The analysis of plant toxins in the feed and food industry

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MSc Chemistry

Analytical Sciences

Literature Thesis

by

Naomi Dam

11811374

August 2020, 12 EC

Supervisor/Examiner:

Examiner:

Dr. Jeroen Kool

Dr. Rob Haselberg

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Page 2 of 103 Literature study Naomi Dam | 2020-07-27

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Abstract

Plant toxins are plant secondary metabolites and protect the plant from harmful effects. With various structures, effects may differ from causing a bitter taste to genotoxicity. Based on their biosynthetic origin, plant toxins can be divided into phenolics, terpenoids, and alkaloids and sulphur-containing compounds. Since plants are a part of the human and cattle diet, it is important that methods are available for analysis of plant toxins, allowing monitoring of toxic compounds. These methods can be applied in the feed and food industry and should therefore be fast, easy to apply, sensitive and possibly applicable for multiple matrices and plant toxins. This literature thesis focused on giving an overview of the method for extraction, possible clean-up and analysis of plant toxins, taking into account pyrrolizidine alkaloids, tropane alkaloids, coumarins, terpenoids, terpenes, saponins, and cyanogenic glycosides. The methods were discussed in the scope of being applied in the feed and food industry, aiming to find similarities and giving a recommendation of the methods that are regarded suitable or not suitable.

For sample extraction, solid-liquid extraction (SLE) was most applied with a broad range of plant toxins and matrices. Volatile terpenes and terpenoids were solely extracted by headspace-related methods, while QuEChERS was mostly applied for alkaloids. Methanol was most applied as solvent for SLE, extracting all kinds of plant toxins. Acidified methanol or aqueous solutions were more applied for extracting alkaloids. In most cases, solely SLE was sufficient before analysis. Mostly for alkaloids a clean-up step was applied, such as solid-phase extraction (SPE) or mixed-mode SPE. Mixed-mode SPE was solely applied for alkaloids, whereas regular SPE was also applied for coumarins, saponins and cyanogenic glycosides to a lower extent. Honey was the only matrix that was dissolved and where strong cation exchange was applied for clean-up. Based on the processed information, it appears that SLE is most suitable for extracting various plant toxins from different matrices, where compounds such as alkaloids can be further purified with mixed-mode SPE. For analysis of plant toxins, liquid chromatography (LC) was clearly most applied. Gas chromatography (GC) was almost solely applied to the volatile terpenes and terpenoids. The different columns and mobile phases applied in LC were discussed and the Acquity UPLC BEH C18 column from Waters was most applied. Other columns with similar properties include the XTerra MS C18 column from Waters and the Zorbax Eclipse Plus and XDB from Agilent. These and other columns were applied for analyses of different plant toxins, making them suitable for applications in the feed and food industry. Regarding mobile phases, it was very clear that water and acetonitrile (ACN) with or without formic acid (FA) were most applied for analysis of various plant toxins. Therefore, a combination of the specific group of columns with water and acetonitrile, possibly acidified with FA, are most likely to be used in the feed and food industry.

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Abbreviations

AA Acetic acid

ACN Acetonitrile

AF Ammonium formate

ASE Accelerated solvent extraction

AUC Area under the drug concentration-time curve

BEH Bridged ethyl hybrid

BSTFA Bis-trimethylsilyltrifluoroacetamide

CE Capillary electrophoresis

Cmax Peak plasma drug concentration

DAD Diode array detection

ddH2O Double distilled water

DHPA Dehydropyrrolizidine alkaloid

DLLME Dispersive liquid-liquid microextraction

dp Particle size

ECL Electro-chemiluminescence

EDTA Ethylenediaminetetraacetic acid

EFSA European Food Safety Authority

ER Estrogen receptors

EtOH Ethanol

FA Formic acid

FLD Fluorescence detection

GABA Gamma-aminobutyric acid

GC Gas chromatography

GCB / GBC Graphitized carbon black / graphitized black carbon

HMW High molecular weight

HPLC High-performance liquid chromatography HPTLC High-performance thin-layer chromatography HRMS High resolution mass spectrometry

HSSE Headspace sorptive extraction

HS-SPME Headspace solid-phase microextraction IPAD Integrated pulsed amperometric detection

