Pyrrolizidine Alkaloid Extraction and Analysis: Recent Updates

Pyrrolizidine alkaloids are natural secondary metabolites that are mainly produced in plants, bacteria, and fungi as a part of an organism’s defense machinery. These compounds constitute the largest class of alkaloids and are produced in nearly 3% of flowering plants, most of which belong to the Asteraceae and Boraginaceae families. Chemically, pyrrolizidine alkaloids are esters of the amino alcohol necine (which consists of two fused five-membered rings including a nitrogen atom) and one or more units of necic acids. Pyrrolizidine alkaloids are toxic to humans and mammals; thus, the ability to detect these alkaloids in food and nutrients is a matter of food security. The latest advances in the extraction and analysis of this class of alkaloids are summarized in this review, with special emphasis on chromatographic-based analysis and determinations in food.


Introduction
Plants and their phytoeffective metabolites are used for medicinal purposes but are also an enormous source of toxic products. Alkaloids contribute considerably to the medicinal and pharmacological activity of natural products while they are also recognized for high potency, a narrow therapeutic index, and, therefore, their toxicity. Alkaloids are produced with high diversity in prokaryotes and eukaryotes and are biosynthesized by many species of bacteria, fungi, marine organisms, insects, plants, and animals [1][2][3].
Pyrrolizidine alkaloids (PAs) and their N-oxides are produced by many flowering plants for protection. Approximately 660 PAs have been characterized in more than 6000 plants, occurring more frequently in the Asteraceae, Boraginaceae, Fabaceae, and Orchidaceae families and to a lesser extent in the Poaceae, Lamiaceae, and Convolvulaceae families [4][5][6][7]. Additional important plant families that contain PAs are Compositae and Leguminosae. PAs and their derivatives are found in many genera, such as Alkanna, Cynoglossum, Heliotropium, Lithospermum, Symphytum, Anchusa, and Borago from the Boraginaceae family and Brachyglottis, Senecio, Tussilago, Cineraria, Petasites, and Eupatorium from the Asteraceae family [6]. Other import genera containing PAs include Amsinckia, Crotalaria, Echium, and Trichodesma [8]. Although PAs are a source of the pharmacological activity in many medicinal plants and are therefore used in folk medicine [9], the toxicity of this class of alkaloids to humans and many animals usually compromises the medicinal benefits.
In this review, different separation methods and chemical analysis of PAs are first presented, followed by a summary of the widest possible range of mass spectrometer specifications used for the analysis of this class of alkaloids.

PA Chemistry
PAs are esters of necine alcohol and necic acids [9] and are described in Figure 1. Necine is a heterocyclic amino alcohol based on a pyrrolizidine nucleus containing two Figure 1. PAs are esters of necine and necic acids. Necine is a pyrrolizidine-based amino alcohol (the structure is shown in the red box) that exists in 4 different forms: platynecine, otonecine, retronecine, and heliotridine. Necic acid (the structure is shown in the upper blue box) exists as three different types: monocarboxylic (aliphatic and aromatic) and dicarboxylic acids separated or forming a macrocycle. One or both hydroxyl groups of necine can be esterified by necic acids, and there are also PAs that lack C7 oxygenation.
PAs are usually found in four different forms according to the N-oxidation and unsaturation levels of the pyrrolizidine ring; three of these forms are tertiary amine structures (saturated and unsaturated and otonecine) and the fourth is an N-oxide. PAs can be divided into different classes depending on the necine base, e.g., retronecine, heliotridine, otonceine, platynecine ( Figure 1). Necic acids are a group of hydroxylated aliphatic acids containing either one or two carboxylic acid groups ( Figure 1). Schramm, et al. [9] further classified PAs according to their overall structure into the following types: senecionine, triangularine, lycopsamine, monocrotaline, phalaenopsine/ipanguline, combined triangularine and lycopsamine, simple PAs, and PAs with unusual linkage patterns (more information can be found in [9]).

Toxicology of PAs
PAs are not intrinsically toxic; however, the 1,2-unsaturated PAs are metabolized in the liver into active pyrrolic metabolites, to which all the hepatotoxicity, including liver cirrhosis and liver failure, is attributed. As reported by Xia, et al. [10], the PA can lead to the formation of five different DNA reactive secondary pyrrolic metabolites. Moreover, it may cause pulmonary hypertension, cardiac hypertrophy, kidney degeneration, carcinogenicity, and genotoxicity, all of which could be fatal. [11][12][13]. The quantity and severity of the toxic metabolites produced by PAs results in different corresponding toxicity and potency levels ( Table 1).  50 ) values of some PAs [14].

