Analysis of botanicals and botanical supplements by LC-MS/MS-based molecular networking: Approaches for annotating plant metabolites and authentication

Abstract

Prior to the advent of modern medicine, humans have used botanicals extensively for their therapeutic potential. With the majority of newly approved drugs having their origins in natural products, plants remain at the forefront of drug discovery. Continued research and discovery necessitate the use of high-throughput analytical methods to screen and identify bioactive components and potential therapeutic molecules from plants. Utilizing a pre-generated plant extract library, we subjected botanicals to LC-MS/MS-based molecular networking to determine their chemical composition and relatively quantify already known metabolites. The LC-MS/MS-based molecular networking approach was also used to authenticate the composition of dietary supplements against their corresponding plant specimens. The networking procedures provided concise visual representations of the chemical space and highly informative assessments of the botanicals. The procedures also proved to define the composition of the botanical supplements quickly and efficiently. This offered an innovative approach to metabolite profiling and authentication practices and additionally allowed for the identification of new, putatively unknown metabolites for future isolation and biological evaluation.

Keywords: Ashwagandha; Black cohosh; Echinacea; LC-MS/MS; Molecular networking.

Figure 1.

Annotation of antioxidant metabolites from plant specimens. Chromatography fractions from plant extracts possessing antioxidant activity were subjected to LC-MS/MS-based molecular networking. Nodes are colored by plant and labeled with their precursor m/z values. Identifications of an anthocyanin from elderberry and aloin from aloe are shown. Node size is relative to ion count.

Figure 2.

Alkylamide molecular network. Pie slices indicate the presence of analytes in various plant parts. The structures of two putative alkylamides are shown near their respective nodes.

Figure 3.

Comparison of black cohosh metabolite content between extract and chromatography fractions in the triterpenoid glycoside cluster. Metabolites observed in fractions that were not initially observed in the crude seed extract are designated by black boxes behind the nodes.

Figure 4.

Distribution of alkylamides in chromatography fractions. Networking shows that the majority of alkylamides were present in the 20% H₂O: 80% CH₃OH fraction.

Figure 5.

Identification of 23-epi-26-deoxyactein in black cohosh botanical supplements. The panel on the left shows the comparison of the authentic standard to both black cohosh supplements extracting M+Na⁺ m/z 683. The panel on the right shows the network of the precursor m/z 683 molecule (black star) clearly present in the standard (orange) and both supplement samples (light blue and yellow) as well as in the black cohosh plant sample (dark blue).

Figure 6.

Analysis of ashwagandha metabolites in supplements and plant parts. The top panel shows LC-MS/MS analysis of the withaferin A standard. The middle panels show the composition of analytes with m/z 471 in an ashwagandha supplement sample. These metabolites were identified as withaferin A, and putatively as withanone and withanolide A. The inset shows retention times for the analytes. The table shows the MZmine filtering process based on m/z values (471) and retention times for these analytes with gray indicating presence and white indicating absence in samples. Std = withaferin A standard; Ash = ashwagandha; Suppl = supplement; BC = black cohosh; Ech = Echinacea.