Analysis of Pyrimidine Synthesis “de Novo” Intermediates in Urine and Dried Urine Filter- Paper Strips with HPLC–Electrospray Tandem Mass Spectrometry

Author:

van Kuilenburg André B P1,van Lenthe Henk1,Löffler Monika2,van Gennip Albert H3

Affiliation:

1. Academic Medical Center, University of Amsterdam, Emma Children’s Hospital and Departments of Clinical Chemistry, Amsterdam, The Netherlands

2. Philipps-University, Institute for Physiological Chemistry, Marburg, Germany

3. Academic Hospital Maastricht, Departments of Clinical Genetics and Clinical Chemistry, Maastricht, The Netherlands

Abstract

Abstract Background: The concentrations of the pyrimidine “de novo” metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC–tandem mass spectrometry. Methods: Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. Results: All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4–3 μmol/L. The intra- and interassay variation for urine with added compounds was 1.2–5% for urines and 2–9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97–106% for urine samples and 97–115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls. Conclusion: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.

Publisher

Oxford University Press (OUP)

Subject

Biochemistry, medical,Clinical Biochemistry

Cited by 33 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3