PA
LD 50  The ingestion of PAs is usually accompanied by toxicity symptoms ranging from nausea, vomiting, jaundice, and fever to hepatic occlusion [15]. According to the time and concentration of the exposure to PAs, alkaloid toxicity can be classified into chronic (long-term exposure with low concentrations of PAs) and acute (short-term exposure with high concentrations of PAs) toxicity, both of which can lead to serious illness, symptoms, and diseases in animals and humans.

Food and Pharmaceutical Products Safety Recommendation Regarding PAs
PAs and their N-oxide derivatives are found in many food products and supplements, particularly tea, herbal products, and honey. The European Food Safety Authority (EFSA) has identified a group of 17 PAs and their N-oxide derivatives that commonly contaminate food, including intermedine/lycopsamine, intermedine-N-oxide/lycopsamine-N-oxide, senecionine/senecivernine, senecionine-N-oxide/senecivernine-N-oxide, seneciphylline, seneciphylline-N-oxide, retrorsine, retrorsine-N-oxide, echimidine, echimidine-N-oxide, lasiocarpine, lasiocarpine-N-oxide, and senkirkine. To better understand the occurrence of PAs in food, PAs other than those mentioned in the 17-PAs list should also be monitored due to chromatographic coelution and structural isomerization problems [16]. As of July 2022, in Europe, the maximum PAs in different tea and herbal products came into effect, as shown in Table 2 [17].  [17].

Foodstuffs Max Sum Level of PAs (µg/kg)
Herbal infusions (dried product) 200 Herbal infusions of rooibos, anise (Pimpinella anisum), lemon balm, chamomile, thyme, peppermint, lemon verbena (dried product), and mixtures exclusively composed of these dried herbs 400 Tea (Camellia sinensis) and flavored tea (Camellia sinensis) (dried product) 150 Tea (Camellia sinensis), flavored tea (Camellia sinensis), and herbal infusions for infants and young children (dried product) 75 Tea (Camellia sinensis), flavored tea (Camellia sinensis), and herbal infusions for infants and young children (liquid) 1.0 Food supplements containing herbal ingredients including extracts 400 Pollen-based food supplements, pollen, and pollen products 500 Borage leaves (fresh, frozen) placed on the market for the final consumer 750 Cumin seeds (seed spice) 400 Borage, lovage, marjoram, and oregano (dried) and mixtures exclusively composed of these dried herbs 1000 EFSA recommends monitoring the concentration of these toxic alkaloids frequently to maintain the lowest possible occurrence in food chains [18]. Some countries, such as Germany, have introduced a limit of 1 µg/day of PAs for pharmaceutical products/medicines used for less than 6 weeks, and of 0.1 µg/day of PAs for consumption exceeding a 6-week period. Previously, the Federal Institute of Risk Assessment in Germany (BfR) recommended a daily intake of not more than 0.007 µg/kg body weight/day [15]. Furthermore, In 2017, and as a reference point for chronic risk assessment, the EFSA panel on contaminants chose a Benchmark Dose Lower Confidence limit for a 10% excess cancer risk (BMDL10) of 237 µg/kg BW per day for an increase in liver hemangiosarcoma incidence in female rats exposed to riddelliine [16].

Analysis of PAs
PA analysis can be divided into three phases: extraction, separation, and identification, the efficiency of which depends on many factors. Table 3 presents the most used gas and high-performance liquid chromatographic methods, including sample preparation, over the last 15 years.

PA Extraction
PA extraction from different samples depends on the form and type of the alkaloid of interest, as well as the complexity of the matrix used to implement the extraction process. The extraction process may involve three stages: sample preparation, PA extraction, and clean up. The preparation process can include simple cutting of a herbal product or homogenization/pulverization of frozen or dried material to increase the surface area for the extraction [83]. As shown in Table 3, the solid-liquid extraction is still the technique most widely used for sample preparation, although other extraction and purification techniques such as solid-phase extraction (SPE) or the QuEChERS procedure are being applied since they allow for cleaner extracts [84]. Extraction from differently prepared samples involves treatment with a specific solvent under suitable conditions to extract the maximum quality and quantity of the target alkaloids. All forms of PAs, including the N-oxides, have slight solubility in nonpolar solvents, i.e., hexane, and are therefore more efficiently extracted with polar solvents, such as methanol or with aqueous dilute acid; therefore, both methanol and dilute aqueous solutions of organic or mineral acids are good extraction solvents for PAs and their N-oxide derivatives [83]. Considering solubility effects, several techniques have been used to extract PAs from different matrices. Some examples of these extraction techniques are maceration [85], refluxing [86], percolation [87], sonication [88], Soxhlet-based extraction [89], supercritical fluid extraction [90], pressurized liquid extraction [91], microwave-assisted extraction [79], and solid phase extraction [92]. For example, These et al. [85] used 25% methanol in 2% formic acid for maceration in a single extraction process, followed by filtration or centrifugation [85]. El-Shazly et al. [93] homogenized herbal components in 0.5 N hydrochloric acid, followed by soaking for 1 h [93]. Mroczek et al. [87] extracted PAs by refluxing with 1% tartaric acid in methanol [87]. The extraction conditions can affect the quality and quantity of the PA yield, e.g., the temperature of the extraction can influence the extraction process; therefore, the prolonged use of Soxhlet extraction under a high reflux temperature has been found to result in a marked decrease in the PA yield [94].
A food matrix could be described as a complex assembly of nutrients and non-nutrients interacting physically and chemically. A food matrix could influences the release, mass transfer, and stability of many food compounds [95]; e.g., in terms of food analysis, there is variation between honey and tea or other herbal product, so a matrix should be considered when attempting to achieve effective extraction results.
Solid phase extraction (SPE) techniques are another option for extracting and cleaning up PAs. The studies in Table 3 showed the utilization of SPE materials, e.g., Ergosil, C18-material, and strong cation exchange (SCX) for herbal products, including tea and spices, and illustrated that using SPE is necessary for many reasons, e.g., switching sample matrices to a form more compatible with chromatographic analyses, concentrating analytes for increased sensitivity, removing interferences to simplify chromatography and improve quantitation, and protecting the analytical column from contaminants. It is noted in most studies, as in Table 3, that there is a need to elute PAs and PA-N-oxides in SCX-based SPE with a basic solution, e.g., dilute NH 4 OH.

