After the LCMS run of plant root extracts, examine the total ion chromatogram of the Q1 and Q3 scans, including the data-dependent product ion scan. Observe for parent mass of abundant ions with tropane alkaloid-like features, including a mass under 500 Daltons for the positively charged ion, typically, an even number. Retention times between two to 22 minutes, and fragments matching master charge values consistent with tropane alkaloids.
Examine the precursor ion scan chromatogram or channel specifically for master charge 124, and determine which peaks or ions at which retention times produce this specific fragment. Click scan by scan, through the chromatogram and review the full MS-MS spectra from the data-dependent product ion scan, especially for lower abundant species. Next, analyze the precursor ion scan chromatograms together for master charge 122 and 140, which indicate disubstituted tropane alkaloids, and master charge 138 and 156, indicative of trisubstituted tropane alkaloids.
Examine the neutral loss scan chromatogram or channels for master charge 160 and 166. Identify which peaks or ions produce neutral losses and note their retention times. Click scan by scan through the chromatogram and compare with the data-dependent product ion scan fragmentation, especially for lower abundant species.
Use the combination of precursor ion scan and neutral loss scan data, supported by data-dependent product ion scan results, to make putative annotations of the observed alkaloids. Start with the smallest tropane mass, then add the neutral loss, and account for any remaining mass. Compare the alkaloid annotations against those reported in the literature to determine the tropane alkaloid substitution pattern.
Additionally, use commercially available standards of common tropane alkaloids for confirmation. The full Q1 scan chromatogram of Datura metel root extract revealed diverse tropane alkaloids with varying abundances. Features for master charge 124, 122, 140, 138, and 156 indicated the presence of tropane alkaloids.
Many of these tropane alkaloids were acetylated, tigloylated, or derived from phenolactic or tropic acid, as evidenced by the signals in the neutral loss scan chromatograms. Spectral data utilization enabled the annotation of specific alkaloids, notably identifying a compound with apparent mass of master charge 224. The method allowed for the deduction of a compound structure through its fragmentation pattern and neutral loss, confirming the presence of tigloyl groups as substitutions.
The examination of a methanol water extract from Datura stramonium seeds yielded a full Q1 scan base peak chromatogram, identifying a highly abundant monosubstituted tropane alkaloid, likely hyoscyamine. The high resolution MS-MS spectrum also allows the identification of new alkaloids from Datura stramonium seeds.
