Mass Spectrometry For Natural Product Discovery: © 2020 Elsevier Ltd. All Rights Reserved

6.
12 Mass Spectrometry for Natural Product Discovery

Katherine B Louie, Suzanne M Kosina, Yuntao Hu, Hiroshi Otani, Markus de Raad, Andrea N Kuftin, Nigel J Mouncey, Benjamin P
Bowen, and Trent R Northen, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, United States
© 2020 Elsevier Ltd. All rights reserved.
6.12.1 Introduction and History 264

6.12.2 Analytical Workflows for Natural Product Discovery 265
6.12.2.1 Mass Spectrometry Workflow 269
6.12.3 Extraction 269
6.12.3.1 Extraction Solvents 269
6.12.3.2 Conventional Extraction Methods 273
6.12.3.2.1 Liquid-liquid, solid-liquid and solid phase extraction (SPE) 273
6.12.3.2.2 Maceration, infusion, percolation and decoction 273
6.12.3.2.3 Reflux extraction 274
6.12.3.2.4 Soxhlet extraction 274
6.12.3.3 Non-conventional Extraction Methods 274
6.12.3.3.1 Ultrasound assisted extraction (UAE) 275
6.12.3.3.2 Microwave-assisted extraction (MAE) 276
6.12.3.3.3 Pressurized liquid extraction (PLE) 276
6.12.3.3.4 Supercritical fluid extraction (SFE) 276
6.12.4 Separation and Fractionation 276
6.12.4.1 Liquid Chromatography 276
6.12.4.1.1 Reverse phase liquid chromatography (RPLC) 277
6.12.4.1.2 Normal phase chromatography (NPLC) 277
6.12.4.1.3 Hydrophilic interaction liquid chromatography (HILIC) 277
6.12.4.1.4 Ion exchange chromatography (IEX) 278
6.12.5 Ionization and Detection of Compounds 278
6.12.5.1 Mass Spectrometry Sources 278
6.12.5.1.1 ESI 278
6.12.5.1.2 APCI 278
6.12.5.1.3 DESI 279
6.12.5.1.4 MALDI 279
6.12.5.1.5 SALDI 280
6.12.5.2 Mass Analyzers 280
6.12.5.2.1 Quadrupole 280
6.12.5.2.2 Ion trap 280
6.12.5.2.3 Time-of-flight (TOF) 280
6.12.5.2.4 Fourier transform-ion cyclotron resonance (FT-ICR) 280
6.12.5.2.5 Orbitrap 281
6.12.5.2.6 Tandem mass analyzers 281
6.12.5.2.7 IM-MS 281
6.12.5.3 Data Collection 281
6.12.6 Detection of Select Natural Product Classes 282
6.12.6.1 Polyketides 282
6.12.6.2 Glycosides 284
6.12.6.3 Nonribosomal Peptides (NRPs) 284
6.12.6.4 Alkaloids 284
6.12.6.5 Flavonoids 287
6.12.6.6 Terpenoids/Terpenes 287
6.12.7 Mass Spectrometry Imaging of Natural Products 287
6.12.7.1 MSI Platforms 289
6.12.7.2 MSI Applications 290
6.12.7.2.1 Natural products from interacting organisms 290
6.12.7.2.2 High-throughput screening 292
6.12.8 Identification and Dereplication of Natural Products 292
6.12.8.1 Considerations in Sample Preparation and Experimental Design 293
6.12.8.2 Stable Isotope Labeling 293
6.12.8.3 Dereplication of Known Compounds 294
Comprehensive Natural Products III: Chemistry and Biology https://doi.org/10.1016/B978-0-12-409547-2.14834-6 2636.12.5

6.12.8.4 Identification of Unknown Compounds 296

6.12.9 Genome Integrated Natural Product Discovery and Analysis 296
6.12.10 Outlook 299
References 300
6.12.1 Introduction and History
Natural products chemistry is focused on small organic molecules produced by organisms, especially microbes and plants,
as secondary metabolites. They have proven to be rich sources of medically- and industrially-important bioactive molecules. The
discovery of penicillin, the first commercially used antibiotic, by Alexander Fleming from the culture broth of Penicillium
chrysogenum in 1929 opened up a decades-long era of natural product discovery.1 Since then, many natural products have been
successfully commercialized including antibiotics, for example, the anti-tuberculosis drug streptomycin, the anti-parasite com-
pound avermectin, the broad spectrum spinosyn insecticides, and artemisinin, a drug against malaria, and many more. The mid-
20th century witnessed the “Golden Age” of antibiotics discovery. Historically, scientists screened culture broths or extracts of
microbes and plants for desired activities, such as antimicrobial properties. This was followed by compound isolation, activity
screening, and structural analysis. Although this approach was effective in finding natural products with desired activities, the
discovery of new natural products became harder and harder due to the increasing rediscovery rate of the same compounds after
laborious screening, purification and identification steps. Nevertheless, recent advancements in genomics technologies have
revealed a treasure trove of natural products that remain to be discovered and characterized. Hence, analytical technologies have
also been developed to unveil the chemical world of living organisms, the “parvome” named by Julian Davies,2 and distinguish
known metabolites from novel compounds.
Mass spectrometry technologies have proven invaluable for characterizing a diversity of organic compounds including natural
products. A major initial application for mass spectrometry was in the petroleum industry. In early days, the major method of
ionization of analytes was by electron ionization (EI). Here, analytes are vaporized under high vacuum and pass through a beam of
high energy electrons, which remove a valence shell electron from the analytes. EI often causes fragmentation of molecular ions and
pure samples are required to obtain useful structural information. Therefore, gas chromatography (GC) was coupled with an EI
mass spectrometer to analyze impure samples. This hyphenated technology (GC-MS) became a powerful tool to identify com-
pounds that were adequately volatile and stable at 300 C. Some polar compounds with hydroxyl groups could also be analyzed
by derivatization of hydroxyl groups. As such, GC-MS is most useful to identify volatile natural products with low molecular weight
such as terpenes.
Although detection of many chemical compounds became faster using GC-MS, a number of natural products were still
overlooked due to the inability of GC to analyze non-volatile compounds. Liquid chromatography (LC; often high pressure liquid
chromatography, HPLC), on the other hand, is a robust technology to separate a wide range of chemical compounds. However,
before the development of approaches to coupling LC with mass spectrometry, liquid chromatography was used with ultraviolet-
visible (UV–vis) spectrophotometry such as a photodiode array detector (LC-PDA). LC-PDA is very useful to detect compounds
containing conjugated double bonds. Unfortunately, many natural products lack such features, and further, UV–vis does not
provide any meaningful structural information.
The advent of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) technologies overcame this
issue. Both technologies are complementary, as discussed in the following section on ionization, and ionize analytes under
atmospheric pressure and often generate protonated (or deprotonated) molecular ions without causing significant fragmenta-
tion. Hence, unlike EI mass spectrometry, these soft ionization techniques provide information that can be used to infer the
chemical composition of intact molecules. The invention of collision induced dissociation (CID) tandem mass spectrometry
(MS/MS) enabled generation and detection of fragment ions from a molecular ion generated by ESI, which can be used to infer
the chemical structure of analytes. By integrating LC and MS/MS, rapid detection of a wide variety of features of natural products
present in a sample together with their molecular ions and fragmentation spectra became feasible. ESI mass spectrometry is often
the primary choice for coupling with LC in an LC-MS system due to its ability to detect analytes over a wider range of masses and
polarity.
The ability to infer the structures of natural products based on the fragmentation patterns is one of the very important aspects of
GC-MS and LC-MS analyses. Both database searches of fragmentation spectra and the mass-to-charge ratio (m/z) of molecular ions
are useful for determining whether the query molecule has already been discovered beforehand (dereplication). This is a significant
advantage of mass spectrometry since researchers can avoid spending tremendous amounts of time and effort on “rediscovery” of
known molecules. In addition, related molecules can be grouped based on the similarity of their fragmentation spectra, and analytes
similar to known molecules can be easily identified. Several algorithms and databases have been developed to do this. Widely used
algorithms include MS-Clustering of molecular networking.3 Global Natural Products Social Molecular Networking (GNPS) has
libraries of fragmentation spectra from about 20,000 compounds and implements the molecular networking analysis for
dereplication.4
Isotope patterns can be diagnostic for elemental composition, especially for high resolution mass spectrometry data. For many
elements, the higher isotopes are much lower abundance than the lowest monoisotopic mass (M0). This is true for carbon,
hydrogen, oxygen and nitrogen, and many others. However, some elements have abundant higher mass isotopes, and this can be
Mass Spectrometry for Natural Product Discovery 265
very helpful in chemical formula determination. For example, the halogen atoms chlorine and bromine each have 2 major isotopes
of high abundance, M0 and M+2. Since a large number of natural products contain these halogen atoms (e.g. chloramphenicol),
they are easily identified by analyzing the isotope distribution of the molecular ions. Owing to the high resolution and mass
accuracy of many modern mass spectrometers, it is possible to directly determine the chemical formula for a given natural product,
especially for small molecules of low mass.
As metabolomics using LC-MS technology has become more common, the need for data analysis tools has also increased since
typical LC-MS analyses involve comparing thousands of signals with m/z and retention time tags, and the most intense signals can
also be associated with MS/MS spectra. Several programs have been developed to deconvolute LC-MS signals into features or peaks
and align features among multiple files. Subsequent to this pre-processing, statistical analyses are often required to prioritize any
LC-MS features or strains/conditions that are of interest. Principal component analysis (PCA) can group strains or cultivation
conditions based on the similarity of metabolite production patterns to prioritize metabolically unique strains/conditions.5 PCA
becomes less effective as the data size grows and is often strongly influenced by largest differences. Kohonen self-organizing map
(SOM) analytics is a complementary method to perform comparative metabolomics with multiplexed data.5 Features with similar
trend of fluctuation are grouped together and spotted onto nearby nodes of a map. SOM is improved by increasing the number of
datasets. By comparing the heatmaps of SOM analyses, metabolically unique strains/conditions can be prioritized for subsequent
experiments.
The more recent development of desorption electrospray ionization (DESI) mass spectrometry, especially nanospray desorption
electrospray ionization (nanoDESI) MS, has advanced in situ detection of secreted metabolites on agar plates.6 Although this was
already feasible with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry imaging (MSI),7
nanoDESI requires no sample preprocessing and unlike MALDI-TOF, can be applied non-destructively to detect local metabolite
signals over the course of time, enabling time course measurements and visualization of the spatial distribution of metabolite
production. One of the applications of this technology is monitoring communication between plants and microbial or fungal
colonies by detecting secreted metabolites, and under certain conditions, result in the detection and identification of produced
natural products.8,9 Another application of this technology is high-throughput metabolomics. For instance, since DESI can analyze
one sample in 2 s with no sample preprocessing,10 high-throughput screening to find natural products with unique structural
features has become faster and easier.
6.12.2 Analytical Workflows for Natural Product Discovery
Mass spectrometry plays a number of critical roles at different stages in the natural product discovery process, ranging from sample
selection, screening and fractionation to identification and dereplication. A generalized analytical workflow for natural product
discovery is shown in Fig. 1, illustrating both a traditional approach and a natural product library approach.10a Both approaches
begin with selecting an appropriate source material with high potential for natural product discovery. Following the traditional
approach, an extracted mixture is screened prior to separation and purification of components, whereas with the library approach,
these steps are reversed as libraries are created from samples and then screened. However, for each, the final step requires
identification of the active compound for potential production or other utility. Both approaches have been successfully applied
to result in discovery of new natural products with desirable properties.
The more traditional approach characterized early natural product discovery efforts in the 1940s to 1970s, or “Golden Age,” and
was based on performing phenotypic and activity screens on extracts from soil bacteria, plants and fungi.11 These extracts could be
either directly screened for activity or fractionated before. An activity screen may include growth inhibition, for instance, by applying
an extract to a susceptible organism’s culture and measuring growth. Extracts found to have desirable activities are then subject to
Screen of Separation into

mixture pure components
Traditional
approach
Mass spectrometry
Sample source LC-ESI/APCI, MS/MS, Identification of active
selection (extracts) MALDI, LC-MS-NMR, MS components
Imaging
Library
approach
Separation into Screen of pure
pure components components
Fig. 1 Role of mass spectrometry in each step of an analytical workflow for natural product discovery. Based on with permission from Gilbert, J. R.; Lewer, P.;
Duebelbeis, D. O.; Carr, A. W. The Central Role of Mass Spectrometry in Natural Products Discovery. In Integrated Strategies for Drug Discovery Using Mass
Spectrometry; Lee M. S. Ed.; Wiley, 2005. https://doi.org/10.1002/0471721034.ch6. Copyright (2005) John Wiley and Sons, Inc.
a variety of techniques including chromatography, further extraction, and other fractionation steps to purify and identify the active
compound(s). A combination of mass spectrometry and NMR is often finally applied for identification of active compounds,
including steps of dereplication, and structure determination. This traditional approach has led to the identification of over 1000
natural products with either antifungal, antiparasitic or antibacterial activity, as well as anticancer drugs based on screens with
cancer cell lines.
Mass spectrometry can also play an important role in determining which samples or extracts to select and subject to an activity
screen or further pursue with treatments to induce natural product production. For instance, to pre-screen and select samples that
have high potential for activity, extracts may be run first through LC-MS or MALDI-TOF.12,13 Spectra can be analyzed to determine
samples that may be prolific in producing secondary metabolites, or compared to control samples or databases of library spectra to
see which samples have new or infrequently occurring compounds.14 In work by Samat et al.,12 PCA was applied to LC-MS data
collected on 267 extracts to prioritize 27 that contained potentially new photosensitizers, leading to the identification of 2 new
compounds with this activity (Fig. 2). Alternatively, spectra can be quickly analyzed to determine if a recombinant technique has
successfully generated production of natural products, or what part of a biosynthetic pathway has been modified.15 In recent work
by Shih et al.,16 a new gene stacking method, jStack, was validated by confirming violacein production in leaves of a plant after
insertion of bacterial genes (Fig. 3).
From the 1970s through today, new strategies have emerged for finding natural products based on advancements in science and
technology including low-cost genome sequencing, synthetic biology (e.g. recombinant DNA), combinatorial biosynthesis, high-
throughput methods for screening and fractionation, and innovations in mass spectrometry. These have led to the ability to easily
create and screen large libraries of purified or semi-purified compounds from hundreds to thousands in size.
Fig. 2 (A) General workflow screening extracts with LC-MS and analyzing with PCA to identify new photosensitizers. (B) Example PCA plot showing outlier extracts,
with unique masses identified in LC-MS data driving group differences targeted for further investigation. (B) Adapted from Samat, N.; Tan, P. J.; Shaari, K.; Abas, F.;
Lee, H. B. Prioritization of Natural Extracts by LC–MS-PCA for the Identification of New Photosensitizers for Photodynamic Therapy. Anal. Chem. 2014, 86,
1324–1331. https://doi.org/10.1021/ac403709a. Copyright (2014) American Chemical Society.
Hsp18 MAS Act2 RbcS Bch1
L-tryptophan VioA VioB VioE VioC VioD

VioA
Indole-3-pyruvic acid (IPA) imine

VioB Violacein standard
Infiltrated leaf
IPA imine dimer Uninfiltrated leaf
Intensity (a.u.)
VioE
Protodeoxyviolaceinic acid
VioD
Protoviolaceinic acid
VioC
Violacein 4.20 4.25 4.30 4.35 4.40 4.45 4.50

