Abstract The methodology for the detection of picogram quantities of nucleotides directly from TLC plates without the use of radioactive labeling has been developed. The method couples thin-layer chromatography (TLC) separation with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) detection. The TLC/MALDI coupling protocol was studied and optimized for the separation and detection of deoxyribonucleotides. Several ammonia based solvents were examined as potential extraction solvents for the TLC/MALDI coupling protocol. It was found that in order to obtain maximum MALDI signal intensity and minimal analyte spreading, the extraction solvent should produce Rf-values for the analytes in the range of 0.30.4. Rf-values above this range led to extensive analyte spreading and those below this range resulted in poor extraction. Various MALDI matrices and co-matrices were investigated, the best results were obtained using 2,4,6-trihydroxyacetophenone (THA) as a matrix. The extraction solvent chosen was an ammonium hydroxide/methanol (100 mM/30%, Rf = 0.280.38) solvent system which was found to provide the best sensitivity, minimal lateral spreading and excellent matrix transfer. Using the optimized coupling protocol, the detection limits for the deoxyribonucleotide monophosphates were established at or better than 10 picograms.
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