LC‐HRMS for the Identification of β‐Carboline and Canthinone Alkaloids Isolated from Natural Sources

β‐carboline and canthinone alkaloids are widely distributed in the Angiosperms. Due to their diverse biological activities, the structures of these alkaloids have been used as important models for the synthesis of novel therapeutic drugs. Combining high‐ performance liquid chromatography (HPLC) with high‐resolution mass spectrometry (HRMS) has provided a valuable tool in the analysis of these alkaloids in, for example, plants, insects, marine creatures, human tissues and body fluids. In this review, we sum‐ marized the main β‐carboline and canthinone alkaloids studied by liquid chromatog‐ raphy high‐resolution mass spectrometry (LC‐HRMS) associated with mass analyzers, molecular weight information, mass fragmentation and biological activities, presenting an overview of increasing interest for carboline alkaloids study by LC‐HRMS.


Introduction
Since ancient times, alkaloids have been used as medicine and in folk medicine for the treatment of different diseases. β-Carboline alkaloids are a group of natural indole alkaloids with different degrees of aromaticity widely distributed in the Angiosperms . Canthinones

Alkaloids and biological activity
Many pharmacological properties attributed to β-carboline alkaloids have been described in the literature, which makes it an important class of natural products. Among them, antimalarial, antileishmanial, trypanocidal, antibacterial and antitumor activities are described [38,44,62].  [39]. Strictosamide, alkaloid glycoside isolated from the crude ethanol extracts of roots and branches of Psychotria prunifolia, has in vitro antiprotozoal activity, especially against promastigotes of Leishmania amazonensis, with IC 50 values of 40.7 μg/mL [29]. The alkaloid (+)-8-hydroxymanzamine A has activity against Leishmania donovani with IC 50 of 2.5 mg/mL and IC 90 of 6.1 mg/mL, whereas (+)-manzamine A is less active, with IC 50 of 11.15 mg/mL and IC 90 of 31.05 mg/mL [44]. Canthin-6-one, isolated from dichloromethane extract of Z. chiloperone stem bark, has antileishmanial activity in BALB/c mice infected with L. amazonensis. The intralesional treatment with canthin-6-one is able to decrease by 15.0% a lesion weight and the parasite load by 77.6% when compared with the group of untreated mice [12].
Antimicrobial activity has also been related to this class of compounds. The alkaloids (+)-8hydroxymanzamine A and (+)-manzamine A are more potent as antimycobacterial than the control ciprofloxacin, with IC 50 values of 0.13 and 0.36 μg/mL against Mycobacterium intracellulare vs. 0.48 μg/mL of ciprofloxacin. However, both substances were inactive against the filamentous fungus Aspergillus fumigatus and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa [44].

Ionization source and mass analyzers
An analysis of a sample comprises ionization where the ion beam is accelerated by an electric field and then a mass analyzer, a region of the mass spectrometer where the ions are separated according to their mass/charge ratio (m/z) [65].
There are many different ionization methods, such as ESI, APCI, FAB, suitable for different applications. Many types of mass analyzers are used according to the type and objectives of the analysis: e.g., dual focus, quadrupole, ion trap, time-of-flight (TOF), Orbitrap and Fourier transform ion cyclotron resonance (FT-ICR) mass analyzers are the magnetic sectors [66]. According to this review, the most used mass analyzers for the analysis of β-carbonyl and canthinone alkaloids are the quadrupole, ion trap, TOF and Orbitrap. The most articles reported TOF as the most used analyzer followed by Orbitrap. TOF is based on the simple idea that the speed of two ions created at the same instant with the same kinetic energy will vary according to the mass of the ion (the lighter ion will be faster), when traveling against the mass spectrometer detector. The main characteristics are as follows: simultaneous analysis of all produced ions, high sensitivity and high mass resolution, which requires very fast data acquisition and detection systems. An Orbitrap mass analyzer is an ion trap comprising a barrel type electrode and an inner coaxial electrode similar to a reel holding the ions in an orbital motion inside the trap [66].

Mass fragmentograms
The observed masses of the fragments in LC-HRMS of the main cited β-carboline and canthinone alkaloids are shown below (Figure 1). The principal peaks are shown in the fragmentograms below. The fragments are based on characteristic alkaloid breaks and/or proposals based on mass spectrometry theory.

