A: Urinary nucleosides
It is known that some modified nucleosides originating from RNA degradation are excreted in abnormal levels in the urine of patients with malignant tumours and they have been proposed as tumour markers. Their measurement could provide a non-invasive diagnostic method, helpful for the identification of different cancers and in the monitoring of therapeutic effects.
In this work the development of a fast on-line multi-column HPLC-ESMS(/MS) system for the selective sample clean up, preconcentration and subsequent analysis of the major and minor (modified) ribonucleosides in human urine will be the main objective. The high sensitivity and selectivity of miniaturised LC-MS(/MS) methods will provide us the means to isolate and detect unknown nucleosides. For the structure elucidation of those compounds MS/MS spectra will be generated and subsequently interpreted. If necessary further (spectroscopic) steps will be undertaken to offer assurance about the proposed structures.
As the proposed analytical method tolerates direct administration of urine samples, it opens the opportunity for a relatively fast screening of samples. So we will use the method to generate urinary nucleoside patterns of both healthy volunteers and cancerpatients. Those patterns will be evaluated in order to discriminate, based on nucleoside concentrations, between healthy men and those suffering from ovarian, prostate or abdominal cancer.
A lot of nucleoside-analogues, which are intracellular metabolized to active nucleotide-analogues, are used as antiviral and cytostatic therapeutics. Therefore higher demands are placed on the technology for the analysis of nucleotides. In addition to this it may be necessary for studies of cell metabolism to separate, determine and quantify mixtures of nucleoside mono-, di- and triphosphates. A lot of chromatographic procedures for the analysis of nucleotides -all based on UV or radiometric detection- were developed in the past. However in order to acquire highly selective structural information the development of a sensitive HPLC-MS(/MS) system is required. Due to the incompatibility of mass spectrometry with the originally proposed separation methods, the development of new LC-MS compatible separations was warranted. Ion-suppression HPLC (-) ESMS and ion-pair HPLC (-) ESMS have been used for the analysis of selected analytes. Yet, a more universally applicable analytical procedure would be useful for future studies concerning the purity and biochemical/medical behaviour of nucleotide-analogues. Special concern will go to a selective on-line sample clean up and preconcentration of the nucleotides. The use of a miniaturized LC-ESMS system equipped with a column-switching technique will result in an amplified mass and concentration sensitivity.