Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1218, Issue 50, Pages 9055-9063Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2011.10.016
Keywords
Restricted-access material (RAM); Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC); On-line sample extraction; Bioanalytical method validation; Urine; Cancer diagnosis
Funding
- Ministerio de Ciencia e Innovacion, Spain [CTQ2008-02200/BQU]
- Junta de Castilla y Leon [GR-65]
- Ministerio de Educacion, Spain
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The present paper describes the development, validation and application of a quantitative method for the determination of endogenous nucleosides and nucleobases in urine based on the on-line coupling of a solid-phase extraction step with hydrophilic interaction chromatography-tandem mass spectrometry. The method combines the use of a highly polar restricted-access material (RAM), based on an N-vinylacetamide copolymer, for efficient analyte extraction and matrix removal, with separation by zwitterionic hydrophilic interaction chromatography (ZIC-HILIC), that revealed a satisfactory retention of the polar analytes studied. Detection using a triple quadrupole analyser allowed reliable identification and high-sensitivity quantitation of the target compounds. The on-line configuration developed, RAM-ZIC-HILIC-MS/MS, provides a convenient approach to automate the application to urine analysis, with minimum sample manipulation. The whole method was validated according to European Legislation for bioanalytical methods. The validation steps included the verification of matrix effects, calibration curve, precision, accuracy, selectivity, stability and carry-over in real samples. The results of the validation process revealed that the proposed method is suitable for the reliable determination of nucleosides and nucleobases in human urine, showing limits of detection from 0.1 to 1.3 ng mL(-1). The application to clinical samples was also checked: the results obtained in analyses of urine samples from healthy volunteers and cancer patients using Principal Component Analysis, Hierarchical Cluster Analysis and Soft Independent Modeling of Class Analogy are also shown. (C) 2011 Elsevier B.V. All rights reserved.
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