Analysis of nucleotides directly from TLC plates using MALDI-MS detection

The methodology for the detection of picogram quantities of nucleotides dir ectly from TLC plates without the use of radioactive labeling has been deve loped. The method couples thin-layer chromatography (TLC) separation with m atrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) det ection. The TLC/MALDI coupling protocol was studied and optimized for the s eparation and detection of deoxyribonucleotides. Several ammonia based solv ents were examined as potential extraction solvents for the TLC/MALDI coupl ing protocol. It was found that in order to obtain maximum TLC/MALDI signal intensity and minimal analyte spreading, the extraction solvent should pro duce R-f-values for the analytes in the range of 0.3-0.4. R-f-values above this range led to extensive analyte spreading and those below this range re sulted in poor extraction. Various MALDI matrices and co-matrices were inve stigated, the best results were obtained using 2',4',6'-trihydroxyacetophen one (THA) as a matrix. The extraction solvent chosen was an ammonium hydrox ide/methanol (100 mM/30%, R-f = 0.28-0.38) solvent system which was found t o provide the best sensitivity, minimal lateral spreading and excellent mat rix transfer. Using the optimized coupling protocol, the detection limits f or the deoxyribonucleotide monophosphates were established at or better tha n 10 picograms.