Retention time (min)
Fig. 3 Synthetic biology technique jStack used to engineer bacterial genes into a Nicotiana benthamiana plant for producing violacein, with production in
leaf confirmed using LC-MS. Modified under public license from Shih, P. M.; Vuu, K.; Mansoori, N.; Ayad, L.; Louie, K. B.; Bowen, B. P.; et al. A Robust
Gene-Stacking Method Utilizing Yeast Assembly for Plant Synthetic Biology. Nat. Commun. 2016, 7, 13215. https://doi.org/10.1038/ncomms13215. https://
creativecommons.org/licenses/by/4.0/.
An advantage of using a purified library compared to a simple extract relates to the complexity and diversity of compounds
found in a sample. When all compounds comprising a sample are tested simultaneously, there is a risk of false discovery. It is
possible that a single compound that is effective may not be present in high enough abundance for activity to be observed, activities
of ubiquitous compounds may interfere with or mask the activity of another, or multiple compounds may have the same type of
activity but at different levels or selectivity.17,18 Libraries make it possible to screen each compound individually (or in small
groups) and over a range of concentrations, overcoming these limitations.
Types of libraries vary depending on how they were created. As already discussed, libraries may be comprised of compounds
fractionated and purified from extracts obtained from natural sources. Creation of these libraries is typically performed using
automated equipment performing a series of steps including separation, fractionation and concentration. For example, SepBox is
one of the most widely used automated technologies for creating fraction libraries.19 Here, a sequential HPLC/SPE/HPLC/SPE
system is applied in which a large volume of extract is introduced into a preparative HPLC column (for separation) coupled to
a solid phase extraction system under high pressure (for concentration), with fractionation from the HPLC to SPE triggered either by
time, UV or evaporative light-scattering detection (ELSD). These SPE are coupled to additional HPLC columns for further
separation, fractionation and then concentration on another set of SPE, followed by final collection with elution off the columns
creating a semi-purified library ready for screening. In an approach like this, 600 fractions can be obtained in under 24 h, and further
selectivity can be achieved by using HPLC columns and SPE cartridges with different chemistries, varying mobile phase solvents and
fractionation trigger parameters. These are discussed in more detail in the following section on separation and fractionation.
Once a semi-purified library from an extract has been created, mass spectrometry is important for determining the purity of the
fraction as well as identifying what compounds are in each. Often, laser desorption MS techniques off specialized surfaces, including
MALDI-TOF, nanostructure-initiator mass spectrometry (NIMS), or desorption/ionization on silicon (DIOS), are used for this type
of testing owing to minimal sample processing steps and amenability for high-throughput.10,20,21 With these techniques, high
quality spectra can be generated in seconds or less from a single spotted sample only a few millimeters in diameter. Further, when
used in combination with acoustic printing of a sample onto target surfaces, amounts as small as only 1 nL may be all that is
required for analysis, conserving sample that may have limited availability and also significantly increasing throughput to make
possible screens of at least 10,000 samples/day (Fig. 4A).22 Often, direct injection into various types of MS sources (e.g. ESI, APCI) is
another option. Although less optimized for high-throughput, use of alternative sources have the ability to detect compounds that
may not ionize as well using other methods.23,24 However, with DESI or nanoDESI, this technique advantageously combines ESI
with surface-based sample preparations (Fig. 4B).25 As will be discussed later, depending on the ionization mechanism, different
MS methods are suitable for detecting different kinds of compounds.
Mass-triggered fractionation is another successful strategy for creating purified libraries from a crude extract or combinatorial
library.26,27 Here, a mass spectrometry system is coupled to an HPLC, supercritical fluid chromatography (SFC) or other separations
technology, and fraction collection triggered by mass detection. Simultaneous collection of UV and ELSD measurements can also be
collected for further characterization of each fraction. This technique is ideal for obtaining high purity samples prior to screening.
Additionally, since the composition of each fraction has already been characterized with mass spectrometry, fraction purity and
compound identification does not need to be determined with further processing steps.
While crude extracts from natural sources are one source of generating natural product libraries, synthetic and genetic techniques
have developed for production of natural products, and the knowledge necessary for creating libraries from combinatorial
biosynthesis. This includes mutasynthesis, in which mutated bacteria can be induced to produce analogs of a known natural
product by making modifications or blocks in specific steps of a synthesis pathway.28 A related technology, precursor-directed
Fig. 4 Desorption based high-throughput MS approaches. (A) Schematic of high-throughput NIMS screening in which samples are acoustically printed and
analyzed by NIMS. (B) DESI source schematic in which a nebulized solvent spray is rastered across a surface and desorbed ions pulled into an MS inlet. Modified
with permission from de Raad, M.; Fischer, C. R.; Northen, T. R. High-Throughput Platforms for Metabolomics. Curr. Opin. Chem. Biol. 2016, 30, 7–13. https://doi.
org/10.1016/j.cbpa.2015.10.012. Copyright (2016) Elsevier.
biosynthesis, relies on disrupting pathways used in synthesis of precursor molecules used in making a specific natural product, and
then adding different precursors to use as a “starting” position resulting in production of analogs.29 With recombinant DNA (rDNA)
technology, genes can be inserted into organisms for production of natural products or other biological modifications.30 Most
recently, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based approaches have been used for accurate
and efficient genome editing of organisms for the discovery, characterization and production of natural products, often by activating
silent biosynthetic gene clusters (BGCs).31–34 As shown in Fig. 5, detection of unique mass spectra peaks present in a modified
(promoter-inserted) versus control (parent) strain leads to identification of new natural products produced upon gene activation.
These and complementary innovations have led to greater understanding of the genes, gene clusters and biochemical pathways
involved in natural product biosynthesis, enabling combinatorial biosynthesis strategies and tools to produce hundreds of
derivatives based on a known structure.35
Whether a library or crude extract, numerous screening techniques are employed to help determine which samples have
desirable activity. Many of these integrate mass spectrometry techniques, either integrated as part of the screen or involved
as part of the fractionation and identification steps. For example, “high resolution screening” (HRS) with MS monitors a change
in fluorescence when molecules elute off an LC column and bind to a target receptor in the effluent stream.36 This active fraction is
directed to the MS for acquisition of spectra for identification and dereplication. Other screening techniques use mass spectrometry
itself as a direct measurement of activity. Many of these rely on detecting mass shifts associated with binding, either covalent or
noncovalent, between target and analyte.37 Multitarget affinity/specificity screening (MASS) is an example of one of these types of
screens, and can be applied for screening large natural product libraries (Fig. 6). Here, when a molecule binds to a target (e.g. ligand
binding to RNA), a new mass, or “shifted mass” resulting from binding to the target, is detected compared to a control solution with
only target or only extract/fraction.38,39
Two other innovative MS-based screening assays include structure-activity relationship (SAR) by MS and detection of
oligonucleotide–ligand complexes by electrospray ionization mass spectrometry (DOLCE-MS). With SAR by MS, a combinatorial
library having a range of classes of motifs may be screened against a target, with binding detected by MS. Swayze et al. used SAR by
MS to find 2 classes of binding motifs with high affinity for a specific RNA target,40 then further performed MS competition
Fig. 5 (A) CRISPR-Cas9 approach used to insert heterologous promoters to activate a silent BGC in a Streptomyces host strain. (B) Mass spectrometry is used
to detect newly synthesized compounds after promoter insertion into Streptomyces (blue and red spectra) that are not present in the parent strain (black spectra).
This is indicated in the spectra shown with unique mass spectra peaks appearing at m/z 405, 780 and 425 in strains with inserted promoter. Modified with
permission from Zhang, M. M.; Wong, F. T.; Wang, Y.; Luo, S.; Lim, Y. H.; Heng, E.; et al. CRISPR–Cas9 Strategy for Activation of Silent Streptomyces Biosynthetic
Gene Clusters. Nat. Chem. Biol. 2017, 13(6), 607. https://doi.org/10.1038/nchembio.2341. Copyright (2017) Springer Nature.Adapted from Zhang et al.
experiments to distinguish separate binding sites for structurally different ligands as well as competitive and concurrent binding of
different ligands. Seth et al. also used SAR by MS to find a new class of small molecules that bind with high affinity to a subdomain
of the Hepatitis C virus.41 Similarly, DOLCE-MS is able to detect non-covalent binding to single-stranded or double-stranded
oligonucleotides by interpreting mass shifts in mass spectra in combination with ratio of detected single- vs. double-strands. This
technique has shown utility as a secondary screen for compounds against viral-associated oligonucleotides, distinguishing whether
compounds from a “primary” viral screen act by either interacting with a target enzyme, or binding DNA or RNA, a mechanism that
may be more likely to be nonspecific and toxic.42 Alternatively, this method is also useful when screening for drug candidates that
specifically target dsDNA, with alternative ionization techniques possible (e.g. laser spray) that are able to maintain stability of the
noncovalent interaction in solution.43
In the following sections, we will discuss in more detail each of these steps in the natural product discovery process, focusing on
the role of mass spectrometry, as well as more recent innovations in mass spectrometry imaging and genomic analysis that are
currently changing the landscape of natural product discovery as new ways to find and detect and understand their biosynthesis.
6.12.2.1 Mass Spectrometry Workflow

Typical mass spectrometry workflows integrate several standard steps, including sample extraction, separation/fractionation, mass
spectrometry data acquisition, and finally data analysis (Fig. 7). The specifics of each of these steps varies depending on the ultimate
goal of the process, chemical properties of the natural products of interest, and often, access to resources and availability.
As discussed, activity screens may occur either upstream or downstream of the mass spectrometry data analysis component.
6.12.3 Extraction
Typically the first step and primary challenge in natural product analysis workflows is sample preparation, which involves the
successful separation of target metabolites from the sample matrix using an appropriate extraction technique. Due to the great
diversity of raw materials and compounds with different chemical properties, a number of different extraction methods have been
developed. Different solvents and methods will be more appropriate for successfully extracting different types of samples. Extraction
methods are highly diverse and range from maceration, simple liquid-liquid extraction, Soxhlet extraction, supercritical fluid
extraction, and even sublimation. Table 1 lists some of these methods and the operating conditions under which they are applied.44
To achieve high yield, good reproducibility, low cost, and efficiency in the extraction process, various parameters in these methods
are optimized for different samples including solvent, temperature, pressure and time.
6.12.3.1 Extraction Solvents

Common to most of the methods listed above is the requirement of a solvent into which compounds will be extracted. For mass
spectrometry preparations, it is generally desirable to use solvents that easily dissolve a range of compounds but are also compatible
(A) (16S + PM)–5

5.0e+07 1849.08
770%
(16Sc)–5
1859.05
Abundance (arbitraty units)

4.0e+07
(16S + 818)–5
1889.69
3.0e+07 212%
2.0e+07
1889 1891 1893 m/z
(16S)–5 (16Sc + PM)–5 (16Sc + 818)–5

1.0e+07 1726.02 1849.08 2022.73
13% 40%
0.0e+00
1700 1800 1900 2000 m/z
Fig. 6 Example of noncovalent binding activity screen of natural product library fractions using MASS. (A) Mass shifts detected in a mass spectrometry assay of
natural product fraction 146 with a synthetic 27-mer 16S rRNA A-site. Peaks are detected at m/z 1627 for 16S RNA, as well as m/z 1849 and m/z 1889
corresponding to a mass shift of 616 for paromomycin (PM) and 818 for a new, unknown molecule. (B) Mass spectra showing m/z of natural products detected in
fraction 146 with two m/z values corresponding to the detected mass shifts associated with 16S RNA. Reprinted with permission from Cummins, L. L.; Chen, S.;
Blyn, L. B.; Sannes-Lowery, K. A.; Drader, J. J.; Griffey, R. H.; Hofstadler, S. A. Multitarget Affinity/Specificity Screening of Natural Products: Finding and
Characterizing High-Affinity Ligands From Complex Mixtures by Using High-Performance Mass Spectrometry. J. Nat. Prod., 2003, 66(9), 1186–1190. https://doi.org/
10.1021/np0301137. Copyright (2003) American Chemical Society.
Fig. 7 Standard steps in a mass spectrometry workflow as part of the natural product discovery process.
Table 1 Methods and associated conditions commonly used for extracting natural products.
Method Solvent Temperature Pressure Time Volume of organic Polarity of natural

solvent consumed products extracted
Maceration Water, aqueous and Room temperature Atmospheric Long Large Dependent on
non-aqueous solvents extracting solvent
Percolation Water, aqueous and Room temperature, Atmospheric Long Large Dependent on
non-aqueous solvents occasionally under heat extracting solvent
Decoction Water Under heat Atmospheric Moderate None Polar compounds
Reflux extraction Aqueous and non-aqueous Under heat Atmospheric Moderate Moderate Dependent on
solvents extracting solvent
Soxhlet extraction Organic solvents Under heat Atmospheric Long Moderate Dependent on
extracting solvent
Pressurized liquid Water, aqueous and Under heat High Short Small Dependent on
extraction non-aqueous solvents extracting solvent
Supercritical fluid Supercritical fluid (usually Near room temperature High Short None or small Nonpolar to
extraction S-CO2), sometimes with moderate polar
modifier compounds
Ultrasound assisted Water, aqueous and Room temperature, or Atmospheric Short Moderate Dependent on
extraction non-aqueous solvents under heat extracting solvent
Microwave Water, aqueous and Room temperature Atmospheric Short None or moderate Dependent on
assisted non-aqueous solvents extracting solvent
extraction
Pulsed electric field Water, aqueous and Room temperature, or Atmospheric Short Moderate Dependent on
extraction non-aqueous solvents under heat extracting solvent
Enzyme assisted Water, aqueous and Room temperature, or Atmospheric Moderate Moderate Dependent on
extraction non-aqueous solvents heated after enzyme extracting solvent
treatment
Hydro distillation Water Under heat Atmospheric Long None Essential oil (usually
and steam non-polar)
distillation
Modified under public license from Zhang, Q. W.; Lin, L. G.; Ye, W. C. Techniques for Extraction and Isolation of Natural Products: A Comprehensive Review. Chinas Med. 2018, 13,
20. https://doi.org/10.1186/s13020-018-0177-x. https://creativecommons.org/licenses/by/4.0/.
with MS instrumentation and do not require extensive drying times, thermal processing or other procedures that may result in
compound degradation. For example, some of the most common solvents used in MS extraction are methanol, ethanol and water.
Both methanol and ethanol are able to extract compounds with both polar and nonpolar properties, do not interfere with signals
when injected into an MS, and can be quickly and easily removed by evaporation or under low vacuum (e.g. SpeedVac) to
concentrate samples or in preparation for further extraction steps. Water is excellent for extraction of polar compounds,
MS-compatible, and is easily removed by freezing and sublimation techniques (e.g. lyophilizer). In contrast, solvents such
as dimethylsulfoxide (DMSO) and dimethylformamide (DMF), while excellent for solubilizing a wide range of compounds of
varying chemistries, are typically not used due to high boiling points (cannot be removed via SpeedVac or lyophilization), and at
higher concentrations, may cause interference with MS signal (e.g. ion suppression) or instrumentation (e.g. compatibility with
PEEK fittings). Acetone, while also an excellent solvent, may contain contaminants that interfere with mass spectrometry especially
when exposed to some plastics during the extraction procedure.
Health and environmental considerations also play an important role in solvent selection. For example, many chlorinated
solvents such as carbon tetrachloride and chloroform have adverse health effects, and methanol is also toxic. More recently, finding
and applying sustainable (“green”) extraction solvents has gained increasing interest.45 Ethanol, for instance, is completely
biodegradable and can be produced from agricultural plants. As a polar organic solvent, ethanol has been used in extraction of
a wide variety of natural product compounds including phenolics, terpenoids, alkaloids, and carbohydrates.46–48 Water, of course,
is considered a green extractant, and ideal for compounds with polar properties, including protein, sugar and organic acids.
However, low-polarity compounds can also be extracted when applied as pressurized hot water extraction (PHWE), or subcritical
water, in which temperature and pressure are raised to the point that extraction behavior more closely resembles that of ethanol.49
Another green alternative is supercritical CO2, which can be applied for applications in which hexane was previously used, and can
extract weakly polar compounds such as carotenoids and triglycerides.50
In consideration of the above, often the first step in solvent selection is making sure the selected solvent has the appropriate
properties to be capable of dissolving, or removing, the compounds of interest from the sample matrix In “liquid-liquid” and “solid-
liquid” extractions, the octanol-water partition coefficient (Kow) of solvent and target molecule, also known as the law of similarity
and intermiscibility (Similia similibus solventur in Latin),51 or like dissolves like, can be used to predict the solubility of a compound
in a particular solvent. While polar solvents easily dissolve ionic compounds and polar compounds, nonpolar solvents tend to
Fig. 8 Isoflavones were extracted using a range of solvent mixtures, with different solvents more effective at extracting specific isoflavones than others. Modified
with permission from Luthria, D. L.; Biswas, R.; Natarajan, S. Comparison of Extraction Solvents and Techniques Used for the Assay of Isoflavones From Soybean.
Food Chem. 2007, 105, 325–333. https://doi.org/10.1016/j.foodchem.2006.11.047. Copyright(2007) Elsevier.
dissolve nonpolar covalent compounds better. Solvents commonly used for natural product extraction that are also suitable for MS
sample preparation include n-Hexane, chloroform, ethyl acetate, ethanol, methanol, and water, listed in order of increasing solvent
polarity. As shown in Fig. 8, different solvent systems will be more effective at extracting particular compounds than others.52 Here,
diadzin and genistin, both glycosylated isoflavones, were both extracted well with 90% methanol. However, this solvent was not
able to extract the non-glycosylated, more hydrophobic version of diadzin, daidzein, which required a less polar solvent of 70%
ethanol for extraction. Solvents that were inefficient at extracting isoflavones included the most polar and most nonpolar solvents
tested, water and 58% acetonitrile respectively, likely since isoflavones had a combination of polar and nonpolar chemical
properties.
Nonpolar solvents n-hexane and chloroform are most effective at extracting highly nonpolar natural products, such
as polyphenolic, lipophilic compounds and certain peptides. For example, the antibacterials pediocin, nisin, and bacillin have all
been recovered from bacterial liquid culture using a chloroform extraction.53 Rapinel et al. reported that lipophilic carotenoids and
aromas could be extracted from carrots and caraway seeds using n-hexane or alternatively, liquefied n-butane.54 For extracting
somewhat less nonpolar compounds, such as flavonoids or phenolics, ethyl acetate is a choice solvent, as well as methanol or
ethanol.55 However, ethyl acetate will extract a lower proportion of polar compounds than either methanol or ethanol, especially
when used in conjunction with water, with which it is immiscible, to form a 2-phase system for removing more polar compounds
due to solubilization in the lower water phase. In comparison, both methanol and ethanol are miscible with water and further
purification steps would be necessary to remove polar compounds from an extract. Therefore, for less lipophilic and more polar
compounds, methanol and ethanol are more appropriate. For instance, iridoids are common types of monoterpenoids and can be
effectively extracted with methanol under 50 C overnight,56 while glucosinolates, anionic secondary metabolites involved in the
plant defense against insects, can be extracted with high efficiency in 70%–80% methanol.57,58
Water is efficient for extracting polar or water-soluble compounds, like amino acids, organic acids, and even phenolic
compounds when under high temperature and pressure.45 As mentioned previously discussing PHWE, temperature may dramat-
ically alter the physicochemical properties of a solvent and improve extraction efficiency. Often, increasing temperature will reduce
the viscosity of a solvent, concurrently raising the solubility and increasing diffusivity of metabolites in solution. Although water is
traditionally a polar solvent, it can be used to recover phenolic compounds, volatile oils, and isoflavones from plant materials when
high temperature and pressure is applied with PHWE.49 In another water extraction example, resveratrol and polydatin were
obtained in one simple extraction step by applying ultrasound-assisted extraction to a cyclodextrin (1.5% w/w) water solution.59
Finally, applying a subcritical water extraction technique to coriander seeds allowed essential oils to be extracted, which also
resulted in concentrating more oxygenated forms.60 One potential drawback of water extracts, however, is that depending on the
sample matrix, desalting steps may also be required if a large amount of salt is extracted with target products. Not only can salt
interfere with MS signal and cause ion suppression, salt can impede efforts to concentrate samples or resolubilization of
a compound in preparation for MS analysis.
For large-scale metabolite profiling, ethanol and methanol are more frequently used as a general extraction solvent since these
provide sufficient solubility over a broad range of compound chemistries, including both polar and nonpolar analytes. Also, further
extraction using either more hydrophobic and hydrophilic solvents can be applied for sample fractionation of dried ethanol and
methanol extracts. For example, in the analysis of plant tricalysiosides, following an initial extraction of Tricalysia dubia leaves with
methanol, concentrated alcoholic extracts were then separated into an ethyl acetate fraction, n-butanol fraction, and water fraction
resulting in the separation of several types of glycosides with differing polarity.61,62
6.12.3.2 Conventional Extraction Methods

