Journal of Chromatography A, 1218 (2011) 9055–9063 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma Development and validation of a hydrophilic interaction chromatography–tandem mass spectrometry method with on-line polar extraction for the analysis of urinary nucleosides. Potential application in clinical diagnosis Encarnación Rodríguez-Gonzalo ∗ , Diego García-Gómez, Rita Carabias-Martínez Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Química, Universidad de Salamanca, 37008 Salamanca, Spain a r t i c l e i n f o Article history: Received 22 July 2011 Received in revised form 22 September 2011 Accepted 5 October 2011 Available online 12 October 2011 Keywords: Restricted-access material (RAM) Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) On-line sample extraction Bioanalytical method validation Urine Cancer diagnosis a b s t r a c t 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 spectrom- etry. 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 identifica- tion 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 Legisla- tion 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 valida- tion 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 vol- unteers and cancer patients using Principal Component Analysis, Hierarchical Cluster Analysis and Soft Independent Modeling of Class Analogy are also shown. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The degradation products of nucleic acids form a particular class of compounds present in human urine [1,2]. Modified nucleosides are widely known as the metabolites of ribonucleic acids (RNA), in particular transfer-RNA (tRNA). Simple structural modifications such as base or ribose methylation, base isomerization, reduction, thiolation or deamination are the most frequent [3]. This primary source of modified nucleosides is enhanced by the presence of dif- ferent types of tumours [3]. These modified nucleosides cannot be reutilized or further degraded but are excreted in the urine as intact molecules. Therefore, any malignant disease, such as can- cer or metabolic imbalances affecting RNA breakdown or turnover, alters the urinary levels of excreted modified nucleosides. More than 93 modified nucleosides have been reported in urine for all forms of RNA [3]. In recent decades, modified nucleosides excreted ∗ Corresponding author. Tel.: +34 923 294483; fax: +34 923 294 574. E-mail address: erg@usal.es (E. Rodríguez-Gonzalo). in human urine have been studied to examine their biomedical sig- nificance as possible biomarkers of cancer and other degenerative diseases [4,5]. Another type of important biomarker for cancers is dam- aged deoxyribonucleic acid (DNA) and its products/metabolites, especially 8-hydroxy-2 ′ -deoxyguanosine (8OH2dG). Among all the different types of DNA damage, oxidative damage by reactive oxygen species is considered to be one of the most important con- tributors to diseases such as cancer, aging, heart disease and other age-related diseases. Of more than 20 known products of oxida- tive DNA damage [6] 8OH2dG has been identified as a biomarker for a few types of cancers, including breast, lung and liver can- cer [7]. Because 8OH2dG is excreted in urine without any further metabolism [8], the determination of urinary 8OH2dG levels has been considered as a non-invasive method for the diagnosis of cancer. Different analytical techniques have been reported for the determination of modified nucleosides, immunoassay [9], capil- lary electrophoresis [10] and, mainly, liquid chromatography [11] being the most widely employed. However, most of the proposed 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.10.016