PA Separation
PA separation is the main step after extraction. Many separation procedures can be used to analyze PAs, among which chromatographic techniques are currently the most utilized due to their ease of use and stability and reproducibility of results. Generally, the chromatographic separation and MS analysis of PAs and their N-oxides is a complex and complicated process owing to large numbers of structural and stereoisomers. This complexity and variation in the chemical structure enforced the utilization of many separations and isolation techniques in an attempt to solve the compound complexity matrix and reduce the problem of compound coelution. Examples of the separation techniques used are high-speed counter-current chromatography and capillary electrophoresis methods. Furthermore, detection techniques such as colorimetric, nuclear magnetic resonance-based, immunological-based, and UV-spectrometry-based or mass spectrometry-based techniques are now widely used to detect PAs, allowing the process of separation and detection and, therefore, sample preparation to be simpler and easier to apply [96]. The most efficient chromatographic techniques that were used to separate PAs were the liquid-gas, liquid-liquid, or liquid-solid techniques. Table 3 shows examples of the most used gas chromatography methods for the analysis of PAs. PA N-oxides are not volatile and therefore cannot be detected by gas chromatography. Consequently (as shown in Table 3), in the reduction in PAs to their cores, retronecine and heliotridine, LiAlH 4 is usually used as a reducing reagent. After reduction, the compounds are subjected to derivatization using N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), heptafluorobutyric acid (HFBA), or other similar reagents. The inability to directly analyze PA N-oxides and the extensive preparation steps, including derivatization, causes the use of gas chromatography techniques to be impracticable for the analysis of PAs. Furthermore, reducing all PAs to their bases does not enable relative amounts of the original individual PAs and the N-oxides to be assessed.

High-Performance Liquid Chromatography Separation of PAs
The use of high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), and liquid chromatography (LC) has been attracting an increasing interest for the separation of PAs, especially as LC-MS instruments become increasingly available (Table 3). LC-MS/MS methods have low detection limits (1 µg/kg or lower) and can be used to detect PAs and PA N-oxides simultaneously in a single run, as well as offering other advantages. Compared with GC, LC-MS offers the high-efficiency separation and detection of Pas without the need for derivatization, which means easier sample preparation. Even so, one of the main challenges in determining Pas or PA N-oxides by LC, HPLC, or UHPLC is the co-occurrence of isomers, which causes coelution, making it difficult to separate these compounds chromatographically and to identify them by mass spectrometry (since they have the same molecular weight and often very similar fragmentation patterns). Moreover, the disadvantage of these analysis techniques is the use of a targeted (non-broad-spectrum) setup, which could result in missing some PAs; furthermore, quantification necessitates the use of certified reference standards that are rare and very expensive [15,97]. Since targeted analysis focuses on specific compounds, it will not identify other compounds during analysis, so it is not effective for discovering new compounds or analyzing unknown samples [98]. In this case, nontarget analysis can reveal more broad information about new compounds [99]. An analysis of Table 3 indicated that the LC-MS methods can be used for both simple and complex matrices by slightly modifying the sample preparation methods to include a cleaning step.
There are some PA isomers recommended to be monitored by the European Commission Regulation 2020/2040, e.g., indicine, echinatine, rinderine (possible coelution with lycopsamine/intermedine), indicine-N-oxide, echinatine-N-oxide, rinderine-N-oxide (possible coelution with lycopsamine-N-oxide/intermedine-N-oxide), integerrimine (possible coelution with senecivernine/senecionine), integerrimine-N-oxide (possible coelution with senecivernine-N-oxide/senecionine-N-oxide), heliosupine (possible coelution with echimidine), heliosupine-N-oxide (possible coelution with echimidine-N-oxide), spartioidine (possible coelution with seneciphylline), spartioidine-N-oxide (possible coelution with seneciphylline-N-oxide), usaramine (possible coelution with retrorsine), and usaramine N-oxide (possible coelution with retrorsine N-oxide) [47]. Chromatographic resolution is fundamental for the differentiation of isomeric PAs such as intermedine, indicine, lycopsamine, rinderine, and echinatine (m/z 300) and their N-oxides (m/z 316) as well as integerrimine, senecionine, and senecivernine (m/z 336) and their N-oxides (m/z 352), [100]. Klein,et al. [100] applied different acidic and alkaline mobile phases and succeeded to differentiate between some of the PA isomers, especially when alkaline conditions were applied. In the same study, the dimension of the C 18 column and its particle size affected the resolution of the PA peaks produced. When a shorter column was used, this allowed for the reduction in sample size and produced a better separation and higher peak resolution. The problem of PA isomer separation will continue to be the most important problem in the analysis of PAs with only partial solutions, which allow for the separation and differentiation of particular groups of these alkaloids.