6.12.3.2.1 Liquid-liquid, solid-liquid and solid phase extraction (SPE)
The most common and basic extractions for mass spectrometry analyses of natural products are liquid-liquid and solid-
liquid extraction. A typical solid-liquid extraction procedure consists of immersing a sample material in solvent (e.g. cell
pellet in methanol), sonication in a water bath, centrifugation to pellet out solids not extracted into solution, and then
removing the supernatant, or extract, to another container for concentration (e.g. drying in a SpeedVac or evaporated under
N2). Often, a solid material will be powderized or homogenized (e.g. bead-beating or mortar and pestle) so that cellular
material is lysed, helping release metabolites, and the extraction solvent can more easily penetrate the material. Aqueous
samples can also be extracted similarly by first freezing then removing water via lyophilization, which leaves behind a solid
residue for extraction.
In comparison, a liquid-liquid extraction often consists of adding an immiscible liquid to the sample liquid. For instance, to
extract nonpolar metabolites from a cell culture media, chloroform can be added 1:1, or in a typical Bligh-Dyer approach, 2:2:1.8
methanol:chloroform:water, vortexed then centrifuged, then the bottom chloroform layer containing extracted products removed
and dried. Ethyl acetate, also immiscible with water, is also commonly used in liquid-liquid extraction for less nonpolar
compounds, and can be added 1:1, with the upper layer containing extracted products removed and dried.
Solid phase extraction (SPE) is a method in which compounds with certain chemical properties (e.g. hydrophobic) are adsorbed
on a stationary phase (solid), allowing other compounds that do not retain (e.g. salts, more hydrophilic compounds) to flow
through. Adsorbed compounds are then eluted with an appropriate solvent allowing separation of adsorbed target compounds
from other types of molecules.63 There is a wide range of commercial bonded phases with different absorption modes (normal
phase, reverse phase, cation exchange, and anion exchange), providing selective retention for analytes with different chemical
properties.
Two commonly used stationary phases for natural product extraction are varieties of bonded silica and ion exchange
resins. A C18 resin, most popular, is often comprised of octadecyl carbon chains bonded to silica resulting in a hydrophobic
phase ideal for sequestering natural products with hydrophobic properties. Numerous variations of this type of resin have
been developed to optimize extractions by tailoring selectivity and modifying polarity, including changing length of alkyl
chain (e.g. C8), cyclization (e.g. phenyl), or adding other functional groups (e.g. cyano, sulfo, amino, pentafluorphenyl).
Amberlite® is a popular solid phase extraction resin typically comprised of a crosslinked polymer co-polymerized with
another compound that provides ion-exchange sites and available in the form of insoluble beads with various porosity.
Amberlite® XAD-2 is commonly used to extract hydrophobic natural products, and consists of beads of crosslinked
polystyrene and divinylbenzene. In comparing extraction of polyphenols and flavonoids using either Amberlite or C18
cartridges, one study found that although C18 cartridges resulted in higher extraction efficiencies overall, Amberlite extracted
a larger number of compounds.64
Depending on the application, a combination of SPE can be applied for optimizing extraction. For example, an explorative SPE
protocol was reported which utilized four types of SPE cartridges to separate bacterial extracts into 15 fractions, allowing the
mapping of microbial natural products across a wide range of pKa values.65 Cutignano et al. developed SPE approaches using HR-X
cartridges to desalt and fragment marine algal extracts allowing a high recovery of alkaloids.66 In addition, SPE can easily be
automated in an online extraction system, which is advantageous for reducing deviation between sample batches, improving
accuracy and decreasing workloads.67 In a recent study, using a platform coupling online SPE to LC-MS, more than 20 hydroxylated
polyunsaturated fatty acids in cells were found that were potential biomarkers for the arachidonic acid cascade pathway.68 Another
application of an online SPE-LC-MS/MS system was performed by Song et al., in which 23 ginsenosides and aconite alkaloids were
quantified in a Chinese medicine preparation known as Shenfu injection.62
Often, multiple extraction methods are combined to more selectively remove target compounds of interest, or create multiple
fractions for analysis. Fig. 9 illustrates a typical sample preparation workflow combining solid-liquid, liquid-liquid and SPE for
extracting and creating fractions of compounds with different chemistries in preparation for LC-MS analysis.69
6.12.3.2.2 Maceration, infusion, percolation and decoction

Maceration, infusion, percolation and decoction are fundamentally similar extraction techniques, varying mainly by time, temper-
ature and certain processing steps.70 Maceration itself is a simple process of exposing a material to solvent for an extended period of
time (usually days), with agitation, for the purpose of softening and breaking apart a material to release soluble metabolites, after
which the material is filtered to obtain extracted compounds. With infusion, exposure time is typically shorter, and with decoction,
Fig. 9 Generalized workflow for extracting compounds in preparation for LC-MS. Here, three different extraction techniques are combined to create multiple
fractions containing different compound chemistries from leaf starting material. After homogenizing samples, three different solvents are added together for
solid-liquid extraction—methanol, chloroform and water. In liquid-liquid extraction, immiscible phases are collected separately, then the aqueous methanol/water
phase is further subject to SPE prior to LC-MS. Here, different fractions are eluted from an SPE by applying solvents with decreasing polarity—water, methanol and
then acetonitrile. Modified with permission from Maia, M.; Monteiro, F.; Sebastiana, M.; Marques, A. P.; Ferreira, A. E.; Freire, A. P. et al. Metabolite Extraction for
High-Throughput FTICR-MS-Based Metabolomics of Grapevine Leaves. EuPA Open Proteom. 2016, 12, 4–9. https://doi.org/10.1016/j.euprot.2016.03.002.
Copyright (2016) Elsevier.
samples are also boiled. Percolation requires a percolator through which hot solvent is dripped at a moderate rate through dried
powdered samples.
6.12.3.2.3 Reflux extraction

Reflux is a typical conventional extraction method in which solvents are evaporated and condensed repeatedly within a heat reflux
extractor. Compared to maceration, which may need 2–3 days to extract natural products under room temperature, this technique
requires much less extraction time (typically several hours).71 However, due to the high temperature of the extraction process, the
yield of thermolabile analytes may be much lower with this approach. In a recent study, reflux extraction using methanol or ethanol
rather than acetone and acetonitrile recovered significantly more polysaccharides, polyphenolics, and flavonoids from Dendrobium
candidum.72 Several studies also showed that compared to other solvents, 60%–80% ethanol proved to be more efficient in
extracting saponins from Panax notoginseng, anthocyanins from grapes, and didehydrostemofoline from Stemona collinsiae root.73–75
6.12.3.2.4 Soxhlet extraction

Soxhlet extractor is a modification of the reflux extractor, allowing recovery of analytes from sample matrix with fresh solvent
continuously, which further increases extraction efficiency.44 Soxhlet extraction is commonly applied in the isolation of phenolic
acids and flavonoids.76 For instance, more than 10 carbohydrates were quantified from ethanolic extracts from woods using Soxhlet
extraction.46 Moreover, Soxhlet can also be applied in the removal of unwanted contaminants from samples.
6.12.3.3 Non-conventional Extraction Methods

There are also a number of non-conventional extraction methods that have been developed for extraction of natural products. For
many of these, specialized equipment is necessary and cost may be higher than conventional methods. System diagrams of some of
these systems illustrate the complexity and special equipment required (Fig. 10A–C).76a However, many advantages are associated
with some of these methods such as less processing time, little or no solvent requirements, more efficient and higher yield extraction
(Fig. 10D), and extraction of compounds not easily performed using conventional methods. Following is a discussion of several of
these techniques.
(A) (B)
(C) (D)
Fig. 10 (A) SFE system (B) PFE system (C) MAE system (D) Graphs showing relative extraction efficiencies of various compounds (Naphthalene; F, fluorene; pH,
phenanthrene; Py, pyrene; BP, benzo(a)pyrene; IP, indeno(1,2,3-cd)pyrene) from coal fly ash (a-lignite, b-bituminous) comparing Soxhlet, SFE and PFE systems and
parameters. Modified with permission from Camel, V. Recent Extraction Techniques for Solid Matrices—Supercritical Fluid Extraction, Pressurized Fluid Extraction
and Microwave-Assisted Extraction: Their Potential and Pitfalls. Analyst 2001, 126, 1182–1193. https://doi.org/10.1039/B008243K. Copyright (2001) Royal Society
of Chemistry.
6.12.3.3.1 Ultrasound assisted extraction (UAE)

UAE uses high frequency (20 kHz) pulses to generate local hotspots at macroscopic scale with high shear stress and temperature by
producing cavitational bubbles.77,78 UAE has advantages of simplicity, and is less time consuming and uses less solvent than other
methods, and can be easily coupled with other extraction techniques. Since this technique can be performed under room
temperature, this can prevent the oxidation and decomposition of target natural products. UAE has been widely applied in the
isolation of different natural products. Ghafoor et al. has successfully extracted phenolic compounds, antioxidants, and anthocy-
anins from grape in <30 min using UAE.79 Extraction time, solvent composition, and input power are the three major factors
affecting UAE extraction efficiency. Optimization of the conditions can be performed using a space curve model.80 Compared to
conventional extraction, polyphenols recovered from Salvia officinalis L. using UAE exhibited 20% higher yield, 3-fold less
processing time, and much less consumption of solvent.81 Microbial natural products have been considered as an important origin
of antibiotics, immunosuppressive chemicals, and other potential pharmaceuticals.82 The carotenoid zeaxanthin, beta-carotene,
and fatty acids were recovered from microalgal (Synechococcus sp.) biomass using UAE with N,N-dimethylformamide as the
solvent.83
6.12.3.3.2 Microwave-assisted extraction (MAE)

There has been an increased interest in using MAE for natural products extraction.84 In this approach, molecules are heated by
microwave through ionic conduction and dipole rotation.85 The water inside the cells, even in dry materials, can be evaporated and
exert enormous pressure on the cell wall, which can lead to the breakup of the cells. The choice of solvent for MAE not only depends
on the solubility of the target compounds but also the efficiency of solvent to convert microwave energy to heat. Polar solvents, such
as water, methanol, 2-propanol typically can absorb microwave energy well.86 Non-polar compounds like hexane is a microwave
transparent solvent, which cannot be directly heated up by microwave, but polar substances (e.g. ethanol and acids) can be added
as a modifier to increase the absorbance of the microwave. Ethanol and water mixtures (60%–90%) proved to be a good solvent
systems for flavonoids extraction from plants.87,88 Dahmoune et al. isolated polyphenols from Myrtus communis L. leaves using MAE
and achieved a recovery of the total phenolic capacity of 162 mg/g dry weight, which was similar to the predicted phenolic capacity
(166 mg/g dry weight).89
6.12.3.3.3 Pressurized liquid extraction (PLE)

PLE, also known as accelerated solvent extraction (ASE), is an extraction technique that operates under high temperature and
pressure.90 The extractants are maintained in a liquid state (subcritical) even at a temperature higher than boiling point if high
pressure is applied.91 The elevated temperature can dramatically increase the solubility and diffusion of analytes resulting in less
extraction time and lower solvent consumption. PLE is becoming increasingly popular in natural products extraction due to these
advantages. A wide range of carotenoids with different polarities were extracted within 5 min under 103 bars using PLE from three
types of fruits, Tunisian kaki, peach, and apricot.92 The property of natural products extracted is also influenced by the pressure used
in PLE. Xi et al. compared the recovery and antioxidant activities of total phenolic compounds extracted from green tea under
pressures ranging from 150 to 450 MPa.93 The extracted phenolic compounds not only exhibited higher yields under 450 MPa but
also stronger antioxidant activities.
6.12.3.3.4 Supercritical fluid extraction (SFE)

Use of supercritical fluids is becoming a popular choice for natural product extraction.94 These are formed as the temperature or
pressure of a substance is raised above the critical point, giving supercritical fluids both liquid- and gas-like properties.95 For
extraction, they have solvent power much like a liquid and penetration power (into a solid) much like a gas. Thus it has much less
extraction time compared to liquids when using supercritical fluid. Carbon dioxide is one of the widest applied solvents in SFE since
it is non-toxic and has a low critical temperature (31 C).94 The relatively low critical pressure (7.3 MPa) of CO2 makes it attractive
in industrial processes.96 In one application, supercritical CO2 (SC-CO2) was applied in the extraction of high valuable phenolic
compounds from lignocellulosic biomass.97 Extraction selectivity of SC-CO2 can be manipulated by modifying temperature and
pressure. Vigano reported a three-step sequential extraction using SC-CO2 in which highly concentrated tocols, fatty acids, and
carotenoids were obtained from passion fruit bagasse.46
6.12.4 Separation and Fractionation
Extracts of natural products are typically complex mixtures consisting of numerous compounds each with different activities,
selectivities and other properties. As discussed earlier, conducting screens with these can be problematic for many reasons including
risk of false discovery, compound interactions or low abundance obscuring activity, and uncertainty in knowing what compound, or
combination of compounds, may be responsible for an effect. For reasons such as these, extracts are often further subjected to
a variety of separation and fractionation techniques, often creating a library, to overcome some of these limitations and enable
screening of individual compounds or smaller groups of compounds. These methods are also commonly used for isolation and
purification of natural products, and many of these are also set up inline with mass spectrometry or NMR systems to characterize
fractions and individual compounds comprising a sample.
6.12.4.1 Liquid Chromatography

Liquid chromatography is the most common separation technology used in combination with mass spectrometry for natural
product discovery. Here, individual compounds in complex mixtures are separated by injecting liquid sample onto a column
containing a stationary phase with chemistry that is selective for compounds with specific types of chemical properties (e.g.
hydrophilic, phenyl groups, hydrophobic, etc.).98 Compounds bound, or interacting with, the column are then eluted off at
different points in time by controlling the solvents (mobile phase) washed over the column, usually by flow rate, composition, and
modifying composition over a time gradient. This separation is useful prior to entering a detection system (e.g. mass spectrometry,
UV, ELSD) to characterize properties of eluting compounds (e.g. mass, absorbance) and generate fractions based on time and other
properties determined by the applied separation parameters.
There are a wide range of liquid chromatography used for mass spectrometry based analysis of natural products. The most
common are described in more detail below. In general, these differ primarily in their selectivity for more hydrophobic or more
hydrophilic compounds, or possession of certain functional groups that determines specific interactions with a column chemistry.
In many cases, a single type of chromatography (e.g. stationary and mobile phase combination) may provide sufficient resolving
power for most metabolites and most sample matrices; however, a combination of different LC modes can be used to further
improve separation of metabolites or for more global profiling of natural products present in a sample.98,99
6.12.4.1.1 Reverse phase liquid chromatography (RPLC)

