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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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

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).

Read this Scientific Article Protocol about Applications of Liquid Chromatography-Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study at JoVE.com

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

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.

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 ...

applications-liquid-chromatography-tandem-mass-spectrometry-natural

Nanyang Technological University

Research

JoVE Journal

Chemistry

634 Views.  Northern Michigan University. 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.

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

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

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

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

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Biochemistry

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

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

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

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

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

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

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

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.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

Using TLC-Direct Bioautography Assay to Identify Natural Products for Biocontrol

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

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Author Spotlight: Discovering New Biopesticides from Bioactive Soil Microbe-Derived Natural Products

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...