LC Liquid chromatography

LLE Liquid-liquid extraction

LMW Low molecular weight

LOD Limit of detection

LOQ Limit of quantification

MAE Microwave-assisted extraction

MALDI Matrix-assisted laser desorption/ionization

MeOH Methanol

MIPs Molecularly imprinted polymers

MISPE Molecularly imprinted solid-phase extraction MMIPs Magnetic molecularly imprinted polymers

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MS Mass spectrometry

MSDP Matrix solid-phase dispersion

NMR Nuclear magnetic resonance

NOAEL No observed adverse effect level

PAs Pyrrolizidine alkaloids

PANOs Pyrrolizidine alkaloids N-oxides

PDA Photodiode array detector

PLE Pressurized liquid extraction

PFP Pentafluorophenylpropyl

PSA Primary secondary amine

QuEChERS Quick, Easy, Cheap, Effective, Rugged and Safe

SD Steam distillation

SDGs Secoisolariciresinol diglucosides SFC Supercritical fluid chromatography

SCX Strong cation exchange

SLE Solid-liquid extraction

SPE Solid-phase extraction

t1/2 Half-life

TFAA Trifluoroacetic acid

tmax Time to maximum plasma concentration

TMS Trimethyl silane

TOF Time-of-flight

UAE Ultrasound-assisted extraction

UHPLC Ultra high-performance liquid chromatography

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1 Introduction

Plants have evolved over the years and have different mechanisms to protect themselves from harmful effects. Part of this defence mechanism are plant secondary metabolites or plant toxins. Also known as phytotoxins, they protect the plants from being eaten or being attacked by other organisms. The plant toxins have different effects that can vary from causing a bitter taste to hepatotoxicity after administration, or even worse, genotoxicity. This defence mechanism could even harm animals or humans after exceeding a certain dosage. Therefore, it is important that plant toxins are not consumed in too high levels or preferably not consumed at all. Since plants are part of the common diet for both humans and animals, the analysis of plant toxins is of importance for human health and for the health of cattle.

Based on their biosynthetic origins, plant toxins can be divided into three major classes: phenolics, terpenoids, and alkaloids and sulphur-containing compounds [1]. The structure of these compounds may differ greatly, as well as their effects. Based on the effects, legislation may be necessary to allow monitoring of the compounds. The European Food Safety Authority (EFSA) is an independent agency that evaluates risks that are associated with the food chain. As risk assessor, EFSA allows risk managers such as the European Commission and the European Parliament to make decisions or set legislation regarding food safety. One of their publications includes the compendium of botanicals, which is a database of botanicals that contain naturally occurring substances that may concern human health when present in food [2]. This compendium should facilitate hazard identification and therefore help with the safety assessment of botanicals and their preparation for e.g. food supplements. Due to the broad range of different properties of plant toxins, specific methods for sample pre-treatment and analysis are required. Companies that are involved in the production of feed and food, but also institutes that analyse or monitor these samples, have to use these methods. In other words, the feed and food industry are required to use a broad range of methods for a broad range of matrices. These methods should be robust, fast, preferably easy and cheap, applicable for different kinds of matrices and compounds, and reproducible. An overview following these criteria for feed and food industry is still lacking. Therefore, this literature thesis will focus on the different methods for the analysis of a broad range of plant toxins, following the earlier described criteria. First, an overview is necessary to determine what is out there and to determine what methods are most applied. Then, a critical view will focus on the criteria, discussing what methods are most applied for a great variety of plant toxins and matrices, obtaining fast and sensitive results. Finally, a recommendation can be given within the scope, regarding which methods are most applied for extracting and analysing different kinds of plant toxins originating from various matrices. In chapter two, different classes and subclasses of plant toxins are explained. A selection is made for specific compounds or compound groups, which will be the focus of this thesis. Then, an overview of the methods is presented in chapter three, focusing first on the sample pre-treatment, followed by the separation and detection. Then, a discussion in chapter four will focus on the methods that were applied most for specific or a broad set of plant toxins and matrices. Furthermore, the obtained results should be sensitive and achieved fast. The discussion is all set in the scope of being applied in the feed and food industry. Finally, a conclusion is given in chapter five, explaining which methods are considered most suitable for extracting and analysing plant toxins in the feed and food industry.

