Name: Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study (Video) | JoVE Journal
Uploaded: 2024-03-08T14:50:00.000+00:00
Duration: 9 min 36 s
Description: Learn LC-MS/MS methods for rapid annotation and classification of tropane alkaloids in plant extracts. This technique enables dereplication and discovery of novel nitrogen-containing compounds with medicinal potential.

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5 minutes

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.

CAUTION: Please consult all relevant material safety data sheets (MSDS) before using the listed chemicals.
1. Sample preparation
CAUTION: Liquid nitrogen can cause cryogen burns. Use cryogen gloves and eye protection in a well-ventilated area. Alkaloid-containing plant samples can be irritating to the skin; always handle them with gloves. Methanol is toxic and flammable and should be handled in a fume hood away from potential ignition sources.
NOTE: In theory, cultivated or wild plant tissue can be used (dried or ground fresh); the below procedure is just that utilized during method development.
<ol>
	<li>If the plant tissue of interest is fresh, freeze it by placing it in a polypropylene conical tube and immersing it in liquid nitrogen for 2-3 min.</li>
	<li>Place the frozen plant tissue into a pre-chilled mortar (in a polystyrene cooler with liquid nitrogen), and using a pre-chilled pestle, grind the tissue to a uniform powder.</li>
	<li>Quickly weigh the desired amount of tissue into a tared polypropylene microcentrifuge tube using a pre-chilled spatula, and immediately add 20% methanol (at room temperature [RT]) at a concentration of 1 mL per every 100 mg of tissue. 
	NOTE: Methanol (20%) is commonly used to extract TAs25. Occasionally, 0.1% formic acid is added, although no difference in extraction efficiency was observed during the development of this method and others.</li>
	<li>Place the capped tubes on a rocking shaker (medium speed) for a minimum of 3 h at RT.</li>
	<li>Centrifuge the tubes at 9464 x g for 10 min. Pipette off the supernatant into an LC-MS autosampler vial, or, if still cloudy, filter through a 0.45 &#181;m syringe filter first. 
	NOTE: The protocol can be paused here, although processed fresh plant samples and extracts should be stored in a -80 &#730;C freezer prior to analysis to avoid any potential alkaloid degradation.</li>
</ol>
2. LC-MS instrument configuration and data collection
CAUTION: Acetonitrile is toxic and flammable; keep away from ignition sources and control vapors using a fume hood. Formic acid is corrosive; avoid skin and eye contact and wear appropriate personal protective equipment.
<ol>
	<li>Use an LC-MS instrument with an electrospray ionization (ESI) source and a reversed-phase HPLC column (C18, 4.6 x 100 mm).</li>
	<li>For HPLC, use 0.1% formic acid in H2O as Solvent A and 0.1% formic acid in acetonitrile as Solvent B; equilibrate the column with 99% A and 1% B. Configure a 30 min gradient of 1%-50% B over 26 min, returning to 1% B at 26.01 min, and holding at 1% B for 4 min. Use a column oven temperature of 45 &#730;C and a flow rate of 0.5 mL/min.</li>
	<li>In the LC-MS method, use the following operating parameters for the mass spectrometer: interface voltage: 4.0 kV, nebulizing gas flow: 3 L/min, heating gas flow: 10 L/min, DL temperature: 250 &#730;C, heat block temperature: 400 &#730;C, interface temperature: 300 &#730;C, drying gas flow: 10 L/min, and collision-induced dissociation (CID) gas (argon) pressure of 17 kPa.</li>
	<li>Build an MS method in ESI positive mode that includes both a Q3 scan and a Q1 scan (100-1000 Da) that functions as a survey event (set to the length of the LC method), with automatic isotope exclusion enabled. Include a product ion scan as a dependent event of the Q1 scan (DD analysis), with a mass window of 50-1000 Da, a collision cell energy of -20 V, and an event time of &#60;0.2 s. 
	NOTE: The count threshold for triggering Q1&#39;s DD product ion scan can be variable, but typically, a level of 7,000-10,000 counts is used. On some instruments, such as the one used to develop the method, the Q3 scan provides greater mass accuracy and sensitivity than Q1 and is included to confirm ions observed in the Q1 scan, although it can be omitted from step 2.4 without issue.</li>
	<li>To the above MS method, add positive-mode PrISs equal to the length of the LC method with collision cell energies of -20 V and event times of 0.75 s. Make sure in all cases, Automatic Isotope Exclude, or De-isotoping functions are enabled. Ensure that the m/z values of interest for TA fragments (in Da, see Figure 1A) are 124.1 (for monosubstituted TAs, mass window of 125-1000 Da), 122.1 and 140.1 (for disubstituted TAs, mass windows beginning at 123 and 141 Da, respectively), and 156.1 and 138.1 (for trisubstituted TAs, mass windows beginning at 157 and 139 Da, respectively). 