RPLC has a long history of use in the analysis of natural products and remains by far the most popular chromatographic
separation mode in metabolite profiling.100 Here, column stationary phases possess a hydrophobic group so that compounds
with hydrophobic properties, typically having partitioning coefficients in octanol/water mixture (log P) ranging from −1 to 7,101
retain easily on these types of columns. In a reverse phase chromatographic system, a polar mobile phase (e.g. water) is first
applied to the column followed by dilution with a more nonpolar mobile phase (e.g. methanol or acetonitrile). Under these
conditions, compounds with more polar attributes elute earlier and more nonpolar later as a result of stronger hydrophobic
interactions with the stationary phase. The most common stationary phase used in RPLC is silica porous particles bonded to alkyl
chains on the surface, which is typically an alkane with 18 carbon backbones (C18) following treatment with an octadecylsilane
(ODS).102 Compared to C18, shorter alkyl chains (C8 or C4) are less retentive for nonpolar compounds and provide unique
selectivity for strong hydrophobic metabolites (log P ranging from 8 to 30), such as cholesta-3,5-diene, coenzyme Q10, and long
chain lipids.103
For compounds with acidic and basic groups, the retention of analyte is strongly dependent on the pH of the mobile phase.104
The relation of eluent pH and analyte pKa determines the charge state of the analyte in the mobile phase. For weak organic acids,
retention on a RP column can be improved by lowering the pH of the mobile phase to avoid deprotonation of acids.
Mass spectrometry with a C18 column is often applied for detection of vitamins.105 For example, 25-hydroxyvitamin D3, one of
the critical indicators for vitamin D metabolism, was derivatized and detected on an HPLC-MS installed with Pro C18 RS column
reaching a limit of detection of 1.5 ng/mL.106 Reverse phase columns can also contain phenyl moieties, more polar than alkyl chains
and exhibiting strong p-p interactions with the aromatic rings of analytes, to provide increased separation resolution for aromatic
and some polycyclic compounds. A phenyl stationary phase has been successfully used in the identification and quantification of
10 steroids and eight sulfated steroids by LC-MS.107
6.12.4.1.2 Normal phase chromatography (NPLC)

Unmodified silica particles also have a long history in the analysis of natural products. Here, polar metabolites elute in order of their
polarity, which is why this is termed normal phase chromatography. The silica materials exhibit high mechanical strength and
tolerance of organic solvents, with the surface of unmodified silica particles consisting of −OH groups.108 The mobile phases used in
NPLC usually consists of solvents such as n-hexane or n-heptane, 2-propanol, ethanol, methanol, etc. One drawback of using NPLC
is sensitivity to the presence of water in the mobile phase, which may strongly influence the resolution and reproducibility of
chromatographic separation.109 An example of using an NPLC-MS/MS platform was in the quantification of four major carotenoids
and tocopherol from several botanical materials, for example, vegetable oils, fruit juices, and wheat bran.110 Heudi et al. reported
using isocratic NPLC with hexane/dioxan/2-propanol (96.7:3:0.3, v/v/v) as the mobile phase to separate vitamin A, D3, and
E simultaneously.111
6.12.4.1.3 Hydrophilic interaction liquid chromatography (HILIC)

Hydrophilic interaction liquid chromatography is a relatively new type of normal phase liquid chromatography that was firstly
introduced by Alpert in 1990 for the retention of polar analytes.112 Similar to RPLC, a wide range of functionalized groups linked to
silica particles are used to create stationary phases with different properties.113 Bonded phases with cyano-, diol-, and amino-
moieties are typical HILIC chemistries exhibiting increasing retention for polar compounds.114 Recently, mixed or zwitterionic
columns are becoming increasingly popular because they are able to retain both positively and negatively charged metabolites.
There are many examples of using this approach in the separation of secondary metabolites and peptides.115–117 Mobile phase
additives (typically weak acids or bases) are added to manipulate the electrostatic interaction of the analytes and the solid phases in
HILIC and to maintain consistent analyte charge states. It should be noted that the pH in the mobile phase can change significantly
during the chromatographic gradient, which may cause peak splitting or tailing, especially in mixed mode. Formic acid, ammonium
acetate, and ammonium formate are common additives when HILIC is used preceding MS detection. Primary metabolites,
including amino acids, amino sugars, and nucleosides, are successfully detected in foods, rhizosphere, and soil by HILIC-MS
system using water/acetonitrile/formic acid as a mobile phase, reaching a quantification limit at nmol/L level.118–120 Strege
proposed a HILIC-MS based analytical platform for the rapid profiling of secondary metabolites, such as nocardicin, oosporein,
and vancomycin, in complex natural product extracts.121 Thanks to the extensive separation resolution of polar compounds, HILIC
is very effective in supporting high-throughput metabolite characterization in natural product drug discovery, enabling identifica-
tion and quantification of hundreds of metabolites from raw plant materials.122
6.12.4.1.4 Ion exchange chromatography (IEX)

Ion exchange chromatography is widely used for separating diverse compounds including carbohydrate and alcohol isomers as well
as sugar phosphates and nucleotides.123,124 A typical stationary phase for ion exchange chromatography is highly crosslinked
styrene or divinylbenzene polymer modified with various functional groups. One common functional modification is the addition
of quaternary ammonium moieties, which are permanently positively charged, making this phase well-suited for anion exchange
chromatography. When carbohydrates are the target for separation, 10–200 mM sodium or potassium hydroxide can be added to
the elution gradient to deprotonate carbohydrates.125 Unfortunately, due to the high concentration of non-volatile salts in the
mobile phase, ion exchange chromatography is typically not directly compatible with MS systems. To address this, an ion
suppressor can be placed between the column and MS. This suppressor acts by exchanging the cations in the mobile phase with
hydronium ions, so the protons will then react with the hydroxide in the mobile phase to form water molecules resulting in very low
interference with the conductivity detector and ionization source of MS.126 An example of this approach is the high performance
anion exchange chromatography used with a Dionex ASRS ion suppressor that removes sodium ions from the gradient and is
directly connected to the electrospray ionization interface of a quadrupole MS. This was used to detect secondary metabolites in the
glycolysis pathway of Saccharomyces cerevisiae and pyrolysis of carbohydrates.127,128 Due to the high water content of the mobile
phase after suppressor, pumping additional organic solvent or modifier prior to the ionization interface of the MS can greatly
increase the ionization efficiency of analytes.
6.12.5 Ionization and Detection of Compounds

6.12.5.1 Mass Spectrometry Sources
For detection by MS, compounds must first be capable of ionization by the mass spectrometry source in use. Often, some
compounds ionize better using one type of source over another, or not at all in some sources. Electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) are two sources commonly used in MS since they are (1) “soft” ionization
methods, leaving molecules largely intact and causing less fragmentation compared to electron ionization (EI), for example, and (2)
capable of ionizing a wide range of molecules. There are numerous other metabolite desorption/ionization methods for natural
product analysis including desorption electrospray ionization (DESI) and matrix-assisted laser desorption/ionization (MALDI),
with some of the more common described here.
6.12.5.1.1 ESI
ESI was first introduced by Fenn in 1989 to generate intact molecular ions for MS analysis.129 In the ESI interface, eluent passes
through a sharp steel needle to which a high voltage, typically 2–6 kV, is applied.130 Charged eluent droplets are created, then
heated and evaporated to form a Taylor cone under the high electric field. Due to increased tension on the droplet surface, Coulomb
fission occurs, and droplets repeatedly decompose to much smaller droplets. Subsequently, gas phase analyte ions are released from
the droplets and carried into the MS analyzer with dry nitrogen. Typically, charged ions generated in the ESI interface are protonated
or deprotonated, [M + H]+ or [M − H]−, respectively, but can also form a range of other adducts with other elements in the solvent
including sodium, potassium, or ammonium. The ability of the MS to detect an analyte, or sensitivity, is highly dependent on the
ionization efficiency of the analyte, that is, how much of the analyte is ionized into detectable gas phase ions. For this reason, even
when two compounds are present at the same concentration in a sample, the MS signal for one may be orders of magnitude higher
than the other.
The ESI response is also affected by various factors including flow rate, solvent additives, and analyte physicochemical properties.
Excessive amounts of salt should be avoided in the mobile phase and removed if possible since this often leads to severe ion
suppression and a characteristically low MS signal.131 Modifying flow rate can also influence ionization, with one ESI MS study
showing that decreasing flow rate enhanced detection limit of peptides by 25–50 fold.132 Another strategy to improve ionization is
derivatization, or attaching a moiety to target analytes to improve ionization, usually by adding a moiety that will more easily
protonate or deprotonate compared to the original chemical structure.133 Recently, a quantitative structure-property relationship
(QSPR) study was performed that correlated compounds properties with ESI sensitivities and found that molecular volume, log P,
pKa, and surface activities most relevant to the ESI ionization efficiency.134 Another significant property influencing ionization in
ESI is a compound’s capability of being multiply charged. Here, large molecules, like proteins and peptides, can be multiply charged
due to having multiple charge carriers (amino and carboxyl groups). This charge state distribution of the analyte can provide critical
information about molecular conformation. For example, myoglobin is a common protein consisting of 153 residues, and when in
the unfolded conformation, shows a broad distribution of charge state ranging from 8+ to 21+, but in the native folded form, only
two charge states (8+ and 9+).135
6.12.5.1.2 APCI
The APCI interface is a similar ionization source to ESI, but here, high voltage is applied to a separate corona discharge needle and
the eluent is sprayed with dry nitrogen.136 In contrast to ESI, the ionization of analyte occurs in a vapor state rather than in liquid
droplets. Briefly, the analyte is first vaporized to the gas phase and the H3O+ and CH3OH+2 vapor typically serves as a proton donor.
Analytes with high ionization efficiency using APCI are typically volatile and have high gas-phase basicity.137 In a metabolomics
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10
0
Phenolic Organic Simple Secoiridoids Flavonoids Lignans Fatty acids Pentacyclic Tocopherols Sterols
acids and acids and phenols and and and triterpenes
aldehydes coumarins derivatives derivatives derivatives
LC-ESI-MS(-) LC-ESI-MS(+) LC-APCl-MS(-) LC-APCl-MS(+) GC-APCl-MS(+)
Fig. 11 Chart comparing the normalized area of different compound classes detected using various combinations of mass spectrometry sources (ESI and APCI),
mode (positive or negative) and chromatography (GC and LC). Modified under public license from Olmo-Garcia, L.; Kessler, N.; Neuweger, H.; Wendt, K.; Olmo-
Peinado, J. M.; Fernández-Gutiérrez, A.; Baessmann, C.; et al. Unravelling the Distribution of Secondary Metabolites in Olea europaea L.: Exhaustive Characterization
of Eight Olive-Tree Derived Matrices by Complementary Platforms (LC-ESI/APCI-MS and GC-APCI-MS). Molecules 2018, 23, E2419. https://doi.org/10.3390/
molecules23102419. https://creativecommons.org/licenses/by/4.0/.
study comparing ESI and APCI, APCI was found to be preferable for the ionization of carbohydrates and tartaric acid in grape
berries.138 In another study using ESI and APCI interfaces, Rebane et al. compared the ionization of 40 compounds across a wide
range of pKa and logP and found that some of the highly non-polar compounds such as pyrene and anthracene could only be
ionized using the APCI source. The data also showed that ionization efficiency was positively correlated with weighted average
negative sigma, logP and molar volume.137 In another study, a global profiling of secondary metabolites in olive trees using ESI and
APCI in both positive and negative mode (Fig. 11) found that polar metabolites, including phenolic acids, flavonoids, and
secoiridoids, demonstrated highest sensitivity in positive mode ESI, while organic acids and coumarin ionized better in negative
mode ESI.139 In comparison, nonpolar sterols and tocopherols ionized best using APCI.
6.12.5.1.3 DESI
DESI is a similar ionization method to ESI but allows for ambient sampling off solid surfaces such as mineral or biological tissue.
Here, solvent spray is emitted from a capillary that flushes analytes on the solid surface. High voltage applied to the capillary allows
direct ionization of the molecules, and these intact ions are directly transported to the MS analyzer. One advantage of DESI is that
the movement of solvent capillary across the sample surface can be programmed via computer, enabling the simultaneous
collection of spatial information of the molecules from the solid surface to create an MS image.140 DESI has been used for the
measurement of sugars, nucleotides, secondary metabolites, peptides, and protein. > 15 alkaloids were identified in situ on the
surface of plant seeds, roots, stems, leaves, and flowers without any pretreatment using DESI coupled to LTQ MS. The measured
spatial distribution of the alkaloids in the plant tissues was in agreement with the conventional extraction and MS analysis
results.141 Wiseman et al. reported an in vivo DESI imaging application on histological tissues including brain, kidney, and lung.
Concentrations of several key metabolites and the distribution of antipsychotic (clozapine) in rat organs were monitored by tracing
the intact ions of the analytes.142
6.12.5.1.4 MALDI
Matrix Assisted Laser Desorption/Ionization is a fairly mature and widely used technology.143 In MALDI-MS, analytes are
co-crystallized with a matrix material (commonly organic acid compounds) that absorbs laser light and transfers energy to the
analyte, releasing it into the gas phase in a process leading to ionization. The ionized analytes are subsequently introduced into
a mass analyzer for detection. The most commonly used mass analyzer with MALDI is time-of-flight (TOF) mass analysis, but
several other mass analyzers have been used including Fourier transform ion cyclotron resonance (FTICR), ion trap Orbitrap and
quadrupole Orbitrap analyzers.144 MALDI is a versatile ionization platform, combining relative high lateral resolution (typically
tens of microns), high detectable mass range, high speed (laser frequency up to 2 kHz), and molecular specificity.145 As MALDI can
be operated under atmospheric pressure or high vacuum, the source pressure has far-reaching influence on, for example, sample
conditions, detectability of volatile components, and molecular ion stability. The major drawback of MALDI is the interference of
matrix ions in the low mass range, which can obscure analytes <500 Da by abundant matrix cluster ions. This has motivated efforts
to develop matrix-free laser desorption ionization (LDI) approaches, including surface assisted laser desorption ionization (SALDI).
6.12.5.1.5 SALDI
A variety of matrix-free SALDI techniques have been developed that are based on nanoscopic structures that efficiently utilize the
laser pulse energy for the production of ions exclusively from the adsorbed analytes.146 The majority of these are based on some
form of nanostructured silicon, including desorption/ionization on silicon (DIOS), nanopost arrays (NAPA) and nanostructure-
initiator mass spectrometry (NIMS).147 While both DIOS and NAPA are based on the direct laser desorption/ionization of analytes
directly from these planar substrates, NIMS uses a unique approach to laser desorption in which a liquid “initiator” is coated onto
a nanostructured surface to aid ionization.148 Although the precise mechanism is not well understood, the laser heats the NIMS
surface, leading to the explosive vaporization of the trapped liquid coat and ionization of the adsorbed analytes on the NIMS
surface.
6.12.5.2 Mass Analyzers

Once the compound has been desorbed/ionized by the source, mass to charge ratio (m/z) measurement occurs by a corresponding
mass analyzer with detector. Many types of mass analyzers have been designed with varying levels of mass accuracy, sensitivity and
other characteristics. Mass analyzer choice often depends on the specific application and a number of factors such as limit of
detection (sensitivity), ability to interface with a particular source, mass accuracy (resolving power), scan rate, mass range, and mass
of target compounds. Hybrid systems take advantage of various properties of each, and more recently, some systems have started
incorporating ion mobility (IM) analyzers, making IM-MS systems, so separation prior to reaching the detector is determined not
only by mass but a molecule’s structural and ionic charge characteristics as well. More extensive overviews are available, but some of
the most commonly utilized analyzers for natural product discovery are described here.149,150
6.12.5.2.1 Quadrupole
One of the most popular and versatile mass analyzers is the quadrupole owing to being relatively low cost, compact, and robust, but
do not generally have very high mass accurace. These are composed of four rods in parallel creating a path through which ions flow
from an initiating source. Typically, both RF and DC fields are applied to the rods to cause oscillation of ions as they flow through.
By changing the RF and DC potentials, ions can be “filtered” by the quadrupole since some ions of particular m/z values will have
unstable trajectories and collide into the rods. Rather than mass filtration, quadrupoles can also be used as an ion transmission
guide (using RF only) to move all ions, for example, from the source into another analyzer. Also, when using only RF and varying
the applied energy, the quadrupole can be used as a collision cell and fragment ions via collision-induced dissociation (CID).
6.12.5.2.2 Ion trap

The Paul Trap, or 3D ion trap, was one of the more common earlier ion traps. Here, a hyperbolic ring electrode is located between
two hyperbolic electrode plates. Similar to the quadrupole, both RF and DC fields are applied, but in this configuration, ions are
trapped between the electrodes; then, by changing RF energy, ions of different m/z are ejected from the trap and detected. The 2D ion
trap, or “linear” ion trap, is now more common and interfaced easily with other analyzers in a system. This is constructed similarly
to a quadrupole but a potential is applied to the ends of the poles to trap ions, which may then be ejected by varying RF energy. The
main advantage of ion traps is increased sensitivity owing to the ability to accumulate ions over time, as well as being small and
affordable. Unfortunately, these analyzers have very low resolving power, making them unsuitable for compound identification in
complex mixtures or determining a molecular formula. As a result, these are commonly used to advantage in hybrid instruments to
accumulate ions and function as a mass filter in tandem with a higher mass accuracy analyzer.
6.12.5.2.3 Time-of-flight (TOF)