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2 Theoretical background

Plant metabolism can be divided into primary and secondary metabolism [3]. The primary metabolism gives rise to compounds that are necessary to maintain cells, such as proteins, lipids, nucleic acids, and carbohydrates. Compounds that are formed by different biosynthetic pathways and are only found in specific groups of organisms, result from the secondary metabolism. Plant toxins are formed by this secondary metabolism and based on their biosynthetic origin, they are further classified into phenolics, terpenoids and alkaloids. A graphical overview of the

inter-relationships between primary and secondary metabolism, including different classes of compounds, is shown in Figure 1.

Since there are so many different kinds of plant toxins, the broad range has been narrowed down for this thesis. To include a great variety of plant toxins with different structures, only terpenoids, alkaloids, cyanogenic glycosides and coumarins are discussed. For alkaloids, a subset was chosen to reduce the number of different alkaloids and for terpenoids the subclasses terpenes and saponins are considered as well, to broaden the class of terpenoids. To obtain better insight in the various properties of the different plant toxins, they are discussed per class in the following chapters. For better understanding, phenolics are discussed in the next section, but are excluded from further discussion in other chapters.

2.1 Phenolics

The phenolics are the largest group of plant toxins. Two metabolic pathways cause the formation of phenolic compounds: the shikimic acid pathway,

resulting in predominantly phenylpropanoids and the acetic acid pathway, resulting mainly in simple phenol [3]. As a result, phenolics consist of at least one aromatic ring and at least one hydroxyl group. In free form they are toxic, which is probably why they are mostly bound to other molecules, such as sugars or proteins. Phenolics range from simple, low molecular-weight molecules to larger polyphenols. Based on the number and arrangement of carbon atoms, phenolics can be classified as shown in Table 1.

Table 1: Classification of phenolic compounds, with corresponding subclasses, basic skeleton, and basic structures. Reproduced from ref. [4]

Class Subclass Basic skeleton Basic structure

Phenolic acids

Hydroxybenzoic acids C6-C1

Hydroxycinammic acids C6-C3

Figure 1: Graphical overview of the inter-relationships between primary and secondary metabolism in plants, reproduced from ref. [3]

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Coumarins C6-C3 Flavonoids Flavonols C6-C3-C6 Flavones C6-C3-C6 Isoflavones C6-C3-C6 Flavanones C6-C3-C6 Flavanols C6-C3-C6 Anthocyanins C6-C3-C6 Chalcones C15 Stilbenes C6-C2-C6 Lignans (C6-C3)n Tannins (C6-C3-C6)n

Phenolics have different functions in plants. For instance, various bonds between flavonoids and the sugar residue cause different colours in flowers. Furthermore, they contribute to the structure, reproduction, and pollination of plants, as well as resisting predators and diseases. Phenolic compounds are even used for their beneficial effects on the human health. Due to their antioxidant or pharmacological properties, they are used to prevent diseases or, such as thymol, as antiseptic. Phenolic compounds with known antioxidant activities include flavonoids, phenolic acids and coumarins.

2.1.1 Flavonoids and phenolic acids

With approximately 4,000 different compounds, flavonoids represent the largest group of all plant phenolics [5]. Flavonoids are polyphenolic compounds and consist of two aromatic rings, connected by a bridge of three carbons [1]. Flavonoids can be further divided into subclasses: flavones, flavonols, flavanols, isoflavones, flavanones, anthocyanins and chalcones. Their structures are shown in Table 1. Looking at the structure in

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general, hydroxyl groups are usually found at the 4, 5 and 7 positions. Flavonoids may also occur as glycosides, where the hydroxyl or sugar groups increase the polarity of the flavonoids. On the contrary, the presence of methyl or isopentyl groups increase the lipophilic properties of flavonoids. Flavonoids can be found in various plants and vegetables, such as onions, tomatoes, garlic, and asparagus. Flavonoids often act as plant pigments, including flavones, chalcones and anthocyanins [5]. Various colours such as purple, blue, red, and pink are mostly caused by the presence of anthocyanins. Although the name anthocyanins suggests that a cyanide-related group is present, its name is derived from the Greek words “anthos”, meaning flower and “kyanos”, meaning blue [6]. The subgroup of isoflavonoids can function as pesticide, insecticide or even piscicide (fish poison). Isoflavones are compounds that are mainly found in soybeans and have been known to exhibit estrogenic properties [7]. They have a chemical structure partly similar to endogenous estrogens, both containing a phenol group [8]. This part of the compound enables the possible interaction with estrogen receptors (ER). Isoflavones are able to bind to ER and regulate gene expression. Furthermore, phytoestrogens can act as agonists or as antagonists in high concentrations, blocking the effects of endogenous estrogens by binding to the ER. Research has shown that isoflavones are related to impaired development of male reproductive organs in mammals [7]. Furthermore, the consumption of soy food results in a lower sperm concentration in men.