	NOTE: During method development, the PrIS were split over two different methods, one for mono- and disubstituted TAs and one for trisubstituted TAs, although they can be split up or combined any way.</li>
	<li>Build a second MS method that includes the parameters in step 2.4.
	<ol>
		<li>To that MS/MS method, add positive-mode NLS equal to the length of the LC method, with collision cell energies of -20 V and event times of 0.75 s. Make sure in all cases, Automatic Isotope Exclude or De-isotoping functions are enabled.</li>
		<li>Ensure that the neutral loss masses of interest for esters on TAs (in Da, see Figure 1B) are 100.05 (for esters derived from tiglic acid, mass window of 110-1000 Da), 60.03 (for acetyl groups, mass window of 100-1000 Da), and 166.06 (phenyllactic or tropic acid esters, mass window of 170-1000 Da).</li>
	</ol>
	</li>
	<li>Download the LC-MS method and create data files for the samples of interest. Once the HPLC column is equilibrated at 45 &#730;C, run the samples of interest in a batch or project file with appropriate extraction solvent blanks between different species or tissue types. A typical injection volume is 10-20 &#181;L. 
	NOTE: At especially high volumes, the mass spectrometer detector may be saturated. If running very concentrated samples, be sure that the ESI source is cleaned and the instrument is tuned regularly. The protocol can be paused here after all data is collected.</li>
</ol>
3. Data analysis
<ol>
	<li>Examine the total ion chromatogram of the Q1 and Q3 scans (and the DD product ion scan), and note the parent mass of any abundant ions which have TA-like features: a) mass &#60;500 Da for the [M+H]+ ion, usually an even-mass, b) typical r.t.s between 2-22 min using the above LC method, and c) fragments from the following list: m/z 93, 124, 142, 140, 122, 138, 156, 174, 110, or 128 Da.</li>
	<li>Examine the PrIS chromatogram/channel for m/z 124, and note which peaks/ions are at which r.t.s (recorded in step 3.1) produce this fragment. Click scan-by-scan through the chromatogram as well as examine the full MS/MS spectra obtained in the DD product ion scan, especially for lower-abundance species. 
	NOTE: A true species containing this fragment will exist for multiple scans and appear in both the Q1 and Q3 scans (if the latter is used). A spreadsheet is attached in the Supporting Information (Supplementary File 1) to aid with&#160;annotations.</li>
	<li>Repeat step 3.2 with the other PrIS chromatograms. As both m/z 122 and 140 are indicative of disubstituted TAs and both m/z 138 and 156 are indicative of trisubstituted TAs, examine these chromatograms/channels together.</li>
	<li>Examine the NLS chromatogram/channels for m/z 100, 60, and 166, and note which peaks/ions at which r.t.s (recorded in step 3.1) produce these neutral losses. As with the PrIS, click scan-by-scan through the chromatogram and compare with the fragmentation obtained in the DD product ion scan, especially for lower-abundance species.</li>
	<li>Using the combination of PrIS and NLS data, supported by the DD product ion scan results, make putative annotations of the observed alkaloids by adding the smallest tropane mass (e.g., 124, 122, or 138) and the neutral loss and then accounting for the remaining leftover mass. 
	NOTE: Typical groups substituting TAs (and their masses in Da) are hydroxyl (18), acetyl (60), propionyl (74), isobutyryl (88), tigloyl (100), saturated tigloyl/2-methylbutyryl (102), or phenyllactate/tropic acid (166).</li>
	<li>Compare annotations for alkaloids to those reported in the literature17 and databases (e.g., MoNA)26 to determine which TA substitution patterns are reported (and which are potentially novel). Additionally, use standards of some common tropane alkaloids (e.g., atropine, littorine, scopolamine) that are commercially available for confirmation.</li>
	<li>For additional structural information for low-abundance samples (the PrIS and NLS may pick up ions below the dependent event thresholds), collect a specific product ion scan (utilizing the mass of interest as the precursor ion) on a more concentrated sample. 
	NOTE: This method was developed on a low-resolution QQQ instrument. For all putative new compounds, a high-resolution MS instrument should be used to obtain accurate mass spectra.</li>
</ol>