At a basic level, TOF analyzers are comprised of a flight tube and acceleration grid to which a pulsed electric potential is applied. This
pulse causes a “packet” of ions to accelerate from the grid through the flight tube to a detector; here, the time taken for an ion to
travel through the tube and reach the detector, or “time of flight,” is a function of mass (since distance traveled and initial kinetic
energy remain constant). Modifications to this basic configuration are often implemented to improve mass resolution, for example,
by adding a reflectron or delaying extraction after ion formation. TOF mass analyzers are very fast and have a large mass range
making them well-suited for interfacing with pulsed ionization sources (e.g. MALDI) often used in performing high-throughput
screening in natural product discovery. The main drawback of these can be limitations in precursor ion selectivity.
6.12.5.2.4 Fourier transform-ion cyclotron resonance (FT-ICR)

Rather than electric fields, FT-ICR analyzers use magnetic fields to trap ions in a device similar to a 3D ion trap. In the magnetic field,
ions circulate and oscillate, then a frequency sweep of an RF field excites ions into a higher cyclotron orbit allowing for detection.
Signal intensities from ions at the receiver plates are digitized to a frequency spectrum by Fourier transform, and the cyclotron
frequency of ions are proportional to m/z. FT-ICR has some of the very highest mass resolution capabilities (1 million and higher),
with resolution increasing with increasing magnetic field strength. However, FT-ICR also has the associated drawbacks of slow
acquisition speed, being very expensive, requiring large amounts of lab space, high maintenance, liquid helium, and implementing
large superconducting magnets. Given these conditions, in natural product discovery, FT-ICR analyzers are typically useful for
determining elemental composition for compound identification, especially when the highest resolving power and mass accuracy
are required for analysis, but are generally not practical for high-throughput or library/fraction screening.
6.12.5.2.5 Orbitrap
The basic orbitrap configuration is a small inner electrode spindle between two hollow concave electrodes to which voltages are
applied. While both orbitrap and FT-ICR analyzers use Fourier transform to convert signals for measuring m/z, orbitraps use
an electric rather than magnetic field to induce oscillation of ions in a trap. Both FT-ICR and orbitraps have very high resolving
power suitable for determining elemental compositions; however, orbitrap resolving power decreases in the low mass range
(decrease proportional to square root of m/z measured) whereas FT-ICR decreases in the higher mass range (e.g. high molecular
weight proteins). Orbitrap analyzers are also advantageous in that they are relatively small and low-maintenance, and commercially
available instruments offer fast scan speeds suitable for processing hundreds to thousands of samples required for many natural
product discovery workflows.
6.12.5.2.6 Tandem mass analyzers

Often, an MS instrument will implement two or more mass analyzers in sequence in order to expand capabilities as well as types of
data collected beyond just a single analyzer. In a simple set-up, the first analyzer will typically be used to select specific m/z ions,
which are then subjected to CID for fragmentation, and subsequent mass analyzer used for analyzing resulting product ions
generated to provide structural information. Some of the most common commercially available tandem MS instruments include
triple quadrupole (QQQ), QTOF (quadrupole-TOF), TOF-TOF and quadrupole-orbitrap.
QQQ is one of the most economical but not typically high resolution. This implements three quadrupoles in sequence, with the
first (Q1) used to either scan a range of m/z or filter to specific m/z, then the second (Q2) used to fragment as a collision cell, and
then the third (Q3) used to analyze the product ions or monitor a specific m/z. These instruments are widely used for clinical
metabolomics given their high sensitivity but are not typically used for natural product research given the difficulty in identification
of unknowns due to their low mass accuracy.
In contrast, the QTOF, in which a TOF analyzer is used on ions filtered first through a quadrupole, takes advantage of the higher
resolution of the TOF while using the speed of the quadrupole to select ions for fragmentation. This results in higher mass accuracy
and scan speed than a QQQ, and a QTOF interfaces easily with ion streams coming from a constant LC stream. In comparison, with
TOF/TOF, two tandem TOFs with CID between them results in very fast acquisition rates making this set-up ideal for ions
originating from a MALDI ion source. However, it is typically difficult to fragment small ions which may be a limitation for natural
product research. Like QTOFs, quadrupole-orbitraps (using an orbitrap as opposed to TOF) are popular instruments for natural
products and as discussed, typically have higher mass accuracy capabilities.
Numerous other hybrid systems have been developed, the extent of which is beyond the scope of this work. In general, analyzer
technologies are continuously trending towards attaining higher mass accuracy at higher scan rates, reducing overall instrument size
and requirements, robustness, lower cost, and of course, setting up tandem acquisition schemes to increase the richness of data that
can be collected. One of the most important for natural product discovery is MSn capability, in which an ion can be fragmented, and
then the fragments fragmented, and so on, for example, in linear ion trap orbitrap instruments. This feature is a requirement for
determining both elemental and more in-depth structural information about a compound.
6.12.5.2.7 IM-MS
More recently, the combination of ion mobility analyzers with mass analyzers has made a large impact on natural product discovery
efforts.151,152 Ion mobility is a type of gas-phase electrophoresis, with ions traveling through an electric field in a carrier gas, with
measured collision cross section (CCS) through the gas based upon structural properties (e.g. charge state, size and shape).
By combining accurate mass m/z measurements with ion mobility “drift time”, co-eluting isomers that do not separate using LC,
for example, may still be distinguished based on varying drift times. In natural product discovery, ion mobility can be important for
prioritization of features in which species can be seen to deviate from predicted mobility trendlines. This may indicate a compound
with unexpected physicochemical properties and a potentially new natural product. Additionally, IM is another feature to add to
traditional MS measurements of m/z, fragmentation and retention time to aid in dereplication, identification and structural
elucidation. MALDI-based systems, specifically, or others used in MS imaging applications and fast high-throughput screening,
are typically lacking LC chromatography retention time-based separation and most susceptible to simultaneous ionization of
isomers with the same m/z. These benefit greatly from integration with IM allowing separation and fragmentation of isomers, and
adding a time-dimensionality to acquired mass spectra increasing the depth of information capable of being collected.
6.12.5.3 Data Collection

Methods applied for LC/MS data acquisition is largely determined by the capabilities of the instrumentation and experimental
requirements. In all cases, acquired spectra typically consists of MS1 containing m/z of ions produced by the ionization source. If the
mass spectrometer has fragmentation capabilities, then MSMS (MS2) data is also collected, and potentially higher levels of MSMS
(MSn) by subsequent fragmentation of fragment ions.
Acquired data will have m/z measurement accuracy and resolution based primarily upon the type of analyzers comprising the MS in
combination with the rate of data collection, and whether or not MSMS is also being collected and at what frequency. On a given
instrument, faster acquisition rates typically have a corresponding decrease in mass accuracy which hinders compound identification
based on exact mass and determining a chemical formula. However, advantageous trade-offs include (1) MS1 chromatograms
(intensity vs. time) for each m/z value having a well-defined peak shape due to a larger number of data points collected during
an ion’s elution and (2) a higher likelihood of having collected associated MSMS fragmentation spectra for compound identification.
Using a longer LC column and longer LC method would allow for data collection at a rate allowing for higher mass accuracy and may
also improve separation between isomers. Unfortunately, this increases the overall runtime for each sample and may be overly time-
prohibitive when hundreds to thousands of samples need to be analyzed in some types of natural product discovery workflows. Given
these considerations, overall sample runtime needs to be balanced with quality and amount of MS and MSMS data capable of being
collected. For example, one strategy, when screening thousands of samples, could be to implement a fast LC method that collects only
MS1 data at a high acquisition rate but no MSMS, and using the MS1 data to determine which samples require follow-up with
a different LC method and MS/MSMS analysis. Or similarly, a fast LC method that collects low resolution MS1 data with low quality
peak shape but very high amounts of MSMS, and use MSMS as the main determinant of which samples require follow-up.
In collection of fragmentation spectra, a collision energy is specified to apply to either an isolated precursor ion or over a given mass
range of precursors, depending on whether the MSMS is data dependent or independent, respectively. Higher collision energies
typically result in a larger number of fragments with smaller m/z values and lower collision energies with less fragments of higher m/z
values. The fragments present at different collision energies are related to the molecule’s chemical and structural composition. These
are characteristic for individual ions leading to identification when compared to a standard, and in the absence of a standard, can also
be used to determine a molecule’s chemical and structural composition. However, sometimes a molecule fragments poorly and higher
collision energies are necessary to achieve any fragmentation at all. For this reason, when analyzing complex samples, more
information can be attained by applying a ramp or stepped collision energy combination (e.g. 10, 20, then 30 eV), and then measuring
the fragmentation from all energies in the same MSMS spectra. Although more informative to collect MSMS spectra for a compound at
each collision energy, this has the drawbacks of being time-consuming, reduces the quality of peak shape and limits the number of
other ions for which MSMS can be collected. As mentioned elsewhere in this article, fragmentation characteristics of ions are important
not only for identification, but for dereplication and feature prioritization in natural product discovery workflows.
For an isolated precursor, fragment ions are ideally generated and detected from only that compound and used for identification.
However, depending on the instrumentation, selecting a compound for isolation and fragmentation is often associated with a mass
window (e.g. 0.01–2 Da) that, if set too wide, may allow in another co-eluting ion with nearby m/z that also fragments with the
target precursor ion and lead to convoluted MSMS spectra, or if set too narrow, results in poor isolation of the precursor ion and
poor quality, low intensity MSMS spectra. This factor is something to consistently take into consideration when interpreting MSMS
spectra of complex samples.
Fragmentation may occur in either a data dependent or data independent manner, or some combination of both. With data
independent acquisition, all ions within a specified m/z range will be fragmented resulting in a multiplexed spectra from multiple
precursors. While it can be challenging to relate specific fragments to the originating precursor, the fragmentation data nevertheless
is unbiased and will be collected for all eluting ions, especially those that may be missed in a data dependent acquisition scheme.
Deconvolution of this spectra may include assigning fragments to precursors with matching elution patterns, for example. Or, in
some cases, specific fragment m/z values can be indicative of a particular class of compounds (e.g. 184 for phosphocholine lipids).
Data dependent acquisition occurs in two main formats. In a “targeted” format, specific ions will be fragmented from a pre-
determined list comprised of attributes including retention time, m/z, minimum intensity, and polarity, for example. In an
“untargeted” format, fragmentation spectra will be acquired based on MS1 spectra, with the goal often to be to acquire MSMS
data on as many compounds as possible without compromising MS1 data quality. Typical implementations are based on
fragmenting, for example, the x most intense ions detected via MS1, and after fragmenting them, adding these to an exclusion
list from fragmentation for y amount of time, and then continuously fragmenting the x most intense ions in MS1 as long as they are
not currently in the exclusion list. This results in not fragmenting the same ion over and over, and provides the opportunity for less
intense ions to also get fragmented. The main advantage of this type of data dependent acquisition is that in a single run,
fragmentation spectra can be obtained for a large number of individual ions for samples with unknown composition. However,
as discussed previously, obtaining higher amounts of MSMS data to include even the low abundance ions is associated with the
trade-offs of having to increase sample runtime, or lower quality peak shape, or lower mass accuracy MS1 measurements overall.
More recently, instrumentation software has been developed to improve MSMS coverage where ions fragmented in one injection are
then added to an exclusion list for the following injection, which is a re-injection of the same sample. This process can be repeated
until essentially all ions, including low abundance, are fragmented. Numerous other types of data dependent acquisition schemes
are possible and can be tailored to experimental design requirements.153,154
This is just a brief overview of some of the factors to consider when setting up the specific data acquisition scheme in a natural
product discovery workflow. More extensive literature discussing these concepts and more are available.155–157 Specific instrument
utilized (and available software) will be a primary factor determining the capabilities and MSMS options available for data
acquisition.
6.12.6 Detection of Select Natural Product Classes

6.12.6.1 Polyketides
Polyketides are a structurally diverse class of secondary metabolites containing alternating carbonyl and methylene groups and are
synthesized in a wide variety of organisms including plants, bacteria and fungi.158 While their roles in nature are thought to be
related to pigmentation, chemical communication/signaling and/or defense, they have been utilized in numerous pharmaceutical
(A) Reduced polyketides

O
OCH3
O
HO O
H3CO OH OH
OH O
H3CO
HO O
O O OH O O O
O
O NH
H3CO
O O N O H3CO
O O OH
HOOC O
O
Rapamycin O
OH Rifamycin S
Monensin A Polyketide-peptide
Polyether antibiotic Ansamycin antibiotic immunosuppressant
OCH3
HO
O OCH3
O
O
O O
HO O O
OH
OH
O OH O
O O O
HO O OH OH OH O OH
COOH O
O
O O
OH OH
NH2
OH
Amphotericin B Lovastatin Avermectin B1b
Polyene antifungal Cholesterol-lowering agent Macrolide antiparasitic
HO OH (H3C)2N O
O OCH3
S N(CH3)2
OH O
HO OCH3
O OCH3
HO
N O O
O O
O
O
O O OCH3
O
O OH O
O OH
Epothilone
Polyketide-polypeptide Erythromycin A Spinosyn A
anticancer agent Macrolide antibiotic Macrolide insecticide
(B) Aromatic polyketides (C) Unclassified

O OCH3
O OH O
OH O N
H
OH
O O
HOOC O O O O COOH
OCH3 O OH O
OH OH
O
O (H3C)2N O O
OH OH
NH2 OH
OH O O O
O
HO OH
Doxorubicin
Cl
Aromatic antitumour Actinorhodin C-1027
agent Aromatic antibiotic Enediyne antitumour agent NH2
Fig. 12 Structural and bioactivity diversity of polyketides. Modified with permission from Weissman, K. J.; Leadlay, P. F. Combinatorial Biosynthesis of Reduced
Polyketides. Nat. Rev. Microbiol. 2005, 3, 925–936. https://doi.org/10.1038/nrmicro1287. Copyright (2005) Spring Nature.
applications such as antibiotics, anticancer drugs and immunosuppressants.159 Polyketides are synthesized by condensation of
acetyl units catalyzed by a multi-domain enzyme, polyketide synthase (PKS), which carries out varying degrees of keto reduction,
dehydration, enoyl reduction and cyclization reactions, followed by post-PKS tailoring reactions.159 The broad substrate specificity
of PKSs for starter and extender units, along with the variety of tailoring of reactions, results in a large diversity of chemical structures
and bioactivities (Fig. 12).159a Mass spectrometry has proven to be a useful tool in the discovery and structural characterization of
novel polyketides.160 PKSs have been utilized in medicinal chemistry for production of novel polyketides using non-natural
substrates and combinatorial biosynthesis strategies where mass spectrometry plays a vital role in rapid identification of novel
products.161,162 Further, while genome mining may identify novel biosynthetic clusters of polyketides, detection of their products
under natural conditions may be challenging due to the low level expression of biosynthetic genes. Thus study designs incorporating
condition screening and mass spectrometry have been beneficial for discovery of novel, natural scaffolds, such as Streptomyces-
produced formicamycins, linearolides and their analogs.163,164 Another challenge with polyketide identification is the vast array of
structural isomers, sometimes produced by a single organism. In this case, tandem mass spectrometry is a robust technique for
comparison of fragmentation patterns between these types of molecules; for example, the identification of two polyketide isomers
(isolasalocid A and lasalocid A) was achieved using MS3 in high resolution Fourier transform ion cyclotron resonance MS with
electron-induced dissociation and collision-activated dissociation.165 Stereochemical isomers, also common in polyketides,166,167
present an additional challenge that can be addressed by mass spectrometry with the combined use of chiral chromatography
and/or derivatization.
6.12.6.2 Glycosides
Glycosides present another diverse class of secondary metabolites, where a sugar moiety is linked to an aglycone molecule by an O-,
S-, N- or C-glycosidic bond.168–170 In some molecules, the glycosidic residue is necessary for bioactivity171 whereas in others, the
glycosides function as storage molecules from which hydrolysis releases an active aglycone172 or a cytotoxic defense chemical such
as hydrogen cyanide.173,174 Commonly, glycosides are classified by the aglycone residue, such as phenolic glycosides, flavonoid
glycosides, etc. or by bioactivity, such as cardiac glycosides, antibiotic glycosides.171 Glycosidic residues typically increase the
aqueous solubility, may alter bioavailability and are thought to control transport of the aglycone in tissues.171 In a recent review of
16,000 natural products from bacteria attempting to cover all currently known natural products, approximately 20% were
glycosylated, thus representing a significant source for discovery of novel therapeutics (Fig. 13).175 Due to the presence of common
residues in glycosides, neutral loss scanning of MS/MS and product ion scanning are logical analytical approaches, both of which
have been successfully applied in glycoside analysis.176,177 The issue of lability variability in complex mixtures of glycosides may be
addressed with collision energy gradients, thus improving structural information and coverage when screening samples with
a variety of classes of glycosides.178 Fragmentation information can elucidate: glycosidic bond types between sugar moieties and
to the aglycone, type and order of monosaccharides, and length of polysaccharide chains.179 For example, fragmentation pathways
were used for the structural identification of cyanogenic glucosides from Phyllagathis rotundifolia.180 Hydrolysis of the glycosidic
bond, prior to analysis, may also provide better structural information due to the ability to selectively fragment the aglycone moiety;
this technique yielded the identification of six cardiac glycosides including two novel compounds from yellow oleander seed.181
A systematic nomenclature for carbohydrate fragmentation has been developed.182 Consistency in the labeling and identification of
fragments has improved the ability to assign partial or complete identities to detected glycosides based fragments reported in prior
literature.177,180
6.12.6.3 Nonribosomal Peptides (NRPs)

