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 conditions^3,4 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 them^6,7. TAs are undesirable in human and animal food⁸ and can taint teas, spices, grains, honey, and herbal supplements^9,10. 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 fields^11,12, 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 plants^{10,13,14,15,16}.

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 process^17,18. 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 networking^19,20 and manual dereplication^18,21,22 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 alkaloids^23,24. 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

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.

1. 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.
2. 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.
3. 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.
4. Place the capped tubes on a rocking shaker (medium speed) for a minimum of 3 h at RT.
5. 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 µm syringe filter first.
   NOTE: The protocol can be paused here, although processed fresh plant samples and extracts should be stored in a -80 ˚C freezer prior to analysis to avoid any potential alkaloid degradation.

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.

1. Use an LC-MS instrument with an electrospray ionization (ESI) source and a reversed-phase HPLC column (C18, 4.6 x 100 mm).
2. 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 ˚C and a flow rate of 0.5 mL/min.
3. 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 ˚C, heat block temperature: 400 ˚C, interface temperature: 300 ˚C, drying gas flow: 10 L/min, and collision-induced dissociation (CID) gas (argon) pressure of 17 kPa.
4. 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 <0.2 s.
   NOTE: The count threshold for triggering Q1'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.
5. 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.
6. Build a second MS method that includes the parameters in step 2.4.
   1. 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.
   2. 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).
7. Download the LC-MS method and create data files for the samples of interest. Once the HPLC column is equilibrated at 45 ˚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 µL.
   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.

3. Data analysis

1. 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 <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.
2. 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 annotations.
3. 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.
4. 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.
5. 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).
6. 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.
7. 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.

Results

To demonstrate the method's effectiveness, a standard mix of TAs (10 µg/mL each of an acetyltropine/acetylpseudotropine mix [monosubstituted], 10 µg/mL each of a mixture of two anisodamine isomers [disubstituted], along with hyoscyamine [monosubstituted], littorine [monosubstituted], and scopolamine [trisubstituted]) was analyzed as a positive control (Figure 2). A full Q1 scan chromatogram (displayed in the base peak chromatogram view) is shown in Figure 2...

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...

Materials

Name	Company	Catalog Number	Comments
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