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

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<li>Christen, P., Cretton, S., Humam, M., Bieri, S., Muñoz, O., Joseph-Nathan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemistry+and+biological+activity+of+alkaloids+from+the+genus+Schizanthus%5BTitle%5D)+AND+%22Phytochem+Rev%22%5BJournal%5D)">Chemistry and biological activity of alkaloids from the genus Schizanthus.</a> Phytochem Rev. 19, 615-641 (2020).</li>
<li>Lounasmaa, M., Tamminen, T. The Tropane Alkaloids. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLounasmaa%2C+M+%22The+Alkaloids%3A+Chemistry+and+Pharmacology%22">The Alkaloids: Chemistry and Pharmacology</a>. , Academic Press. New York, NY. (1993).</li>
</ol>

The authors have no conflicts of interest to disclose.

Although&#160;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 how the method is used. &#34;Deep&#34; metabolic profiling or dereplication should use a larger injection volume (15-20 &#181;L was used in this study), while simple detection of alkaloids could use smaller volumes. Care should be taken when using large sample volumes to (a) keep the ion source cleaned regularly, (b) monitor the HPLC column for pressure changes or clogs (an appropriate guard column should always be used), (c) prevent carry-over between samples, and (d) examine chromatograms for potential &#34;isotope leakage&#34; as described above. The interface temperatures and voltages should not be changed significantly from those specified in the method to avoid premature in-source fragmentation. Additionally, although a collision cell voltage of 20 V is recommended, it may not provide optimal chromatogram signal (or diagnostic DD product ion scan fragments) for all TAs. Some MS instruments and functions have a &#34;collision energy ramping&#34; feature, or the ability to collect scans at multiple or optimized collision energies, which could also find utility in a method like this30.
This MS method itself is also readily modifiable: while tiglic, acetic, and phenyllactic/tropic acid esters are common in Datura species, the TA ester substitution patterns may be different in other plants or samples. The NLS masses can be changed to detect other ester groups (e.g., a loss of 74 Da for propionic esters) or potentially detect glycosylations (e.g., a loss of 162 Da for hexoses). The PrIS masses can also be changed. For example, a TA in both Datura samples (r.t. = 12.5 min; see Supplementary File 1 and Supplementary File 2) were annotated as norhyoscyamine or norlittorine (hyoscyamine/littorine minus the tropane N-methyl group) based on its DD product ion scan fragments (m/z 110 and 93) and match to the literature spectra31; this TA did not appear on any of the tropane fragment PrIS channels. A channel could be added for these two masses to detect nortropanes. The m/z 93 fragment is also present in most monosubstituted TAs17; adding this channel could provide additional confirmation for a monosubstituted TA&#39;s presence and help reduce false positives.
