Low nanogram detection of nucleotides using fast atom bombardment-mass spectrometry

Abstract

The effect of trimethylsilyl (TMS) derivatization on detection limits of mononucleotides in fast atom bombardment-mass spectrometry (FAB-MS) was examined. FAB-MS methods were developed to optimize sensitivity using adenosine 5'-monophosphate as a model compound and then applied to reference standards of two clinically important nucleotides: tricyclic nucleoside-5'-monophosphate (TCNMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The detection limit for the TMS derivative of TCNMP was 2.5-5 ng/microliters and less than 2.5 ng/microliters for FdUMP as its TMS derivative. This is greater than two orders of magnitude more sensitive than the FAB-MS analysis of the corresponding free compounds. These low detection limits for the TMS derivatives were obtained using a narrow scan range, signal averaging, detection in the negative ion mode, and 3-nitrobenzyl alcohol as the matrix. Hydrolysis of one or more of the labile TMS groups did occur, with the extent of hydrolysis being greatest in the more protic matrices.

Development of fast atom bombardment mass spectral methods for the identification of carcinogen-nucleoside adducts

“…The utility of each component approach for the analysis of these or related compounds by mass spectrometery has been previously reported. The method incorporates the use of CNL scans for screening [30,42], TMS derivatization for sensitivity enhancement (31)(32)(33), and daughters of the BH! ion for structure characterization (31].…”

“…Moser and Wood [24] and Weng et aI. [32] have reported comparable detection limits (250-500 ng) for unmodified nucleotides by FAB/MS using p-toluenesulfonic acid/glycerol or glycerol as matrix compounds.…”

“…This ion is attributed to loss of the disilylated deoxynbose moiety from those bissilylated adduct protonated molecules having both TMS groups attached to the sugar. In a related study, Schram and co-workers [32] reported a method for the analysis of low nanogram levels of clinically important nucleotide-monophosphates by FAB/MS. Derivatization was accomplished in capillary tubes by using small volumes of BSTFA in pyridine.…”

“…For example, the adduct reported in ref 27, N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was correctly identified after FAB/MS analysis of the silylated derivative but not by using EI/MS. Recent results from Schram's laboratory [32,33] show surprisingly low level detection for silylated derivatives of related compounds, including ribonucleosides and ribonucleotide monophosphate derivatives by FAS/MS. This approach is analogous to the electrophore derivatization methods used to enhance the detection of alkylated nucleosides and bases using NIO mass spectrometry [34], or electron capture GC [35].…”