Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants

Abstract

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Fig. 1

Structures of compounds.

Fig. 2

LC/MS and MS/MS analysis of plant natural products and detection using different ionization modes and polarities. A, Total ion current obtained with a mixture of authentic standards in ESI(pos) (upper panel), ESI(neg) (second panel from top), APCI(pos) (third panel from top) and APCI(neg) (lower panel). The elution order of the compounds in the standard mixture were (note that these are not the compound numbers used in the text): (1) isoalantolactone, (2) (6b-acetyl-13a-hydroxy-4a,6a-dimethyl-13-oxo-1,2,3,4,4b,5,6,7,8,9,10,10a,11,11a,11b,12-hexadecahydroindeno[2,1-a]phenanthren-2-yl) acetate, (3) 2-[1-(1H-benzimidazol-2-yl)-3-(4-methylphenyl)propan-2-yl]-1H-benzimidazole, (4) 2-[1-(1H-benzimidazol-2-yl)-3-(2-methoxyphenyl)propan-2-yl]-1H-benzimidazole, (5) skimmianine, (6) evoxine, (7) bucharaine, (8) foliosidine, (9) 9-methoxy-2,2-dimethyl-6H-pyrano[3,2-c]quinolin-5-one, (10) methyllycaconitine, (11) 2-piperidin-1-ylethyl 10-acetyloxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1H-picene-2-carboxylate, and (12) [1,3,4a-trimethyl-4,4a,9,9a-tetrahydro-1H-9,2-(epoxymethano)xanthen-2(3H)-yl]methyl phenylcarbamate. B, MS/MS spectra of peak # 5 (skimmianine) at different collision energies; 10 eV (upper panel), 20 eV (middle panel); 40 eV (lower panel). An annotation of detected fragments is provided in the MS/MS spectral panels.

Fig. 3

Flow diagram for the development of an accurate mass – time tag (AMT tag) database. For details see text. Acronyms: LC, liquid chromatography; MFG, molecular formula generation; MS, mass spectrometry.

Fig. 4

Flow diagram for the searching of an accurate mass – time tag (AMT tag) database using Spektraris or MassBank. For details see text. Acronyms: LC, liquid chromatography; MFE, molecular feature extractor; MS, mass spectrometry.

Fig. 5

Workflow of accurate mass – time tag (AMT tag) database search using the Spektraris tool (http://langelabtools.wsu.edu/amt/). (A) Screenshot showing the search options and a sample data set (bottom). Executing the query brings up a new window with a summary of search results (B). Clicking on an individual compound hit opens a window with relevant compound information in MassBank format (C).

Fig. 6

Quantitation of (A) andrographolide (26) in tissue extracts (harvest date shown as days after germination) obtained from Andrographis paniculata and (B) forskolin (13) in tissue extracts obtained from mature Plectranthus barbatus plants. Photos of representative plants are provided (C, Andrographis paniculata; D, Plectranthus barbatus).