In addition to its adaptability for different types of TAs, one major advantage of this protocol (over existing alkaloid annotation methods) is its speed. Depending on how the MS method is configured (and the HPLC run time used), all MS/MS data for a sample can be collected within 60-90 min. For example, during our studies, the mono- and di-substituted TA PrISs were built into one method, the trisubstituted TA PrISs were in another method, and the NLSs were in a third (all methods contained the DD product ion scan), for a total of three injections at thirty minutes apiece. Complete annotation of the sample utilizing the combined MS/MS data is much faster than manual examination of every peak or collecting individual product ion scans for numerous peaks of interest. This method can be performed on any instrument with product ion scan, PrIS, and NLS modes and can be used for rapid qualitative assessment (i.e., are TAs present or absent?), for distinguishing or &#34;fingerprinting&#34; different plant genera, species, varieties or cultivars, organs, or growth stages, and for comparing observed TAs to those reported (as part of a dereplication workflow). As demonstrated, the protocol can also be used for the discovery of new TAs as leads for isolation, purification, or bioassay, as well as identification of candidate species and tissues to isolate them from. This method could be used in the natural products and metabolomics fields, including to aid in understanding plant biochemistry and for early-stage drug discovery investigation. Additional applications could be found in food safety or forensics (e.g., detection of TAs in food, supplements, or biofluids).
The method does have several drawbacks that warrant discussion. First, depending on the instrument used and the purpose of employing the protocol, a large sample injection volume could be required, which could cause instrument performance issues and the potential for false positives in some cases (as described above). Second, this method was developed on a low-resolution QQQ instrument, and false positives could result from the low resolution (e.g., the feature containing a m/z 124 fragment in the negative&#160;control that is not a TA). A higher-resolution instrument would provide better diagnostic ability, and accurate-mass MS/MS spectra should be obtained for any proposed new alkaloid. Additional drawbacks to this method are simply the disadvantages of MS/MS analysis as a whole. While MS/MS is useful for proposing structures (&#34;annotation&#34; as designated by the Metabolomics Standards Initiative32), it does not provide concrete assignments of molecular connectivity (&#34;identification&#34;). For example, in both Datura samples, we found&#160;several isomers of a m/z 306 TA, which seemed to have both m/z 124 and m/z 140 fragments (Supplementary File 2), thus making definitive assignment of the substitution pattern difficult. This could represent a yet-undiscovered fragmentation mechanism, and another technique (such as nuclear magnetic resonance spectroscopy) may be helpful in resolving ambiguities. MS/MS-based methods frequently struggle with isomerism: the phenyllactic acid ester littorine (or its derivatives) is difficult to distinguish from hyoscyamine (tropic acid ester), and other Solanaceous plants contain TAs substituted with the isomeric tigloyl, senecioyl, and angeloyl groups33,34; in the absence of standards, these compounds might be difficult, if not impossible, to tell apart. The method also will not differentiate enantiomers (e.g., 3,6- vs. 3,7-dihydroxytropane; see Figure 1 for numbering) or regioisomers (e.g., distinguishing 3-acetyl from 7-acetyl without a standard). We have assigned most monosubstituted tropanes as 3-substituted based on prior patterns reported in the genus Datura and other Solanaceous plants17,35.&#160;Ergo, any biochemical knowledge or literature about the organism studied (if available) should always be consulted alongside any instrumental analysis.

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.

