Title

High-throughput Intracellular Pteridinic Profiling by Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry

Abstract

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reproducibility (2.1-5.4% RSD), method detection limits (0.1-3.0µgL-1) and limits of quantitation (0.1-1 µg L-1), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.

Department(s)

Chemistry

Keywords and Phrases

A549; HPLC-QTOF MS; Pteridine Extraction; Pteridines; Pteridinic Profiling; Diagnosis; Drug Products; Extraction; High Performance Liquid Chromatography; Liquids; Limits Of Quantitations; Liquid Chromatography-quadrupole Time-of-flight Mass Spectrometries; Method Detection Limits; Non Small Cell Lung Cancer; Pteridines; Pteridinic Profiling; Mass Spectrometry; 6 Biopterin; 6 Formylpterin; 6 Hydroxylumazine; 6 Hydroxymethylpterin; 6 Methylpterin; 6,7 Dimethylpterin; 6,7 Dimethyltetrahydropterin; 7 Hydroxylumazine; 7,8 Dihydroxanthopterin; Biolumazine; Dihydrobiopterin; Dihydropterin; Isoxanthopterin; Lumazine; Pteridine Derivative; Pterin; Pterin Derivative; Sepiapterin; Tetrahydrobiopterin; Tetrahydropterin; Unclassified Drug; Xanthopterin; Pteridine Derivative; A549 Cell Line; Cell Culture; Cell Lysate; Concentration (Parameters); Extraction; High Performance Liquid Chromatography; Human; Human Cell; Limit Of Detection; Limit Of Quantitation; Non Small Cell Lung Cancer; Quadrupole Mass Spectrometry; Quantitative Analysis; Reproducibility; Time Of Flight Mass Spectrometry; Validation Process; Chemistry; High Performance Liquid Chromatography; Intracellular Space; Isolation And Purification; Procedures; Tandem Mass Spectrometry; Tumor Cell Line; Chromatography, High Pressure Liquid; Humans; Intracellular Space; Pteridines; Tandem Mass Spectrometry

International Standard Serial Number (ISSN)

0003-2670

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2015 Elsevier, All rights reserved.


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