Advantages and disadvantages of the LC-HRMS as analytical tool
The natural product research requires the development of fast and robust techniques for the difficult identification of substances in samples of plant extracts. Actually, GC-MS and LC-MS/MS are more used techniques than LC-HRMS for the identification of plant metabolites. However, the advantages of LC-HRMS and the chemical complexity of plant extracts can justify the investment in that newer technique.
Compared with gas chromatography (GC), techniques involving liquid chromatography (LC) have the advantage of being applicable to a wider variety of chemical classes of compounds.
In GC, the analytes must be in gaseous form, and some substances must be hydrolyzed or derivatized to lower polarity and increase volatility to be analyzed. In LC, the analytes must be soluble in the liquid mobile phase and works well with polar substances. LC-MS/MS also has higher sensitivity than GC-MS [65,69].
GC-MS has a single quadrupole mass detector, whereas LC-MS/MS has two quadrupole detectors in tandem. In MS/MS, only one ion from the first detector, frequently the molecular ion is fragmented in the second detector. The selected-ion monitoring (SIM) mode can be applied for GC-MS to increase sensibility and consists of the selection of three of the more abundant ions from the mass spectrum to be measured by the spectrometer and the comparison between the abundance relative ratio of these ions with the predetermined ratio for the suspect substance. The presence of contaminants affects the ion ratio hinders the identification. The selected reaction monitoring (SRM) mode is applied for LC-MS/MS and consists of the selection of some ions fragmented in the second detector. Thus, LC-MS/MS has more specificity than GC-MS, because two substances with the same nominal mass will exhibit different fragmentations in the second detector. Therefore, SIM or SRM is suitable only for targeted substances. GC-MS and LC-MS/MS can also be employed for the analyses of unknown compounds, but only in the full-scan MS mode and with lower sensitivity. Both GC-MS and LC-MS/MS have resolution of 1 atomic mass unit (amu) [65].
The LC-HRMS has the characteristics of the accurate mass measurement of the analytes, which confers many advantages as compared to other techniques of analysis traditionally used. The mass resolution is about 2 ppm, which represents an error of 0.0006 amu for substances of 300 amu [65]. The exact molecular ion mass is associated with an exact molecular formula of the analyte, a valuable structural information. The exact mass is a calculated parameter, while other techniques depend on experimental results for comparison. Therefore, the main advantage of LC-HRMS is that it allows the identification of a wider number of analytes, including unexpected substances in the sample, and does not require reference standards or preexisting MS libraries for comparison [70]. Additionally, LC-TOF/MS can be applied to a larger range of molecular masses (up about 20,000 amu), while LC-MS/MS is indicated for substances up to about 3000 amu [65].
Besides the high mass resolution, the LC-HR/MS has other important advantages. A previous chromatographic treatment of the sample is not required, and a robust method for qualitative analysis can be applied for different and unknown samples, even for the identification of minority substances. Thus, analyses are faster than in LC-MS/MS, because the time in the development of the method is saved. It is especially interesting in natural product studies which frequently are related to complex mixtures, as in metabolomics, extract authentication and screening studies [70][71][72][73][74].
However, given the high complexity of many substances of plant origin, it is important to carry out analyzes using different ionization modes and both polarities. Most alkaloids are detectable in positive mode, either for ESI or APCI, but the matrix interference is more pronounced. The formation of adducts is possible, more specifically, cationization in positive mode may lead to the formation of alkali adducts, with the formation of multimers that add ions to the mass spectrum [73].
The LC-QTOF/MS adds the high mass resolution to mass fragmentation, which provides higher confidence in identification, although with higher cost. Comparing LC-QTOF/MS to LC triple quadrupole linear ion trap (QqLiT), the first leads to fewer false positives, but the latter has slightly lower detection limits in most situations [74].
Besides the high cost, LC-HRMS has the disadvantage of not differentiating structural isomers, which is important in phytochemistry since substances with more than one stereocenter are common. In those cases, it is necessary to complement with other information, such as retention time and spectroscopic data [73]. Another disadvantage is the rapid saturation of the detector, which requires work with more diluted samples [65]. It is expected that these equipments will become less costly, so that the technique will gain wide use.