NRPs are peptide secondary metabolites mainly produced by bacteria and fungi.183 These peptides are synthesized by enzymes
called nonribosomal peptide synthetases (NRPSs) unlike canonical polypeptides such as proteins.184,185 Many of them are used
clinically as antibiotics and immunosuppressants, such as daptomycin, vancomycin, and ciclosporin.183 Unlike ribosomally-
synthesized peptides, NRPs mainly consist of non-proteinogenic amino acids, which are far more diverse (>500).186,187 In
addition, only approximately one-fourth of known NRPs are linear structures, while most of them have branches or cycles in
their structures.187 Due to the high diversity and complexity of NRPs, advanced LC-MS/MS-based analysis is a powerful tool in
characterizing and discovering novel NRPs.186 In a rational NRPs engineering study, secreted and intracellular benzylpenicillin were
quantified from engineered Saccharomyces cerevisiae using LC-MS/MS platform.188 Paenilamicin is an antibiotic produced by a novel
nonribosomal peptide biosynthetic pathway in Paenibacillus larvae. Müller et al. utilized LC-MS and GC-MS to identify and analyze
four paenilamicin, of which structure was further characterized by multidimensional NMR.189 In addition, high resolution mass
spectrometry can aid NRP discovery by integrating the in silico fragmentation into informatic search algorithm to detect the
fingerprint of unknown NRPs.186 Ng et al. developed an algorithm to compare similar but not identical multistage MSn spectra of
known cyclic NRPs; this can be utilized to predict the novel NRPs which are similar to identified NRPs.190 Finally, database, such
as NORINE, can help in the prediction and identification of novel NRPs.191
6.12.6.4 Alkaloids
Alkaloids are a class of chemical compounds that contain nitrogen in a ring structure, such as isoquinoline and piperidine, and
typically derived from amino acids. They are widely distributed in various plants, animals, and microorganisms, and are bioactive
even in extremely low doses.192 Alkaloids have a long history of being used as drugs. Examples include vinblastine and vincristine,
which can be used as an anticancer agent by prohibiting the assembly of microtubules.193 Most alkaloids can be easily dissolved in
organic solvents rather than in water. They are thermally stable and can be easily extracted using conventional techniques with
methanol or ethanol.194,195 Six alkaloids which can inhibit acetylcholinesterase activities were extracted using ethanol from the
Fig. 13 Neutral loss and fragment analysis for identification and structural elucidation of glycosides. Modified with permission from Barnaba, C.; Dellacassa, E.;
Nicolini, G.; Nardin, T.; Serra, M.; Larcher, R. Non-targeted Glycosidic Profiling of International Wines Using Neutral Loss-High Resolution Mass Spectrometry.
J. Chromatogr. A 2018, 1557, 75–89. https://doi.org/10.1016/j.chroma.2018.05.008. Copyright (2018) Elsevier. Representing the carbohydrate fragmentation
nomenclature as defined by Domon and Costello.182
stems of a native Brazilian plant.195 Although alkaloids are typically weakly basic molecules, they are very diverse with their
polarities. Thus, RPLC, NPLC, HILIC, or IEX have been all successfully applied to the separation of alkaloids depending on the
physicochemical properties of the target compounds. RPLC can be performed under either acidic pH by using formic acid as a buffer
or under basic pH with ammonium.48,196 Alkaloids are mainly in ionic form under lower pH, which may significantly decrease the
retention time and better sharp peaks can be expected.197 Bagheri et al. tested the separation performance of five opium alkaloids on
HILIC columns with five different stationary phases, that is, bare silica, ZIC-HILIC, amino-, diol-, and cyano-using acetonitrile and
water with 60 mM ammonium acetate as mobile phase. Longest retention of the five alkaloids was obtained on the cyano-HILIC
column, but also led to broad peak widths and low peak heights. Best chromatographic responses (signal to noise ratio) was found
on diol-HILIC column and demonstrated a LOD of 13 pg (Fig. 14).198 Isolation of psilocin, psilocybin, morphine, codeine, and
thebaine was achieved using ZIC-HILIC with acidic pH.199,200 IEX is an ideal tool for separating highly polar compounds, and
an orthogonal 2D-HPLC consisting of RPLC and IEX was proposed recently to purify hydrophobic alkaloids and highly polar
alkaloids. This 2D analytical system demonstrated a high degree of orthogonality and the IEX effectively removed the non-basic
coeluted components in the samples. In total, nearly 20 alkaloids were identified from Scopolia tangutica Maxim extracts, including
one novel alkaloid compound. Although the mobile phase may not be ideal for MS detection due to the presence of sodium
phosphate, the 2D-HPLC proved to be an emerging technique for isolation of alkaloids (Fig. 15).201
8000
2
3
7000 2
1
4
5 E
6000
2 3
5000 1 4
mAU
5 D
2
4000
2
3
1
3000 4 5 C
3
2000
1
4
5
B
1
1000
4 5 A
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Minutes
Fig. 14 Chromatographic separation of opium alkaloids using HILIC columns with five different types of stationary phase: (A) bare silica, (B) ZIC-HILIC, (C) amino,
(D) diol, and (E) cyano. Although elution order remained the same for noscapine, papaverine, thebaine, codeine and morphine, stationary phase chemistry changed
retention time and amount of separation between compounds. Modified with permission from Bagheri, M.; Taheri, M.; Farhadpour, M.; Rezadoost, H.;
Ghassempour, A.; Aboul-Enein, H. Y. Evaluation of Hydrophilic Interaction Liquid Chromatography Stationary Phases for Analysis of Opium Alkaloids. J. Chromatogr.
A 2017, 1511, 77–84. https://doi.org/10.1016/j.chroma.2017.06.072. Copyright (2017) Elsevier.
0.5
0.4
0.3
0.2
0.1
0
0 50
2 45
4 40
6 35
30
8 25
10 20 CX
XCha 12 ge S
rge C
18 14 10
15
XChar
16 5
18 0
Fig. 15 HPLC-UV chromatography of alkaloids separating by a 2D-C18 RPLC/SCX system. Modified with permission from Long, Z.; Guo, Z.; Xue, X.; Zhang, X.;
Liang, X. Two-Dimensional Strong Cation Exchange/Positively Charged Reversed-Phase Liquid Chromatography for Alkaloid Analysis and Purification. J. Sep. Sci.
2013, 36, 3845–3852. https://doi.org/10.1002/jssc.201300863. Copyright (2013) John Wiley and Sons, Inc.
6.12.6.5 Flavonoids
Flavonoids, while structurally diverse, contain a conserved core, the phenyl benzodihydropyran (flavan) as shown in Fig. 16A where
R groups are commonly hydroxy, methoxy, prenyl or glycosyl groups.76 Flavonoids are pigments produced in plants and function
as visual cues for pollination, as chemical deterrents to herbivory, and as UV-protectants.76 They exhibit pharmacological desirable
properties including anticancer, antioxidant, anti-inflammatory, and anti-bacterial activity.203 A range of liquid and solid phase
extraction techniques have been reported and reviewed in the literature.76,204 Flavonoid analysis from crude extracts may require
hydrolysis release free flavonoids from their glycosylated conjugates.76,205 Additionally, both reverse and normal phase chroma-
tographies have been developed for the separation and retention of flavonoids.204,206,207 Flavonoids absorb UV-vis light in two
bands: Band II between 220 and 280 nm is due to the benzoyl group (A ring) and Band I between 300 and 450 is due to the
cinnamoyl group (B and C rings) thus making incorporation of a spectrophotometer a useful tool for improving detection and
identification.204,208,209 In mass spectrometry, both APCI and ESI are commonly used ionization methods.210 A nomenclature for
flavonoid product ions produced from C-ring fragmentation events was developed by Ma et al., in 1997 (Fig. 16B),202 with
additional aglycone fragmentation events described in detail,211 while the nomenclature of flavonoid glycoconjugates follows the
conventions of Domon and Castello182 and has been expanded upon by Cavaliere et al.212 In fragmentation of aglycone flavonoids,
loss of functional groups and retro-Diels Alder reactions produce unique fragments that are useful in identification of either the
flavonoid or class of flavonoid.204 Additionally, flavonoid glycosides, produce unique fragments due to rearrangement events,
glycoside residues with predicted fragments, and sometimes depend on the type of aglycone at the flavonoid core.213 Recently,
a database of predicted fragments from approximately 7000 know flavonoid structures along with an annotation tool, Flavonoid-
Search, has been developed for annotation of flavonoids by Akimoto et al.214
6.12.6.6 Terpenoids/Terpenes
Terpenes are organic compounds consisting of isoprene, a five-carbon building block. A monoterpene is a cyclic molecule
composed of two isoprene units. Terpenoids are a class of secondary metabolites derived from terpenes with multiple cyclic groups
and oxygen. Both are derived from isopentenyl diphosphate and dimethylallyl diphosphate. Volatile terpenes are believed to play
critical roles in plant defense.215 Moreover, terpenes and terpenoids can be used as flavors, drugs, and fragrances. Terpenes are
typically volatile, while terpenoids may be non-volatile or semi-volatile as they normally contain other polar moieties.216 The
extraction and analysis protocols of terpenoids and terpenes largely depend on their polarities. Volatile or non-polar terpenes are
best extracted with hydrophobic solvent and further measured with GC-MS, but polar terpenoids are suggested to be analyzed with
LC-MS (Fig. 17).216 Unlike alkaloids and flavonoids, hexane is the major solvent used in the terpenes extraction. Terpenes have been
quantified from woods and leave hexane extracts using GC-MS in several studies, and liquid alkane hydrocarbon (dodecane,
pentadecane, and hexadecane) is utilized as an internal standard for quantification.217–219 An offline combination of C18 SPE and
solid phase micro extraction was developed to enable detection of low abundant terpene in the polar fraction.220 Sun et al. reported
a highly sensitive LC-MS based analytical method to quantify the bioactive polar terpenoid lactones in Ginkgo biloba tablets, in
which LOD is 35 fmol for Ginkgolide A and 4.9 fmol for Bilobalide.221 Polar terpenoids can also be identified using similar
analytical methods like flavonoids, for example, C8 or C18 RPLC-ESI-MS under acidic pH.47,222
6.12.7 Mass Spectrometry Imaging of Natural Products
Rather than going through the complex process of extraction and separation, mass spectrometry imaging (MSI) techniques have
been developed allowing direct examination of the spatial distribution of biological molecules within a material and even construct
two-dimensional (2D) and three-dimensional (3D) visualizations of these. MSI was popularized by Caprioli in the 1990s when he
demonstrated the spatial mapping of m/z values, using a MALDI MS system, to generate ion images of molecules comprising various
mammalian tissue sections.223 Since then, MSI has been used extensively to study the spatial distribution of biomolecules within
tissues, in microbial colonies, in chemical arrays, and other biological media, including the discovery of novel natural
products.147,224–226
MSI enables the analysis of low abundance molecules that may be obscured in bulk analyses and can correlate ions with
observed phenotypes, which may hint at the function of the compound. With MSI, mass spectra are obtained at discrete spatial
points, usually in a raster format, with a mass spectrum measured at each coordinate. From the collected mass spectra an ion image
can be created by calculating the cumulative or maximum intensity over a selection of ions for each position in the dataset, where for
example, each ion is represented by a unique color. MSI datasets can be analyzed by a wide variety of software tools, the choice of
which is determined by the nature of the research. An up-to-date overview of the most common tools for MSI data analysis has been
developed.227 Since MSI datasets are typically quite large, specialized software tools have been developed to enable visualization
and analysis. For example, OpenMSI is a powerful tool that allows for not only the storage of raw MS imaging data, but a web-based
interface with access to high-performance computing resources to efficiently visualize and analyze MSI datasets.228 Recently,
a complementary resource was developed, OpenMSI Arrayed Analysis Toolkit (OMAAT), comprised of a software package specific
for analyzing large arrays of samples acquired as an MS image.229 Together, these provide a mechanism to uncover novel natural
products in materials as well as rapidly generate and screen metabolite profiles in high-throughput for natural product discovery.
(A)
(B)
(C)
Fig. 16 (A) Common classes of flavonoids and representative compounds with R group substitutions indicated. (B) Mass spectrometry fragmentation in flavonoids.
An example retro-Diels-Alder reaction producing the 1,3A+ and 1,3B+ fragments commonly seen in flavonoids. (C) Additional C-ring fragmentation events
produce additional unique fragments to flavonoids. (A)–(C) modified with permission from de Rijke, E.; Out, P.; Niessen, W. M.; Ariese, F.; Gooijer, C.;
Udo, A. T. Analytical Separation and Detection Methods for Flavonoids. J. Chromatogr. A 2006, 1112, 31–63. https://doi.org/10.1016/j.chroma.2006.01.019.
Copyright (2006) Elsevier. Based on the nomenclature set forth in Ma et al.202
Fig. 17 A schematic representation of key terpenes and terpenoids differed in volatility and polarity. With increasing of polarity and decreasing of volatility,
compounds are preferably analyzed with LC-MS. Modified under public license from Jiang, Z.; Kempinski, C.; Chappell, J. Extraction and Analysis of Terpenes/
Terpenoids. Curr. Protoc. Plant Biol. 2016, 1, 345–358. https://doi.org/10.1002/cppb.20024. https://creativecommons.org/licenses/by/4.0/.
MSI has become more accessible and increasingly popular in the last 2 decades with the emergence of a large number of
ionization techniques and new sample preparation strategies. A wide array of MSI platforms are now available for the detection of
specific chemical compounds. Each MSI platform has unique benefits and drawbacks including variations in analysis time, what
sample types are amenable to imaging, and a wide range of sample preparation methods. The following section provides
an overview of MSI techniques with a highlight on applications geared towards analyzing natural products. These are summarized
in Table 2.
6.12.7.1 MSI Platforms

