analytical strategies for the identification and characterization of tropane alkaloids in plant samples using lc-ms

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Although fully synthetic molecules have become more prominent in drug discovery in recent decades, nearly two-thirds of all approved drugs from the last 39 years are natural products or natural-product-inspired,1,2&#160;underscoring the continued importance of natural products research. Alkaloids, certain nitrogen-containing natural products, are especially prized for their medicinal properties. Tropane alkaloids (TAs) containing the [3.2.1.]-bicyclic nitrogen-containing system, are produced mostly by plants in the Solanaceae (nightshade), Erythroxylaceae, and Convolvulaceae families. Examples include atropine, scopolamine, and cocaine; multiple semi-synthetic or synthetic tropanes are also used clinically3. TAs and their derivatives are used to treat many conditions3,4 and several of these drugs appear on the WHO&#39;s 2023 List of Essential Medicines5. Because of their potent activities, TAs are also used recreationally (as stimulants or deliriants) and can cause poisoning upon ingestion of plants (or preparations) that contain them6,7. TAs are undesirable in human and animal food8 and can taint teas, spices, grains, honey, and herbal supplements9,10.&#160;Because of both their medicinal promise and ability to poison, analytical methods that can aid in the discovery of new TAs (and identification of known TAs) are useful.
In tandem mass spectrometry (MS/MS), &#34;mass filters&#34; (e.g., quadrupoles, time-of-flight tubes) are coupled together physically (&#34;in-space&#34;), or an instrument employs additional &#34;in-time&#34; reaction/separation steps. In-space MS/MS uses different modes to select and fragment different ions at the different mass filters (e.g., the quadrupoles of a triple-quadrupole or QQQ instrument). These different modes can be used to determine which specific fragments are made by a given ion (product ion scan), which ions in a sample yield certain fragments (precursor ion scan or PrIS) or undergo losses of a characteristic mass (neutral loss scan or NLS), or which specific compounds possess which specific fragments (multiple reaction monitoring). MS/MS, therefore, provides fragments that are useful for proposing structures for new compounds or confirming an existing compound&#39;s presence. MS/MS is increasingly used in the drug discovery, natural products chemistry, and metabolomics fields11,12,&#160;and has been used to profile alkaloid-containing species (for phytochemical characterization or chemotaxonomic analysis) and to detect and quantify specific alkaloids in food or medicinal plants10,13,14,15,16.
Despite the many mass spectrometry techniques available, there are challenges in finding new alkaloids. In addition to finding a candidate organism to screen, a full structural confirmation of an alkaloid is an arduous process that may include many different analytical techniques. Additionally, researchers could isolate a compound that is already known, wasting labor, time, and resources. This is especially difficult for TAs, where hundreds, if not thousands of TAs, many of which are isomeric with one another, are reported. The process of &#34;identifying the knowns and distinguishing them from the unknowns&#34; is known as dereplication. Databases of the retention times (r.t.s) and mass fragments of different TAs and other compounds are published to aid with this process17,18.&#160;Nonetheless, dereplication is laborious; merely annotating (i.e., assigning putative structures to) the alkaloids in a sample&#39;s entire LC-MS/MS chromatogram is time-consuming. Recently, both molecular networking19,20 and manual dereplication18,21,22 have been used for benzylisoquinoline, monoterpene indole, and tropane alkaloids, and PrISs have been used for &#34;structural filtering&#34; of spectra to identify pyrrolizidine and solanine-type alkaloids23,24.&#160;There are no specific methods or workflows available for rapid LC-MS/MS-based dereplication of TA-containing samples, however, even though TAs possess common, easily-identifiable fragments (Figure 1). The method described here uses a combination of data-dependent (DD) product ion scans, PrISs, and NLSs to annotate and classify TA structures in plants based on both the distinct fragmentation patterns for mono-, di-, and trisubstituted tropanes (Figure 1A) and the losses of common ester groups found in these alkaloids (Figure 1B). The study organisms are several species in the nightshade genus Datura. A rich source of diverse TAs, Datura has been used&#160;throughout the world&#39;s history for medicinal and cultural purposes17- and is a challenging matrix to dereplicate because of its numerous, structurally similar TAs, providing us with appealing samples upon which to test our method.

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

In the simplest form of our research, we are trying to discover new molecules in plants. Plants are chemically complex, and some of these chemicals may be medicinally useful. We're especially interested in finding new alkaloids, which are certain molecules that contain nitrogen.Recently, we have so many new different alkaloid instruments and techniques available, so people discover more and more new molecules every day. With these instruments, we can answer questions like what chemicals are present, when are they present, and what part of the plant can they be found? Mass spectrometry as an analytical technique has really improved over the past 50 years.Instruments are more powerful, less expensive, and have higher resolution. And these techniques allow us to not only identify more plant molecules like alkaloids but also be more sure in our identification. This method is fast and sensitive.We were able to collect a full set of data on nightshade plants containing many tropane alkaloids in 60 to 90 minutes, and assign proposed structures to even though low upon those alkaloids. We're very interested in developing tandem mass spectrometry-based filtering methods for other classes of alkaloids and using methods like these to study how plant chemistry differs between different species, different plant parts, and different growth stages.

Analytical Strategies for the Identification and Characterization of Tropane Alkaloids in Plant Samples Using LC-MS

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.

analytical-strategies-for-identification-characterization-tropane

Research

JoVE Journal

Chemistry

54 Aufrufe. Untersuchen Sie nach dem LCMS-Lauf von Pflanzenwurzelextrakten das Gesamtionenchromatogramm der Q1- und Q3-Scans, einschließlich des datenabhängigen Produktionenscans. Beobachten Sie die Muttermasse von reichlich vorhandenen Ionen mit tropanalkaloidähnlichen Eigenschaften, einschließlich einer Masse unter 500 Dalton für das positiv geladene Ion, typischerweise eine gerade Anzahl. Retentionszeiten zwischen zwei und 22 Minuten und Fragmente, die mit den We...