Analysis of alkaloids in body samples by LC-HRMS
Plant species that contain β-carboline alkaloids, including canthinone alkaloids, are widely employed therapeutically or even as a drug of abuse. Given the diversity of the biological activities already described for these alkaloids, including neurological effects, it is necessary to develop techniques for the detection and quantification of these alkaloids and their metabolites in biological fluids and tissues, as a tool for toxicological analysis and pharmacokinetic studies. This knowledge may also be the starting point for the development of new drugs with potential commercialization.
LC-HRMS is promising in toxicological and analytical studies of metabolism, where substances are often unexpected, and the sample is available in small amount. In addition, it provides rapid analysis and the possibility of using a general method for a wide variety of substances [65,[74][75][76]. To date, there are few studies using LC-HRMS for the analysis of alkaloids, including β-carboline alkaloids in biological samples, possibly because of the still very high equipment prices. Frequently, LC-MS/MS or GC/MS is used previously, and only after the high-resolution mass is obtained for confirmation.
A large variety of phase I metabolites of β-carboline alkaloids, formed by N-decarbonylation, oxidation and methylation, and phase II metabolites, formed by conjugation, such as glucuronides, sulfates and N-acetylcysteine derivatives, are present in body samples. For analysis of phase I metabolites, β-glucuronidase and/or arylsulfatase enzymes can be added to the sample for cleavage of conjugates and to avoid interferences of phase II metabolites [43,53,68,76].  [75]. The oven temperature is set at 30°C [43], 35°C [53] or 40°C [75]. After pretreatment, samples are frequently diluted in methanol or in mobile phase before injection in LC systems. The mobile phase is frequently a gradient from formic acid (0.05 or 0.1%) in water to acetonitrile, with or without formic acid [43,68,76]. This aqueous phase may be replaced by an aqueous solution containing ammonium formate buffer (2.5 or 10 mM) with 0.1% (v/v) formic acid [53,75]. The organic phase may be 0.1% formic acid in acetone:acetonitrile 20:80 [67]. The solution B of the method developed by Kolmonen et al. [75] consists of 2.5 mM ammonium formate and 0.1% formic acid in 90% acetonitrile. The flow rate varies from 300 μL/min [68] to 1 mL/min [43]. The total run time varies from 8 min [75] to 67 min [53].
In general, the MS analyzer, TOF or Orbitrap, employs electrospray ion source. For this class of substances, the positive ionization mode is the most applied (ESI+) [43,53,67], although it is more appropriate to use both positive and negative ionization modes in screening analyses to cover more substances [68,75]. Capillary voltage varied from 3 to 4.5 kV, [43,53,67,68,75] and resolution varies from 7500 to 60,000 [46,53]. After the analysis, data processing is necessary with suitable software to help in the identification of metabolites.
Although LC-HRMS has been more used to confirm identification, the technique can be used alone successfully in screening, as the methodology proposed by Kolmonen et al. [75]. The methodology uses LCTOFMS for the search of doping agents in human urine. The method is applicable to at least 207 analytes, including the indole alkaloid strychnine, and may even be used for quantitative analyzes for many of this substances. After an SPE sample pretreatment, the analysis run time is 8 min for each ionization mode, with a total time of 16 min.
Some β-carboline alkaloids and their metabolites have been identified in biological tissues and fluids, such as tetrahydro-β-carboline derivatives-present in plant species and also considered LC-HRMS for the Identification of β-Carboline and Canthinone Alkaloids Isolated from Natural Sources http://dx.doi.org/10.5772/68075 an endogenous alkaloid; [43,67,68] speciogynine-isolated from Mitragyna speciosa, a plant species used as drug of abuse [53]; 1-methyl-3-carboxy-β-carboline-found in cow milk probably derived from the diet and metabolism [76]. The technique is still expanding, and the few works found in the literature indicate a great potential not yet explored.

Summary
An important class of natural products found in Angiosperms, β-carboline and canthinone alkaloids, has various pharmacological properties and toxic effects. Coupled chromatographic and mass spectrometric techniques can be used to identification of these alkaloids. In this chapter, an approach overview of LC-HRMS applied to chemical complexity of plant extracts and forensic samples containing β-carboline and canthinone alkaloids can be a good choice technique to analyze and elucidate this kind of compounds. In addition, the HRMS/MS fragments of some important β-carboline and canthinone alkaloid are shown in mass fragmentograms schemes. Among important advantages of LC-HRMS, the main one is that it allows the identification of a wider number of analytes, including unexpected substances in the sample, and does not require reference standards or preexisting MS libraries for comparison. This technique can be used alone successfully in screening since it provides rapid analysis and the possibility of using a general method for wide variety of substances.