As described earlier, in desorption electrospray ionization (DESI), electrosprayed solvent is used to desorb molecules from the
sample surface for subsequent introduction into the source (Fig. 4B). In an MSI platform, the sample is raster-scanned with the DESI
interface. A modification of DESI is nanoDESI in which the sample is scanned with a droplet of solvent fed into an electrospray
ionization source. Both techniques are performed at atmospheric pressure and nanoDESI can be used to image microbial colonies
that are growing on hydrated nutrient agar directly from a Petri dish without sample preparation.233 Both DESI and nanoDESI have
been successfully applied for chemical profiling of natural products in a wide variety of organisms, ranging from single bacterial
colonies to zebrafish.251
Samples can also be imaged using laser ablation with subsequent electrospray ionization (laser ablation electrospray ionization,
LAESI). A modified version of LAESI is laser ablation atmospheric pressure photoionization (LAAPPI), where the electrospray
ionization source is exchanged for a nebulizer chip with an anisole and heated nitrogen gas flow to improve ionization. Like DESI
and nanoDESI, LAESI can be performed at ambient conditions thus allowing imaging of living microorganisms.252 However, LAESI
is a destructive method and will typically ablate the sample.
Table 2 Highlights from published literature showing the application of MSI for the analysis of natural products.
MSI platform Sample types Known chemical family/compounds visualized Refs.
Desorption electrospray ionization (DESI) Microbial colonies and Rhamnolipids, siderophore [230]
fungi
Algae Bromophycolides, and callophycoic acids [231]
(diterpenoids)
Plants (seed and fruit) Chromone alkaloid [232]
nanoDESI Microbial colonies NRPS [233]
yeast, fungi and bacteria Phenazines, quinolones, rhamnolipids, cyclic [234]
lipopeptides
Laser ablation electrospray ionization (LAESI) Microbial colonies Antibiotics (Streptomycin) [235]
Flower petals Anthocyanins, polyphenols [236]
Laser ablation atmospheric pressure photoionization (LAAPPI) Plant leaves Terpenes and terpenoids [237]
Matrix-assisted laser desorption/ionization (MALDI) Fungal and bacterial Actinomycins, macrolides [238]
colonies
Plants (root/stem/leaf/ Glucosinolates, polyphenols, flavonoids among [145]
flower/fruit/seed) others
Plant leaves Sesquiterpene lactones [239]
Laser desorption ionization (LDI) Leaves and pollen Naphthodianthrones (hypericin and [240]
pseudohypericin) and flavonoids
Roots and nematodes Phenylphenalenones [241]
Symbiotic bacteria on Antibiotics (streptochlorin and piericidin [242]
insect cocoons derivatives)
Desorption/ionization on silicon (DIOS) and nanowire-assisted laser Marine molluscs Brominated indoles [243]
desorption ionization (NALDI)
Nanostructure-initiator mass spectrometry (NIMS) Tissues, bacterial colonies Secondary metabolites [244,245]
Cation-enhanced NIMS Plant (stems) Sugars [246]
Secondary-ion mass spectrometry (SIMS) Bacterial colonies Surfactins [247]
Bacterial colonies Pigmented antibiotics [248]
Bacterial biofilms Alkyl-quinolone and alkyl-quinoline N-oxide [249]
signaling molecules
Bacteria colonizing roots Cyclic lipopeptides [250]
The highest resolution imaging approaches use secondary ion mass spectrometry (SIMS), which is based on the rastering of
a focused ion beam. This primary beam bombards the sample, ejecting and ionizing molecules from the sample surface that are
subsequently analyzed in a mass spectrometer.253 As SIMS is operated at high vacuum, samples must be dry and vacuum stable.
SIMS has been applied to visualize secondary metabolites in bacterial colonies, tissues and insects.249,253
Laser desorption ionization is another common technique used in MSI platforms with MALDI being the most widely used.
As previously mentioned, one challenge with MALDI is that abundant matrix ions (<500 Da) can interfere with analysis. However,
due to advances in laser beam technology and application of novel matrices, matrix interference can often be minimized.
Commonly, MALDI-MSI will be performed under high vacuum; however, applications of MALDI at atmospheric pressure have
been reported.144,254 MALDI has been used to image natural products in microorganisms, plants and animal/human
tissues.144,145,255
Other laser desorption ionization (LDI) approaches typically use the laser to directly ablate compounds from the sample surface.
Unlike MALDI, LDI does not require an externally applied chemical matrix and circumvents the issues associated with matrix. Direct
LDI can be performed when the molecules of interest contain chromophores that are capable of absorbing the wavelength of energy
produced by the laser, to assist in the desorption/ionization process.226 As many natural products contain conjugated double-bond
systems like aromatic/heteroaromatic rings and show strong UV absorption at 337 or 355 nm; both levels are emitted by the most
common UV lasers.252 Using LDI, antibiotics, toxins and pigments have been imaged in microorganisms and plants.226,252
A wide-range of matrix-free, surface assisted laser desorption/ionization (SALDI) approaches can be used for MSI. Major SALDI
approaches include NIMS, described earlier, desorption/ionization on silicon (DIOS), NAPA and nanowire-assisted laser desorp-
tion ionization (NALDI). The majority of these approaches are based on the direct laser desorption/ionization of compounds
directly from nanostructured substrates.10 All these SALDI platforms have been applied to study secondary metabolites in plants,
tissues, biological fluids and marine snails.146,148,243
6.12.7.2 MSI Applications

6.12.7.2.1 Natural products from interacting organisms
Synthesis of many natural products by microorganisms (or plants) often only occurs in response to the presence and signals from
other microorganisms or plants. In nature, microbes are part of polymicrobial communities (far from the monocultures in the lab)
where studies have shown interactions through direct contact and communication via various secondary metabolites, or natural
products. For example, compounds such as acyl-homoserine lactones (AHLs), oligopeptides and autoinducers have been elucidated
as fundamental for processes such as quorum sensing, quorum quenching, kin selection and pathogenicity.256 MSI has proven
a powerful tool for exploring in situ the production and discovery of new natural products by interacting organisms through spatial
mapping of detected ions.
Co-cultivation of different microorganisms in close proximity on a solid surface allows for either direct or indirect sampling by
MSI platforms. Ambient ionization based MS techniques, including DESI, nanoDESI and LAESI, can be used to probe the
microorganisms and their environment directly without any disturbances (e.g. directly from cultures on agar). MS techniques
operated under vacuum, such as MALDI, SIMS and SALDI, typically require sample desiccation prior to MSI analysis. Alternatively,
MSI can be performed indirectly by imprinting or blotting analytes onto a MS compatible surface.245,257
Traxler et al. used both nanoDESI and MALDI MSI to investigate the chemical interactions of the model bacterium Streptomyces
coelicolor with five other actinomycetes.258 In each interaction, the majority of secreted compounds associated with S. coelicolor
colonies were unique, including an extended suite of at least 12 different desferrioxamines with acyl side chains of various lengths.
The production of these natural products was triggered by siderophores made by neighboring strains. In another MSI study using
DESI MSI on seeds, the alkaloid rohitukine could be traced both spatially and temporally throughout seed development, including
modified forms of the compound (Fig. 18A).232 An example of an indirect MSI technique is “replica-extraction-transfer” NIMS,
where a solvent-laden gel is first used to extract metabolites from a microbial sample, such as a biofilm or agar culture, and then the
metabolites are replica “stamped” onto the NIMS surface.245 This approach was used to analyze the metabolite composition of
interacting Shewanella oneidensis and Pseudomonas stutzeri colonies (Fig. 18B).
Fig. 18 Application of MSI. (A) Ion images, specified by m/z, of D. binectariferum seeds during different developmental stages acquired using DESI MS. The ion m/
z 306.2 corresponds to rohitukine, a chromone alkaloid. Figure adapted from Mohana Kumara et al.232 (B) Schematic of a typical REX-NIMS workflow (left), and ion
images comprised of different m/z, indicated by color, of interactions occurring in a microbial co-culture on agar (right). (A) Modified with permission from
Kumara, P. M.; Srimany, A.; Ravikanth, G.; Shaanker, R. U.; Pradeep, T. Ambient Ionization Mass Spectrometry Imaging of Rohitukine, a Chromone Anti-Cancer
Alkaloid, During Seed Development in Dysoxylum Binectariferum Hook.f (Meliaceae). Phytochemistry 2015, 116, 104–110. https://doi.org/10.1016/j.phytochem.
2015.02.031. Copyright (2015) Elsevier. (B) Modified and reprinted with permission from Louie, K. B.; Bowen, B. P.; Cheng, X.; Berleman, J. E.; Chakraborty, R.;
Deutschbauer, A.; et al. ‘Replica-Extraction-Transfer’ Nanostructure-Initiator Mass Spectrometry Imaging of Acoustically Printed Bacteria. Anal. Chem. 2013, 85,
10856–10862. https://doi.org/10.1021/ac402240q. Copyright (2013) American Chemical Society.
6.12.7.2.2 High-throughput screening

As described above, the main application of MSI has been the examination of the localization of biological molecules. However,
MSI enables the comparison of thousands of spatially defined samples allowing high-throughput characterization of biological
samples. Desorption/ionization based MSI methods can achieve 10,000 samples/day.10 However, often MSI platforms are
separation-free (e.g. without chromatographic or electrophoretic separation), so analyte coverage is often limited to the most
abundant and easily ionized compounds in complex sample compositions.10
DESI MSI has been applied to screen compound libraries in complex biological sample matrices and MALDI MSI has been used
to screen carotenoids in crude extracts from plant tissues.259,260 Further, by combining NIMS with acoustic sample deposition,
which is a contact-free liquid transfer approach, Greving et al. was able to print 10,000 samples onto a NIMS surface (Fig. 4A).22
Although separation-based MS platforms are the primary approaches for natural product analysis, high-throughput MSI platforms
can be complementary to existing MS screening methods, for example, by pre-screening large numbers of samples for subsequent
LC-MS/MS analysis. Recently, a new method was developed combining IMS and high-throughput elicitor screening (HiTES), or
HiTES-IMS.261 In this work, silent BGCs were activated by exposing genetically unmodified cultures with compounds from a natural
product library and testing 500 different conditions. To find new NPs, extracted supernatants were rapidly screened using IMS with
LAESI to create mass spectrometry profiles across all conditions as the readout for analysis of secondary metabolites and resulting in
the discovery of 9 new compounds, including a new glycopeptide, with potentially therapeutic benefit.
6.12.8 Identification and Dereplication of Natural Products
Medicinal use of natural products has been documented as early as the 30th century BCE.262 However, natural products chemistry
rapidly developed with the introduction of modern extraction, purification and analytical approaches such as mass spectrometry
and NMR.263 In the past decade, there has been increasing use of combinatorial libraries and synthesis, but natural products
research still plays a major role in the discovery of novel therapeutic drugs, a process which commonly relies on ethnobotanical
studies, discovery of novel taxonomies and high-throughput screening of microbial/plant/fungal natural products
collections.264,265
Central to the discovery of novel active compounds is the accurate identification of compounds. While mass spectrometry
provides a robust tool for achieving this goal, often complementary methods such as NMR, IR spectroscopy, X-ray diffraction and
crystallography may be used in conjunction to confirm molecular structure, formula, chirality and other chemical properties of
novel compounds. However, in many cases, the molecular formula and either partial or full structural details can be deduced using
only chromatography with mass spectrometry. A stepwise approach employing dereplication strategies and statistical analysis can
be employed to reduce the total number of possible candidates for identification so that the focus remains on identification of novel
natural products (Fig. 19).
Fig. 19 Steps for the identification of novel natural products after dereplication of knowns.
6.12.8.1 Considerations in Sample Preparation and Experimental Design

When working with biological materials, the number of ions detected in a mass spectrometry dataset can number in the tens of
thousands. Thus it is imperative to consider approaches to both select and process samples to reduce the final number of
compounds that would be selected for further investigation. Bioactivity analyses combined with MS data analyses can be used to
prioritize experimental conditions and help focus identification efforts to the most relevant features.266,267 Appropriate design of
experiments268 and use of multivariate statistical analysis269 is paramount to success in metabolomics analyses. When possible,
purification or fractionation can reduce the complexity of analysis and dereplication steps. Typically, an initial crude extraction
should be performed to reduce the background signal and convert the material into a format suitable for analysis (e.g. solid plant
tissue may be homogenized to release intracellular metabolites, followed by a solvent immersion to extract metabolites and analyze
using LC-MS) (Fig. 20).
Two general approaches may be taken for pre-processing of samples prior to MS analysis: a small-scale targeted or large-scale
screen. A targeted approach is appropriate when looking for a natural product from a small subset of materials with a single known
biological activity. Here, compounds may be extracted and/or purified, tested for activity and this process repeated until a somewhat
“pure” sample is obtained for MS/NMR identification. With a larger scale screening approach, hundreds of thousands of biological
materials can be evaluated for compounds that have natural product-like chemical features, and activity of novel compounds may
be tested following MS analysis. Preliminary crude extractions may be used for purification of classes of molecules. Without
fractionation and purification, a well thought-out and suitable control may be required to rule out features that do not confer
bioactivity so that the focus remains on those determined as significant by differential analysis.270 Additionally, no single method
will detect all natural products in a sample. For example thermally labile and/or poorly volatile compounds would be unsuitable for
GC-MS without derivatization. When chemical properties are known, selection of analytical methods is possible, otherwise,
screening multiple methods may be required for compound detection.
6.12.8.2 Stable Isotope Labeling

Using stable isotopes to probe metabolic pathways is widely used in proteomics, metabolomics, and metabolic flux measure-
ments.271 In the context of natural products research, stable isotope labeling can be used to help determine the identity of
an unknown molecule and better understand the mechanism of how a molecule is assembled. Often, a labeled compound, or
substrate, will be either uniformly or positionally labeled, and “fed” to a culture so that compounds then synthesized in the culture
will also be labeled (if that substrate is utilized). In the case of acetate, for example, substrates could be (1,2-13C2)acetate, (1-13C),
and (2-13C)acetate. Tracking a polyketide assembly process could yield detection of positional 13C labels on the resulting molecule
Fig. 20 Workflow for analysis of biological materials for detection of novel natural products. When possible, incorporation of activity screening, fractionation and
heterologous expression will improve identification capabilities.
as well as precursors that would inform steps during the assembly process. Using a mass spectrometry instrument with high
resolving power, the products can be observed with a mass difference between the 13C and 12C isotopes (which is n 1.003355 Da
where n is the number of atoms that have been substituted). In another stable isotope labeling application, labeling can be
performed with deuterated solvents to aid in compound identification.272,273 Here, labile hydrogens (e.g. those that can rapidly
exchange with the solvent) equilibrate with a deuterated solvent. A molecule’s detected m/z will shift by the number of labile
hydrogens multiplied by the mass of a hydrogen neutron. Combined with fragmentation studies, both of these approaches can yield
rich insight into both biosynthetic mechanisms and compound identity.
6.12.8.3 Dereplication of Known Compounds

In 2002, US Secretary of Defense Donald Rumsfeld gave a now famous address, wherein the terms “known knowns”, “known
unknowns”, and “unknown unknowns” were popularized. These same concepts have been applied to mass spectrometry based
metabolomics274 where different strategies are utilized for the identification of known or unknown types of metabolites. While
Rumsfeld’s usage referred to one’s awareness of one’s own knowledge, Little et al.,274 in 2011, applied the terms in the context of the
intersection of knowledge between the researcher and the literature. To expand upon this, we can subset all mass spectral features
into one of the four intersections, each requiring a different approach for identification with the goal of moving the features on the
right into the categories on the left: the known unknowns become known knowns and the unknown unknowns become unknown
knowns (Fig. 21). What the researcher is left with are the knowns and the remaining unknowns.
An untargeted analysis will produce a large list of features, typically containing information on retention time, m/z, intensity and
fragmentation spectra. Relative mass defects can be used to clean up the feature list such that isotope, adduct and fragment peaks are
annotated and the molecular ion and its associated neutral monoisotopic mass is determined. Care should be taken to avoid
misannotation of co-eluting in-source fragments with parent molecular ions.275 To obtain a list of features containing the
“unknown unknowns”, the list should be annotated with identifications of compounds known to the researcher and in the
literature or within free/commercial spectral databases. It is important to complete these steps, even if compounds are not of
Fig. 21 Metabolite identification strategies. The knowledge of molecular identification between the researcher and the literature as represented in a Johari
window.
Table 3 Mass spectral databases with importance in natural product dereplication.
Database name NP source Information Link Refs.
Golm Pure reference standards, also contains Free, online: 29,590 spectra (EI-MS, March 25, http://gmd. [276]
Metabolome metabolomics profiling data of plants/ 2019) mpimp-golm.
Database cyanobacteria mpg.de/
(GMD)
AntiBase— Focused on antimicrobials Commercial: 41,048 MS records (May 2017, https://www. [277]
Wiley ISBN:978-3-527-34359-1) wiley.com/
Spectral Mainly commercial chemical reagents 25,497 MS spectra (March 25, 2019) https://sdbs.db. [278]
Database for aist.go.jp
Organic
Compounds
(SDBS)
GNPS Third-party libraries (e.g. MassBank), spectral Free, online: 72941 spectra (March 25, 2019) https://gnps. [4]
libraries created for GNPS and spectra from the 221,000 MS/MS reference library spectra from ucsd.edu
NP community 18,163 compounds and 8853 MS/MS community
spectra from 5568 unique compounds
ReSpect and Phytochemicals from literature reports and Online (9017 spectra, Dec 14, 2018) http://spectra. [279]
AtMetExpress authentic standards psc.riken.jp/
Human Human metabolites from chemical library (HMDB) Online, free: 318,249 predicted mass spectra; http://www. [280]
Metabolome and third-party spectral databases with 29,665 experimental mass spectra (April 22, 2019) hmdb.ca/
Database experimentally collected, high-resolution MS/MS
(HMDB) and EI spectra
MassBank General small molecules related to life sciences 52,819 spectra, April 29, 2019 http://www. [281]
submitted by community contributors massbank.jp/
MassBank of Spectra from experimental and in-silico libraries, 271,335 spectra, from 77,409 compounds (April 28, http://mona. NA
North America and user contributions 2019) fiehnlab.
(MoNA) ucdavis.edu/
Metlin lipids, amino acids, carbohydrates, toxins, small Experimental spectra from >20,000 compounds and https://metlin. [282,283]
peptides, and natural products, and other small their isotopologues, in-silico spectra additional scripps.edu/
molecule classes known molecular structures contained in the
database(ref )
Spektraris Plant natural compounds Spectra from 500 compounds (May 01, 2019) http:// [284]
langelabtools.
wsu.edu/amt/
Yeast Yeast metabolites 50,598 MS spectra (includes experimental and http://www. [285]
Metabolome predicted MS/MS) from 15,979 compounds (May ymdb.ca/
01, 2019)
Fiehn GCMS Commercial libraries through Agilent and Leco
mzCloud Drugs, cosmetics and commercial chemicals, 4,739,606 spectra from 8779 compounds (includes https://www. NA
endogenous metabolites, natural products and in-silico, experimental spectra) mzcloud.org/
toxins
DrugBank Collection of drug related metabolites, spectrum EI spectra https://www. [286]
data from SDBS and NIST drugbank.ca/
E. coli E. coli metabolites 19,294 NMR and MS spectra (experimental and http://ecmdb.ca/ [287]
Metabolome predicted) for 3098 compounds
Database
(ECMDB)
LIPID MAPS and Lipids—standards, reported in the literature and 30,000 structures, a subset have MS/MS and https://www. [288,289]
LipidFinder computationally generated structures in silico predictions available lipidmaps.org/
NIST Reference standards Commercial: SRD spectra from 267,376 compounds https://www. [290,291]
(EI MS) and 13,808 compounds (MS/MS). Free: nist.gov/srd,
Mass spectra for >33,000 compounds (May 02, https://
2019) webbook.nist.
gov/
interest, as this will reduce the number of features of interest requiring investigation in downstream steps when attempting
identification of unknown unknowns.
A robust dereplication strategy uses a combination of targeted analysis (known knowns) and semi-targeted analysis (knowns
unknowns). A number of online databases and tools are available to support these steps (Table 3) and provide mass spectral
information. Many have significant overlap, either due to automated aggregation from other databases, or duplicate analysis of
compounds.292 For additional databases, including NMR spectral databases, see also Johnson and Lange.293 Chemical and pathway
databases such as PubChem (http://pubchem.ncbi.nlm.nih.gov/), Super Natural II,294 Dictionary of Natural Products (http://dnp.
chemnetbase.com), MarinLit (http://pubs.rsc.org/marinlit/), MetaCyc,295 and KEGG296 are useful tools for interpretation of natural
product mass spectra results. Additionally, numerous natural products repositories/collections are available online: e.g. https://dtp.
cancer.gov/organization/npb/introduction.htm.
For a recent review of metabolomics analysis tools and resources, see Misra and Mohapatra297 and for a recent review of
metabolomics analysis tools and resources, see Misra and Mohapatra.297
A targeted analysis is used to annotate expected or commonly detected metabolites that are produced by the organism(s) using
metabolite atlases.298,299 Pure reference standards analyzed using the same methods should be used for comparison of adducts,
retention times and fragmentation spectra with sample data. Next, features that are known in the literature but are not known or
present in the researchers own library (known unknowns) are annotated.300,301 Exact mass measurements, collected from high
resolution accurate mass instruments, compared with chemical databases produces a list of possible structures.274 Orthogonal
measurements such as retention time and MS/MS can be used to confirm identification. Retention order prediction302 and retention
indices303 are used to improve confidence of an identification. Comparison of measured sample MS/MS with spectral data-
bases293,304 allows the researcher to predict the most likely structures but may not differentiate between isomers. Comparison of
fragmentation spectra using molecular networking for identification of known compounds additionally provides information of
related analogues that may be useful for the researcher to include in future structural searches (Fig. 22).305 In silico fragmenta-
tion306,307 software is used to predict MS/MS spectra for a predicted structure. Lastly, fragmentation trees308,309 and evaluation of
neutral losses, isotope abundance ratios, and adducts are useful for piecing together substructures and more accurately predicting
identifications. These techniques are also used in the identification of unknown unknowns.
6.12.8.4 Identification of Unknown Compounds

