Fig. 4 The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the number of matrix layers (A) and the spraying nozzle height (B) for the positive ionization mode, rat spinal cord tissue slices (matrix: 25 mg/ml DHB (50% MeOH, 0.2% TFA))
Considering the ions intensities, the highest increase in those parameters was observed between 10 and 12 layers (see: Fig. 3,4). The parameters for the 12 and 14 layers were similar and did not lead to the further spectra enhancement. The same trend is observed with the s/n value. Thus, we decided that 12 layers would be optimal for the measurements since it allows to save the chemical reagents and time for tissue preparation. The spraying nozzle height of 5 mm gives the highest intensity for observed peaks. For s/n ratio the 5 mm and 10 mm positions give the maximum value, but the intensity itself is higher for 5 mm. Thus, considering both parameters, the 5 mm position was recommended for the DHB matrix in the positive ionization mode.
Norharmane (6 mg/ml matrix (chloroform: MeOH:H2O) – positive ionization mode)
Norharmane works differently than DHB matrix. For s/n ratio, 12 and 14 layers show high value, but 14 matrix layers are optimal considering the peaks intensities only (see: Fig. 5,6). Regarding the position of the spraying nozzle above the measured sample, the most intensive peaks are obtained both at the highest (z=1) and the lowest position of the spraying nozzle (Z=25), preferably with the lowest position. The same is valid for the s/n ratio.