclusion of the new method into the standard newborn screening procedure did not alter the results for acylcarnitines or amino acids; the total time per analysis, however, was increased from 1.15 to 1.85 min. OA levels of 707 unaffected newborns ranged from 0.28 to 3.73 μmol/L. Five newborns with OTC-D showed concentrations of 89.7-211.1 μmol/L. In newborns with severe citrullinaemia we found values in the range of 4.99-127.7 μmol/L.

CONCLUSIONS

This new method can be used as a standalone measurement of OA but it can also easily be implemented into standard newborn screening techniques as a useful supplement. In this case the method allows detection of newborns with OTC deficiency without an extra analytical run.