The first step for the identification of unknown unknowns is determination of the neutral mass of the molecular ion. This may be
complicated with soft ionization methods which produce adducts or analysis of labile compounds that fragment in the ionization
source eliminating detection of the parent ion. With high resolution mass spectrometry, molecular formula is predicted from
neutral mass, however as mass increases the number of possible molecular formulas310 increase thus requiring additional
techniques to narrow down the possibilities. A set of rules has been developed, “the seven golden rules”, which apply additional
criteria to reduce the number of possible molecular formulas.311
Some structural isomers may not be chromatographically or spectrally resolvable, depending on the methods chosen for
analysis. It may be necessary to employ non-MS based but complementary methods for structural confirmation. Additionally,
sequencing and genome mining for biosynthetic gene clusters (BGC) can be used for prediction of chemical structure of
products,312,313 which could help narrow down the search to specific features. With MS2 or MSn fragmentation spectra, partial
and in some cases full structural determination may be made by manual evaluation of fragmentation spectra, comparison with
in silico fragmentation spectra, and molecular networking for comparison of spectra to those from similar classes of molecules.
While in silico comparisons are typically used for dereplication, construction of all possible structures from mass or formula using
software such as MOLGEN,314 allows for input of structures not found in chemical databases into in silico fragmentation
software.315 Further substructure annotation by analysis of fragment ions and neutral losses further narrows down the possibilities.
While manual analysis of the spectra may be feasible for small numbers of unknowns, software such as MS2Analyzer316 should be
used for analysis of large numbers of unknown spectra. Lastly, molecular networking has been used for the identification of novel
compounds with similar structures to those in databases,317,318 such as GNPS, which contain built-in tools for molecular
networking analysis.318
Essential to a metabolomics analysis is strict adherence to production of high quality data. Minimum reporting standards for
novel metabolites319 and metabolomics data analysis320 have been defined to ensure high quality of data in published literature.
Once a putative identification has been made, in addition to in vitro and in vivo bioactivity analysis, in silico predictions,321 or
in vitro and in vivo testing for the absorption, distribution, metabolism, excretion, and toxicity may be necessary to determine the
usefulness of the discovered compound in human medicine. Chemical synthesis of the novel natural product and/or cloning and
expression of the suspected biosynthetic gene cluster may be pursued for a reliable source of the compound.
6.12.9 Genome Integrated Natural Product Discovery and Analysis
Often, along with the discovery of a novel natural product, the corresponding genes or biosynthetic cluster is reported.322 Following
from the central dogma of biology, enzyme complexes made from proteins and encoded by genes catalyze reactions that transform
metabolites including natural products. Thus, natural products are connected to genes via reactions and in some cases conserved
pathways.323,324 This linkage can facilitate integration of transcriptomics data, lead to expression of the biosynthetic cluster in
another organism, and allow the broad propagation of the learned function by sequence homology.325 In some cases, the mapping
between an observed natural product and the genes responsible for its synthesis is straightforward, but more often it is a laborious
process involving an iterative combination of genetic manipulation, screening, and bioinformatic techniques. Some of the
computational techniques pursue this challenge in a “gene-forward” manner: predict gene function and consequently the products.
Others pursue this challenge in a “compound-forward” manner: predict reactions necessary for synthesizing a product and search
Fig. 22 Molecular networking combined with MS/MS spectra used to identify and dereplicate two natural products in the same family as the known “seed”
compound carmabin A. Reprinted with permission from Yang, J. Y.; Sanchez, L. M.; Rath, C. M.; Liu, X.; Boudreau, P. D.; Bruns, N.; et al. Molecular Networking
as a Dereplication Strategy. J. Nat. Prod. 2013, 76, 1686–1699. https://doi.org/10.1021/np400413s. Copyright (2013) American Chemical Society.
Compound score MAGI score Homology score

+ +
O + +++
+
-- HO OH
+ +
O
-\+
++ O OH + + +
O
-\+ + +++ ++
HO
HO +
+
O
+++ OH
HO + + +
Metabolomics Data Reactions with Reactions catalyzed
Compound structures MAGI algorithm Enzyme Gene Sequence
(Mass Spectrum, m/z, nuetral mass) detected compound by enzyme
Flexible compound inputs Integrated output Flexible enzyme inputs
Fig. 23 This workflow is described by Erbilgin et al. specific to the Metabolite, Annotation and Gene Integration (MAGI) workflow, but other algorithms that link
metabolites to genes operate with a similar overall architecture. With MAGI, metabolomics data is associated with compounds and consequent reactions and genes
(compound forward) or genes are associated with reactions and consequent expected metabolites (gene forward). Algorithms like MAGI attempt to integrate and
reconcile these two approaches. Reprinted with permission from Erbilgin, O.; Ruebel, O.; Louie, K. B.; Trinh, M.; Raad, M. D.; Wildish, T.; et al. MAGI: A Method for
Metabolite Annotation and Gene Integration. ACS Chem. Biol. 2019. https://doi.org/10.1021/acschembio.8b01107. Copyright (2019) American Chemical Society.
for enzymes capable of performing those reactions. In some cases, simultaneous gene and compound scoring is performed
(Fig. 23).325a
The total diversity of natural products is likely much larger than the current listings in chemical databases. Yet, there are many
times that a sample will contain expected, well characterized natural products and several tools are available for mapping these
metabolites to genes and pathways. Unfortunately, many natural products are typically not easily integrated in this way because of
poor gene annotations and the lack of relevant reactions in databases.326,327 Since reactions serve as the pivotal connection between
metabolites and genes, these issues severely limit direct, explicit integration of metabolomics data with genomic data.
To attempt to overcome these challenges, several computational strategies have been developed to expand reaction space and
chemical databases. MyCompoundID,328,329 IIMDB,330 MINES331 and the ATLAS of biochemistry332 all use methods to enzymat-
ically enlarge compound space, though the enzymatic steps are quite general, for example, using the most basic transformations to
modify compounds (e.g. methylation at any functional group). While this method is quite effective at biochemically enlarging
biochemical compound space, the transformations used are not connected to an explicit reaction (e.g. methylation of cytosine), and
therefore are not connected to an explicit gene and thus not assessed in the context of a given genome. Retrorules333 and rePrime334
enlarge compound space by generating and using generalized reaction “rules” constructed from specific reactions in databases, and
thus are linked to explicit reference gene sequences that may plausibly catalyze the novel reaction, though these strategies have been
developed for retrosynthesis and pathway construction purposes,334,335 and are not currently easily used for interpreting mass
spectrometry data.
In addition to the strategies listed above, chemical networking has emerged as a valuable approach to addressing the dearth of
metabolites represented in reactions. Chemical networking relies on the similarity between metabolites. This similarity can be used
to expand reaction-space based on the probability that a new molecule can be substituted for a well-studied molecule in a chemical
reaction.305,332,336–338 By doing this, chemical networking bridges reaction databases with expansive metabolite databases to aid
integration. Once metabolites are associated with a reaction and a reaction is associated with a gene then putative metabolite
identifications can be assessed using the predicted metabolism of an organism.
The remaining challenge of connecting metabolites with specific gene products is that (like metabolite annotations) gene
annotations are often wrong. This is predominantly due to functional assertions being based on homology to reference sequences
unsupported by experimental validation.339 Specifically, annotation services296,340 attempt to annotate a gene product with
a specific biochemical function, sometimes choosing among equally probable but mutually exclusive functions or leaving them
unhelpfully vague. This practice can lead to false conclusions in the absence of biochemical experiments, since some enzymes can
have multiple substrates, are multifunctional, or have similar homology to several different reactions. Additionally, some annota-
tions are incorrect due to propagation of false annotations.341
Conducting one or more metabolomics experiments and then linking observed metabolites to gene sequences can provide direct
biochemical evidence for a gene product’s biochemical function, bolstering existing bioinformatic-based annotations, correcting
wrong annotations, and making vague annotations more specific. Recently, new approaches have been introduced that bridge the
gap between metabolomics and genomics and take into account enzyme promiscuity. The genomes-to-natural products (GNP)
platform was developed specifically for discovering new nonribosomal peptides (NRPs) and polyketides, and using a gene-forward
strategy, predicts possible chemical structures of NRP and polyketide synthases and then generates predicted MS/MS spectra based
on these (Fig. 24); predicted MS/MS spectra are then used to mine MS data.342 Pep2Path was also developed exclusively for NRPs
and post-translationally modified peptides (RiPPs), and using a Bayesian approach, scores putative NRPs and RiPPs based on gene
Fig. 24 The GNP. Modified under public license from Johnston, C. W.; Skinnider, M. A.; Wyatt, M. A.; Li, X.; Ranieri, M. R.; Yang, L.; et al. An Automated Genomes-
to-Natural Products Platform (GNP) for the Discovery of Modular Natural Products. Nat. Commun. 2015, 6, 8421. https://doi.org/10.1038/ncomms9421. http://
creativecommons.org/licenses/by/4.0/.
sequences in the assayed organism.343 Finally, a more general approach has been developed where major differences in the mass
spectrometry profiles of a mutant library are assayed, and these differences are then manually annotated using human intuition.344
Metagenomics and single-cell sequencing have enabled, for the first time, glimpses into the vast metabolic potential of Earth’s
collective biological systems. Yet, for the most part we cannot accurately predict nor identify the products of most biosynthetic
pathways. Most of what we know of microbial biochemistry is based on characterization of a few model microorganisms, and these
findings have been extended through sequence correlations to the rest of sequence space. Unfortunately, these extrapolations have
questionable validity for the vast majority of environmental microbes and therefore requires fundamentally different approaches
for directly linking novel sequences to their biochemical functions. The high-throughput and large scale application of genetic
techniques, product screening, and integrated informatic tools promises to deliver a detailed mapping of natural products, their
associated synthetic routes, and experimentally validated gene annotations.
6.12.10 Outlook
We see a very bright future for mass spectrometry approaches for discovery and analysis of natural products. The sensitivity and
specificity of mass spectrometers continues to dramatically improve as well as innovations in sample processing. Coupling mass
spectrometry to microfluidics has tremendous potential to increase throughput and reduce sample volumes. Such devices can be
integrated with DNA synthesis, systems biology tools, and microfluidic natural product production systems to create a relatively low-
cost platform for exploring natural product space. Equally important will be the development of computational tools that can quickly
sift through mass spectrometry data to identify novel natural products. While these will initially use pathway abstraction and chemical
networking approaches as we have described, increasingly they will shift to computational chemistry-based approaches that directly
predict structure de novo from fragmentation spectra. With these integrated capabilities mass spectrometry will continue to be a vital
tool in enabling researchers to explore biochemical space to discover urgently needed new natural products.
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6.

12 Mass Spectrometry for Natural Product Discovery

6.12.1 Introduction and History 264

Comprehensive Natural Products III: Chemistry and Biology https://doi.org/10.1016/B978-0-12-409547-2.14834-6 2636.12.5

6.12.8.4 Identification of Unknown Compounds 296

6.12.1 Introduction and History

6.12.2 Analytical Workflows for Natural Product Discovery

Screen of Separation into

Hsp18 MAS Act2 RbcS Bch1

L-tryptophan VioA VioB VioE VioC VioD

Indole-3-pyruvic acid (IPA) imine

Violacein 4.20 4.25 4.30 4.35 4.40 4.45 4.50

6.12.2.1 Mass Spectrometry Workflow

6.12.3.1 Extraction Solvents

(A) (16S + PM)–5

Abundance (arbitraty units)

(16S)–5 (16Sc + PM)–5 (16Sc + 818)–5

Method Solvent Temperature Pressure Time Volume of organic Polarity of natural

6.12.3.2 Conventional Extraction Methods

6.12.3.2.2 Maceration, infusion, percolation and decoction

6.12.3.2.3 Reflux extraction

6.12.3.2.4 Soxhlet extraction

6.12.3.3 Non-conventional Extraction Methods

6.12.3.3.1 Ultrasound assisted extraction (UAE)

6.12.3.3.2 Microwave-assisted extraction (MAE)

6.12.3.3.3 Pressurized liquid extraction (PLE)

6.12.3.3.4 Supercritical fluid extraction (SFE)

6.12.4 Separation and Fractionation

6.12.4.1 Liquid Chromatography

6.12.4.1.1 Reverse phase liquid chromatography (RPLC)

6.12.4.1.2 Normal phase chromatography (NPLC)

6.12.4.1.3 Hydrophilic interaction liquid chromatography (HILIC)

6.12.4.1.4 Ion exchange chromatography (IEX)

6.12.5 Ionization and Detection of Compounds

LC-ESI-MS(-) LC-ESI-MS(+) LC-APCl-MS(-) LC-APCl-MS(+) GC-APCl-MS(+)

6.12.5.2 Mass Analyzers

6.12.5.2.2 Ion trap

6.12.5.2.3 Time-of-flight (TOF)

6.12.5.2.4 Fourier transform-ion cyclotron resonance (FT-ICR)

6.12.5.2.6 Tandem mass analyzers

6.12.5.3 Data Collection

6.12.6 Detection of Select Natural Product Classes

(A) Reduced polyketides

(B) Aromatic polyketides (C) Unclassified

6.12.6.3 Nonribosomal Peptides (NRPs)

6.12.7 Mass Spectrometry Imaging of Natural Products

6.12.7.1 MSI Platforms

MSI platform Sample types Known chemical family/compounds visualized Refs.

6.12.7.2 MSI Applications

6.12.7.2.2 High-throughput screening

6.12.8 Identification and Dereplication of Natural Products

6.12.8.1 Considerations in Sample Preparation and Experimental Design

6.12.8.2 Stable Isotope Labeling

6.12.8.3 Dereplication of Known Compounds

Table 3 Mass spectral databases with importance in natural product dereplication.

Database name NP source Information Link Refs.

6.12.8.4 Identification of Unknown Compounds

6.12.9 Genome Integrated Natural Product Discovery and Analysis

Compound score MAGI score Homology score

Flexible compound inputs Integrated output Flexible enzyme inputs

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