The application of HPLC/ESI tandem mass spectrometry on urine-soaked filter-paper strips for the screening of disorders of purine and pyrimidine metabolism

2000 ◽  
Vol 23 (4) ◽  
pp. 434-437 ◽  
Author(s):  
T. Ito ◽  
A. B. P. van Kuilenburg ◽  
A. H. Bootsma ◽  
A. J. Haasnoot ◽  
A. van Cruchten ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Filter Paper ◽  
Tandem Mass ◽  
Pyrimidine Metabolism

scholarly journals Rapid Screening of High-Risk Patients for Disorders of Purine and Pyrimidine Metabolism Using HPLC-Electrospray Tandem Mass Spectrometry of Liquid Urine or Urine-soaked Filter Paper Strips

2000 ◽  
Vol 46 (4) ◽  
pp. 445-452 ◽  
Author(s):  
Tetsuya Ito ◽  
André B P van Kuilenburg ◽  
Albert H Bootsma ◽  
Anja J Haasnoot ◽  
Arno van Cruchten ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Filter Paper ◽  
Screening Method ◽  
Urine Samples ◽  
Rapid Screening ◽  
Specific Method ◽  
Tandem Mass ◽  
Pyrimidine Metabolism ◽  
Inherited Disorders

Abstract Background: A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips. Methods: Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available. Results: Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96–107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89–110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency. Conclusions: HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.

scholarly journals Rapid diagnosis of MCAD deficiency: quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry

1997 ◽  
Vol 43 (11) ◽  
pp. 2106-2113 ◽  
Author(s):  
Donald H Chace ◽  
Steven L Hillman ◽  
Johan L K Van Hove ◽  
Edwin W Naylor
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Newborn Screening ◽  
Chain Length ◽  
Filter Paper ◽  
Tandem Mass ◽  
Mcad Deficiency ◽  
Medium Chain ◽  
Blood Spots ◽  
Medium Chain Length

Abstract We report the application of tandem mass spectrometry to prospective newborn screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. MCAD deficiency is diagnosed from dried blood spots on filter paper cards from newborns on the basis of the increase of medium chain length acylcarnitines identified by isotope dilution mass spectrometry methods. A robust and accurate semiautomated method for the analysis of medium chain length acylcarnitines as their butyl esters was developed and validated. Quantitative data from the analyses of 113 randomly collected filter paper blood spots from healthy newborns showed low concentrations of medium chain length acylcarnitines such as octanoylcarnitine. The maximum concentration of octanoylcarnitine was 0.22 μmol/L, with the majority being at or below the detection limit. In all 16 blood spots from newborns with confirmed MCAD deficiency, octanoylcarnitine was highly increased [median 8.4 μmol/L (range 3.1–28.3 μmol/L)], allowing easy detection. The concentration of octanoylcarnitine was significantly higher in these 16 newborns (<3 days of age) than in 16 older patients (ages 8 days to 7 years) with MCAD deficiency (median 1.57 μmol/L, range 0.33–4.4). The combined experience of prospective newborn screening in Pennsylvania and North Carolina has shown a disease frequency for MCAD deficiency of 1 in 17 706. No false-positive and no known false-negative results have been found. A validated method now exists for prospective newborn screening for MCAD deficiency.

scholarly journals Comprehensive Detection of Disorders of Purine and Pyrimidine Metabolism by HPLC with Electrospray Ionization Tandem Mass Spectrometry

2006 ◽  
Vol 52 (6) ◽  
pp. 1127-1137 ◽  
Author(s):  
Susen Hartmann ◽  
Jürgen G Okun ◽  
Christiane Schmidt ◽  
Claus-Dieter Langhans ◽  
Sven F Garbade ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Electrospray Ionization ◽  
Multiple Reaction Monitoring ◽  
Dihydropyrimidine Dehydrogenase ◽  
Urine Samples ◽  
Tandem Mass ◽  
Pyrimidine Metabolism ◽  
Phosphoribosyl Transferase ◽  
Ionization Tandem Mass Spectrometry

Abstract Background: Clinical presentation and disease severity in disorders of purine and pyrimidine metabolism vary considerably. We present a method that allows comprehensive, sensitive, and specific diagnosis of the entire spectrum of abnormalities in purine and pyrimidine metabolism in 1 analytical run. Methods: We used reversed-phase HPLC electrospray ionization tandem mass spectrometry to investigate 24 metabolites of purine and pyrimidine metabolism in urine samples from healthy persons and from patients with confirmed diagnoses of inherited metabolic disorders. Urine samples were filtered and diluted to a creatinine concentration of 0.5 mmol/L. Stable-isotope–labeled internal standards were used for quantification. The metabolites were analyzed by multiple-reaction monitoring in positive and negative ionization modes. Results: Total time of analysis was 20 min. Recovery (n = 8) of a compound after addition of a known concentration was 85%–133%. The mean intraday variation (n = 10) was 12%. The interday variation (n = 7) was ≤17%. Age-related reference intervals were established for each compound. Analysis of patient urine samples revealed major differences in tandem mass spectrometry profiles compared with those of control samples. Twelve deficiencies were reliably detected: hypoxanthine guanine phosphoribosyl transferase, xanthine dehydrogenase, purine nucleoside phosphorylase, adenylosuccinate lyase, uridine monophosphate synthase, adenosine deaminase, adenine phosphoribosyl transferase, molybdenum cofactor, thymidine phosphorylase, dihydropyrimidine dehydrogenase, dihydropyrimidinase, and β-ureidopropionase. Conclusion: This method enables reliable detection of 13 defects in purine and pyrimidine metabolism in a single analytical run.

scholarly journals Defects in Pyrimidine Degradation Identified by HPLC-Electrospray Tandem Mass Spectrometry of Urine Specimens or Urine-soaked Filter Paper Strips

2000 ◽  
Vol 46 (12) ◽  
pp. 1916-1922 ◽  
Author(s):  
Henk van Lenthe ◽  
André B P van Kuilenburg ◽  
Tetsuya Ito ◽  
Albert H Bootsma ◽  
Arno van Cruchten ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Electrospray Ionization ◽  
Filter Paper ◽  
Degradation Products ◽  
Degradation Pathway ◽  
Urine Samples ◽  
Tandem Mass ◽  
Pyrimidine Degradation ◽  
Ionization Tandem Mass Spectrometry

Abstract Background: Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. Methods: We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N-carbamyl-β-aminoisobutyric acid, and N-carbamyl-β-alanine. 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 degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4–4 μmol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3–12% for liquid urines and 1.0–10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3–11% (100–200 μmol/L). Recoveries were 89–99% at 100–200 μmol/L and 95–106% at 1 mmol/L in liquid urines, and 93–103% at 100–200 μmol/L and 100–106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. Conclusions: HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.

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

2004 ◽  
Vol 50 (11) ◽  
pp. 2117-2124 ◽  
Author(s):  
André B P van Kuilenburg ◽  
Henk van Lenthe ◽  
Monika Löffler ◽  
Albert H van Gennip
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Filter Paper ◽  
Urea Cycle ◽  
De Novo ◽  
Degradation Products ◽  
Urine Samples ◽  
Tandem Mass ◽  
Urea Cycle Defect ◽  
Ionization Tandem Mass Spectrometry

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.

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