To show the capabilities of mass spectrometric off-line reaction monitoring, kinetic parameters for the enzymatic cleavage of minimal substrates (dinucleoside monophosphates and 2',3'-cGMP) by RNase T1 were acquired. Reactions were conducted using a fixed enzyme concentration and several different substrate concentrations. The time-concentration data obtained as described in 3.1.2 was converted into rate curves using the MARQUARDT-LEVENBERG non-linear least square fitting algorithm of gnuplot (see 2.1.3.2). To obtain MICHAELIS-MENTEN-parameters, the rate-substrate concentration data were fitted to the MICHAELIS-MENTEN equation using the same algorithm. Each assay was accompanied by a control sample without enzyme to exclude autohydrolysis or a contamination by a hydrolytic enzyme.