Phenolics are a part of the human’s diet and approximately one-third of that includes phenolic acids [3]. The structure of phenolic acids comprises at least one aromatic ring with one or more hydroxyl groups. These compounds and derivatives, such as hydroxycinnamic acid and caffeic acid, can have high antioxidant properties. Besides the antioxidant properties, phenolic acids may cause a bitter or astringent taste of vegetables, such as in soybeans and carrots [5]. Based on their parent compound, phenolic acids may be further divided into two classes, benzoic acids and cinnamic acids. Benzoic acids have seven carbon atoms, arising from the C6-C1 structure they have in common, while cinnamic acids have nine, arising from an aromatic structure with a three-carbon side chain (C6-C3) [4]. The antioxidant activity can be determined by the number of hydroxyl groups of these compounds. In general, hydroxylated benzoic acids are less effective than the cinnamic acids. One of the derivatives of cinnamic acid, are coumarins.

2.1.2 Coumarins

Coumarin was first isolated from the tonka bean, also known as ‘coumarou’. They can be found in essential oils, such as cinnamon bark oil and lavender oil, but also in fruits and green tea. Regarding families of plants, they can be found in Rutaceae, Apiaceae, Fabaceae and Moraceae [9]. Coumarins are mainly synthesized in the leaves and act as phytoalexins [10]. Phytoalexins are formed as a reaction to e.g. traumatic injury or plant diseases. Therefore, coumarins act as repellents against certain insects or inhibit growth of pathogens, by accumulating on the surface of leaves or seeds. As a result, the highest contents of coumarins can be found in fruits, and in lower degrees in the roots and stems.

The coumarin family can be divided into four sub-types: the (simple) coumarins, furanocoumarins, pyranocoumarins and pyrone-substituted coumarins [11]. The simple coumarins include the hydroxylated, alkylated and alkoxylated derivatives of coumarin, as well as their glycosides. A five-membered furan ring attached to the coumarin nucleus is specific for the furanocoumarins, where pyranocoumarins have a six-membered ring. Some examples of structures of coumarins are shown in Figure 2. As for pharmacokinetics in humans, coumarin is completely absorbed in the gastrointestinal tract after oral administration [12]. With half-lives of around 1.5 hours,

Figure 2: Examples of structures of coumarins, reproduced from ref. [11]

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coumarin is metabolized fast in the liver. Effects that have been known, are carcinogenesis and hepatotoxicity [13]. Hepatotoxicity has been observed in many mammals, from rodents to Beagle dogs. In Beagle dogs, a dosage of 25 mg/kg body weight daily would result in hepatotoxic effects. In contrast to being genotoxic, coumarin’s carcinogenic property is related to toxicity. Coumarin does not bind covalently to DNA, but toxicity in the target organ leads to the formation of tumours. As a result, a no observed adverse effect level (NOAEL) could be determined for coumarin, which was (based on the Beagle investigation) determined to be 10 mg/kg body weight daily. When in 2005 a coumarin content of 22 mg/kg was detected in cookies, a political debate resulted in new coumarin limits for cinnamon-containing foods. In the European Regulation (EC 1334/2008) is described that coumarins may not be added to food products and their natural content may not exceed the following limits: 50 mg/kg for traditional and/or seasonal bakery, such as cinnamon start; 20 mg/kg for breakfast cereals such as muesli; 15 mg/kg for fine bakery products; 5 mg/kg for desserts that contain cinnamon.