<table><tbody><tr><td>Acetonitrile, For UHPLC, suitable for mass spectometry</td><td>Sigma-Aldrich</td><td>900667</td><td>HPLC solvent</td></tr><tr><td>Argon gas</td><td>AirGas</td><td>AR UHP300</td><td>CID gas</td></tr><tr><td>Formic acid, 99% for analysis</td><td>Thermo Scientific</td><td>AC270480010</td><td>HPLC additive</td></tr><tr><td>Guard column holder</td><td>Restek</td><td>25812</td><td></td></tr><tr><td>HPLC, Shimadzu LC-2030C 3D Plus</td><td>Shimadzu</td><td>228-65802-58</td><td>HPLC column</td></tr><tr><td>LCMS, Shimdazu LCMS-8045</td><td>Shimadzu</td><td>225-31800-44</td><td>Mass spectrometer; we ran LabSolutions software, which is standard for Shimadzu instruments</td></tr><tr><td>Liquid nitrogen</td><td>AirGas</td><td>NI 180LT22</td><td></td></tr><tr><td>Methanol, for HPLC/UHPLC/LCMS</td><td>VWR</td><td>BDH 85800.400</td><td>For making extraction solvent</td></tr><tr><td>Microcentrifuge&amp;nbsp;</td><td>VWR</td><td>2400-37</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.5 mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Mortar&amp;nbsp;</td><td>Fisher Scientific</td><td>FB961C</td><td>For grinding plant tissues</td></tr><tr><td>Pestle</td><td>Fisher Scientific</td><td>FB961M</td><td>For grinding plant tissues</td></tr><tr><td>Pipette 1000 mL</td><td>Gilson&amp;nbsp;</td><td>F144059M</td><td></td></tr><tr><td>Pipette tip 1000 mL</td><td>Fisher scientific</td><td>02-707-404</td><td></td></tr><tr><td>Plant tissues</td><td>Various sources</td><td>N/A</td><td>Can be anything wild or cultivated</td></tr><tr><td>Polypropylene conical tubes, 15 mL</td><td>Fisher Scientific</td><td>05-539-4</td><td></td></tr><tr><td>Polystyrene cooler</td><td>ULINE</td><td>S-18312</td><td>The type of coolers that reagents for molecular biology are shipped in would be appropriate</td></tr><tr><td>Roc C18 3 &amp;micro;m, 100 mm x 4.6 mm</td><td>Restek</td><td>9534315</td><td>HPLC column</td></tr><tr><td>Roc C18, 10 mm x 4 mm</td><td>Restek</td><td>953450210</td><td>Guard column</td></tr><tr><td>Rocking shaker</td><td>Themo Scientific</td><td>11-676-680</td><td></td></tr><tr><td>Screw thread vial convenience kit (9 mm)</td><td>Fisher scientific</td><td>13-622-190</td><td>LCMS autosampler vials</td></tr><tr><td>Syringe, 3 mL</td><td>Fisher Scientific</td><td>03-377-27</td><td></td></tr><tr><td>Syringe filter 0.45 &amp;micro;m&amp;nbsp;</td><td>Avantor/VWR</td><td>76479-008</td><td></td></tr><tr><td>Water, for use in liquid chromatography and mass spectrometry</td><td>JT Baker</td><td>9831-03</td><td>For making extraction solvent</td></tr><tr><td>Water solution, contains 0.1% v/v formic acid, For UHPLC, suitable for mass spectometry</td><td>Sigma-Aldrich</td><td>900687-1L</td><td>HPLC solvent</td></tr></tbody></table>

applications of liquid-chromatography tandem mass spectrometry in natural products research: tropane alkaloids as a case study

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

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).

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.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

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Research

JoVE Journal

Chemistry

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

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Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

This study developed a high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ-MS) method to identify and quantify tropane alkaloids (hyoscyamine, anisodamine, scopolamine) in Anisodus tanguticus. The method proved rapid, accurate, and reliable for high-throughput analysis of medicinal plants.

An Efficient and Rapid HPLC-QQQ-MS Method for the Quantitation of Tropane Alkaloids in Medicinal Plants

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism.  This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile.  This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue.  A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

Learn LC-MS/MS methods for rapid annotation and classification of tropane alkaloids in plant extracts. This technique enables dereplication and discovery of novel nitrogen-containing compounds with medicinal potential.

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

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