Table 1.
Optimized MS/MS settings of purines and related metabolites.
Table 2.
Optimized MS/MS settings of pyrimidines and related metabolites.
Table 3.
Assay validation using the ERNDIM „Control Purines and Pyrimidines” external quality control samples (EQC; LOT used for method development: 2015.010) (n = 10).
Table 4.
Cutoff values of the measured analytes.
Fig 1.
Results of the ERNDIM Diagnostic Proficiency Testing (DPT) samples (n = 10).
Numbers on the cylinders show how many times a given result exceeded the respective cutoff for that biomarker. PP: purine-pyrimidine disorder. Further abbreviations are provided in the List of Abbreviations Section. Note that orotidine is not pathognomonic for HGPRT (sample E04) but is accumulated as a result of allopurinol treatment.
Fig 2.
Results of urine samples from patients with known diagnosis (n = 10).
Numbers on the cylinders show how many times a given result exceeded the respective cutoff for that biomarker. Abbreviations are provided in the List of Abbreviations Section. The Y-axis has been cut at 45 for a better visibility. Note that orotidine is not pathognomonic for HGPRT (sample P05) but is accumulated as a result of allopurinol treatment.
Fig 3.
LC-MS/MS chromatograms of A: the Cal06 calibrator (16 μM) and urine specimens from B: a healthy proband; C: a patient with ADSL deficiency; D: a patient with HGPRT deficiency; E: a patient with UPB1 deficiency; and F: a patient with APRT deficiency. All intensities (Y-axes) have been normalized to 1.5*108 cps for better comparability and have been plotted against retention time (X-axes). Analytes have been numbered as follows: 1. URA; 2. DHU; 3. Thy; 4. DHT; 5. BUP; 6. ADN; 7. Hypo; 8. ALLOP; 9. BUIB; 10. Xan; 11. DHA; 12. dAdo; 13. AICAr; 14. ADS; 15. HMU; 16. Oro; 17. dUrd; 18. THD; 19. PSU; 20. dIno; 21. dGui; 22. Ino; 23. Guo; 24. Ord; 25. SAICAr; 26. SAdo. The ISs have been marked by an asterisk (*) after the number of the respective unlabeled analyte. Note that guanosine-IS has been measured in both ESI+ and ESI-. ADSL: Adenylosuccinate lyase deficiency; HGPRT: Hypoxanthine-guanine phosphoribosyltransferase deficiency; UPB1: beta-Ureidopropionase deficiency; APRT: Adenine phosphoribosyltransferase deficiency. Further abbreviations are provided in the List of Abbreviations Section. Chromatograms were generated by MassLynx 4.1 (Waters, Milford, MA, USA) and modified using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).