Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study
Summary
We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.
Abstract
Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known – a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).
Introduction
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 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'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. 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), "mass filters" (e.g., quadrupoles, time-of-flight tubes) are coupled together physically ("in-space"), or an instrument employs additional "in-time" 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's presence. MS/MS is increasingly used in the drug discovery, natural products chemistry, and metabolomics fields11,12, 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 "identifying the knowns and distinguishing them from the unknowns" 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. Nonetheless, dereplication is laborious; merely annotating (i.e., assigning putative structures to) the alkaloids in a sample'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 "structural filtering" of spectra to identify pyrrolizidine and solanine-type alkaloids23,24. 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 throughout the world'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.
Protocol
Representative Results
Discussion
Although the instrument parameters provided in the protocol allow for satisfactory performance, the successful use of this method may require careful attention to or optimization of several critical steps. While the HPLC solvent gradient provided in step 2.2 is generally appropriate for tropane alkaloids, it may need to be modified depending on the tropane alkaloid profile of the sample or plant species being examined. The sample injection volume can also be changed depending on the sensitivity of the instrument and…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by a Faculty Research Grant (Northern Michigan University, awarded to M.A.C.), an undergraduate research fellowship (Northern Michigan University, awarded to J.C), and the Department of Chemistry. The authors wish to thank John Berger (NMU) for assistance with plant tissue preparation, Hannah Hawkins (NMU) for LC-MS maintenance and troubleshooting assistance, and Dr. Ryan Fornwald and his CH 495 (Natural Products Synthesis) students for their preparation the acetyltropine mix. The authors also wish to thank Dr. Daniel Jones (Michigan State University) for acquiring high-resolution MS/MS spectra.
Materials
| Acetonitrile, For UHPLC, suitable for mass spectometry | Sigma-Aldrich | 900667 | HPLC solvent |
| Argon gas | AirGas | AR UHP300 | CID gas |
| Formic acid, 99% for analysis | Thermo Scientific | AC270480010 | HPLC additive |
| Guard column holder | Restek | 25812 | |
| HPLC, Shimadzu LC-2030C 3D Plus | Shimadzu | 228-65802-58 | HPLC column |
| LCMS, Shimdazu LCMS-8045 | Shimadzu | 225-31800-44 | Mass spectrometer; we ran LabSolutions software, which is standard for Shimadzu instruments |
| Liquid nitrogen | AirGas | NI 180LT22 | |
| Methanol, for HPLC/UHPLC/LCMS | VWR | BDH 85800.400 | For making extraction solvent |
| Microcentrifuge | VWR | 2400-37 | |
| Microcentrifuge tubes, 1.5 mL | Fisher Scientific | 05-408-129 | |
| Mortar | Fisher Scientific | FB961C | For grinding plant tissues |
| Pestle | Fisher Scientific | FB961M | For grinding plant tissues |
| Pipette 1000 mL | Gilson | F144059M | |
| Pipette tip 1000 mL | Fisher scientific | 02-707-404 | |
| Plant tissues | Various sources | N/A | Can be anything wild or cultivated |
| Polypropylene conical tubes, 15 mL | Fisher Scientific | 05-539-4 | |
| Polystyrene cooler | ULINE | S-18312 | The type of coolers that reagents for molecular biology are shipped in would be appropriate |
| Roc C18 3 µm, 100 mm x 4.6 mm | Restek | 9534315 | HPLC column |
| Roc C18, 10 mm x 4 mm | Restek | 953450210 | Guard column |
| Rocking shaker | Themo Scientific | 11-676-680 | |
| Screw thread vial convenience kit (9 mm) | Fisher scientific | 13-622-190 | LCMS autosampler vials |
| Syringe, 3 mL | Fisher Scientific | 03-377-27 | |
| Syringe filter 0.45 µm | Avantor/VWR | 76479-008 | |
| Water, for use in liquid chromatography and mass spectrometry | JT Baker | 9831-03 | For making extraction solvent |
| Water solution, contains 0.1% v/v formic acid, For UHPLC, suitable for mass spectometry | Sigma-Aldrich | 900687-1L | HPLC solvent |
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