Nils Janzen1, Michael Terhardt2, Stefanie Sander2, Mübeccel Demirkol3, Gülden Gökçay3, Michael Peter2, Thomas Lücke4, Johannes Sander2, Anibh M Das5. 1. Screening-Labor Hannover, Hannover, Germany; Department of Neuropediatrics, Children's Hospital, Ruhr University of Bochum, Bochum, Germany; Department of Clinical Chemistry, Hannover Medical School, Hannover, Germany. Electronic address: Janzen.Nils@mh-hannover.de. 2. Screening-Labor Hannover, Hannover, Germany. 3. Children's Hospital, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey. 4. Department of Neuropediatrics, Children's Hospital, Ruhr University of Bochum, Bochum, Germany. 5. Paediatric Metabolic Medicine, Clinic for Paediatric Kidney, Liver- and Metabolic Diseases, Hannover Medical School, Hannover, Germany.
Abstract
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.
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.