PA Identification
Colorimetric, nuclear magnetic resonance-based (NMR), immunological, UV-spectrometrybased, and capillary electrophoresis methods have been used to analyze PAs as detection techniques, and NMR is used for structure identification [83] as well. The identification of PAs separated by LC procedures using MS-generated data remains challenging due to the high diversity and relative complexity of PA structures. Many characteristic mass fragments for the different types of PAs have been determined (Table 4) [85]. For example, Joosten, et al. [101] described the pyrrolizidines in Jacobaea vulgaris where 25 PAs were identified based on typical mass spectral transitions and retention time [101]. Lu et al. [102] performed a study on pyrrolizidines in the Senecio species and identified two mass ions at m/z 120 and 138 indicating the presence of retronecine-type PAs, as well as fragments at m/z 122, 150, and m/z 168 distinguishing otonecine-type PAs. Lu et al. [102] also identified fragments 122, 140 m/z as characteristic for the platynecine type of PAs. Moreover, PA N-oxides were found to produce a neutral fragment at m/z 44 [102]. Zhou et al. [103] developed a coupled precursor ion scan (PIS) and multiple reaction monitoring (MRM) approach to improve PA identification. Ruan et al. [104] studied the fragmentation pattern of some PA N-oxides and their related PAs. Retronecine-type PA N-oxides were found to produce two characteristic fragment clusters at m/z 118-120 and 136-138, which were not detected in the parent retronecine-type PAs. Likewise, fragmentation of the platynecine-type PA N-oxides was found to produce two characteristic ion clusters at m/z 120-122 and 138-140.    a-precursor ion, b-product ion, c-declustering potential, d-entrance potential, e-collision energy, f-collision cell exit potential, NOs (PA N-oxides).

Conclusions
Pyrrolizidine alkaloids are compounds with different toxicity symptoms that should be detected in food and feed materials. PAs can be extracted similarly to other members in the class of alkaloids by acid-base, liquid-liquid, or liquid-solid extraction. Different techniques can be used to separate PAs and their N-oxides, of which the most common are LC-MS or GC-MS. GC-MS cannot be used to identify PA N-oxides directly and requires extensive sample preparation; consequently, GC-MS is generally considered to be impracticable for PA separation. On the other hand, LC-MS and LC-MS/MS are currently the most applied techniques for the separation and identification of PAs and their N-oxides because of numerous advantages, including effective separation, the potential for a wide range of compounds to be identified, and simple sample preparation. Nowadays, there are methods for detecting and identifying PAs from MS/MS traces, but these methods still need to be improved in the future in order to reduce the time and to distinguish between PA isomers more accurately. On the other hand, nontargeted PA detection needs more development to increase the specificity and sensitivity of the process to more accurately identify these alkaloids. Further clinical studies are recommended to assess the pharmacodynamic and pharmacokinetic effects of Pas on humans and animals in more detail. Finally, studies on Pas require a high safety level and detailed analyses.  Acknowledgments: The authors would like to thank the Saudi Food and Drug Authority (SFDA) for their assistance and valuable support.

Conflicts of Interest:
The authors declare no conflict of interest.

Disclaimer:
The views expressed in this paper are those of the authors and do not necessarily reflect those of the SFDA or its stakeholders. Guaranteeing the accuracy and validity of the data is the sole responsibility of the research team.