No abstract

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.

An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS 4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H 2 O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H 2 O by particular fragments of guanosine was experimentally proven by using argon, saturated with H 2 18 O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H 2 O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed. , and nucleoside levels in urine have the potential to act as cancer biomarkers [2][3][4][5]. As their occurrence is both ubiquitous and quantitatively varying [6,7] their unequivocal characterization is crucial.For guanine alone over 20 natural variants are known to occur in RNA [6], which makes structural identification a challenging task. In our earlier LC-ESI-MS/MS experiments of urinary nucleosides [8], unexpected product ions of guanosine and its methylated derivatives were detected. The presence of these ions prompted us to perform additional mass spectrometric studies since their origin was not straightforward. This report provides an in-depth study of the fragmentation behavior of guanosine under lowenergy CID conditions, revealing H 2 O addition to specific fragments.We also wish to demonstrate the use of a Q-TOF mass spectrometer in the collection of the equivalent of MS 4 data, a level of information normally acquired only in ion trap-or FT-MS n experiments [9,10]. This was achieved by making optimal use of "in

The development of an on-line automated SPE-HPLC--ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.