Abstract
Objectives
To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals.
Methods
Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS).
Results
The established method is precise (intra-day CV: 1.4–6.3%; inter-day CV: 1.3–15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61–121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: −5.6 to 7.4%), are stable in thymol (variation: −8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206).
Conclusions
The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.
Funding source: National Institute for Neurological Research
Award Identifier / Grant number: LX22NPO5107
Acknowledgments
The authors would like to thank Dr. Brian Fowler (University Children Hospital Basel, Basel, Switzerland), Dr. Jorgen Bierau (Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands) and Dr. George Ruijter (Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands) for kindly providing confirmed pathological samples for method validation.
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Research funding: The work of Václava Škopová and Marie Zikánová was supported by the Cooperation Program, research area Medical Diagnostics and Basic Medical Sciences and the programme National Institute for Neurological Research (Programme EXCELES, ID Project No. LX22NPO5107) funded by the European Union – Next Generation EU.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: The local Institutional Review Board deemed the study exempt from review.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2022-1236).
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