Furanocoumarins

Citrus are members of the Rutaceae family and are able to synthesize furanocoumarins [9]. As previously mentioned, furanocoumarins contain an additional furan ring that is attached to the coumarin nucleus. Examples of furanocoumarin structures are shown in Figure 3. Although the coumarins may have beneficial effects in plants, they can be harmful for humans. Furanocoumarins (and

coumarins) are potential photosensitizers, allowing them to make a chemical change in another molecule under the influence of light. This means that exposure to UV light after dermal contact to or ingestion of furanocoumarins, may result in a sever phototoxic inflammatory reaction known as Phyto photodermatitis. This causes the skin to turn red, feel itchy or burned, followed by large blisters being formed. Since citrus is widely used in cosmetics and perfume, this is a problem. Moreover, the monomers and dimers of furanocoumarins are able to inhibit cytochrome P450 (CYP) from different families. For instance, CYP3A4 is an important enzyme for humans: it plays an important role in the metabolism of drugs by oxidation. In addition, CYP3A represents half of the human P450 enzyme pools and is able to metabolize many drugs from almost every drug class [14]. In the body, furanocoumarins are mainly metabolized in the intestine by CYP3A4, and the formed intermediate is able to covalently bind to the active site of the enzyme [15–17]. This means that furanocoumarins may cause suicide inhibition of the enzyme, resulting in damaging CYP3A4 by accelerating the degradation process of the enzyme. This causes irreversible inactivation of the CYP enzyme, resulting in a shift of the metabolism from the intestine towards the liver. As a result, drugs that are administered orally

and simultaneously with furanocoumarins, are more dependent on the systemic clearance by CYP3A4 in the liver [16]. Other research showed that when the drug felodipine, a calcium antagonist, was administered orally together with grapefruit juice, the peak plasma drug concentration (Cmax) and area under the drug concentration-time curve (AUC) were increased [18]. However, the time to maximum plasma concentration (tmax) was not changed for the calculated half-life (t1/2). A corresponding graph is shown in Figure 4, comparing the concentration of felodipine when administered orally versus intravenously, with and without simultaneous intake of grapefruit juice. The graph shows that when grapefruit juice and felodipine are administered orally, the mean plasma concentration is increased strongly, compared to absence of grapefruit juice. For intravenous administration, there is no difference in the mean concentration of felodipine with and without presence of grapefruit juice. To summarize, the oral administration of furanocoumarins resulted in an increased concentration of felodipine by causing suicide inhibition of the CYP3A4 enzymes. The role of furanocoumarins in the metabolism as described here is visually summed in Figure 4, together with the graph as reproduced from Lundahl et al. [18].

Figure 3: Structures of

furanocoumarins, reproduced

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The inhibition of CYP3A4 is a serious problem and over 80 different drugs can suffer from augmented oral bioavailability when combined with grapefruit, including different kinds of anti-cancer, anti-infective, cardiovascular and central nervous system agents, as well as estrogens (see Bailey et al. for the original article and corresponding appendix for the list of grapefruit interacting drugs [15]).

2.2 Terpenoids and terpenes

The terms terpenoids, terpenes and isoprenoids are used to describe the terpenoids. The name terpene is derived from the Greek name for the terebinth tree [19]. Terpenoids are a very large class of plant secondary metabolites, having over 36,000 members that differ in structure and functional groups [20]. What they have in common, is the fusion of C5 units that have an isopentenoid structure. In this class, the isoprene rule states that all terpenoids are derived from the junction of head-to-tail joining of isoprene units. Exceptions to this rule apply to the pyrethrins, where no head-to-tail joining exists. Based on the number of isoprenoid units, a classification is followed. Varying from two isoprenoid units (monoterpenes) up to eight (tetraterpenes), these include the largest categories. The classification is shown in Table 2.

Figure 4: Summary of the metabolism of felodipine and the effect of grapefruit juice, containing furanocoumarins. At the bottom, a graph is shown with the mean plasma concentration of felodipine, comparing oral and intravenous administration with and without presence of grapefruit juice, as reproduced and adjusted from ref. [18]

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Terpenes or terpenoids may have many different properties, such as antimicrobial properties or attracting symbiotes for e.g. dispersal of seeds or pollination [21]. This is achieved by (airborne) chemical signals providing flavours and scents. Another property of terpenes is that some may function as both deterrent and attractant. For instance, the monoterpene 1,8-cineole is toxic to some flies, harmless to honeybees and attracts insects that ensure pollination. Many monoterpenes play a role in the defence against pathogens, where sesquiterpenes act as phytoalexins or as antifeedants to deter herbivores [22]. Abietic acid is a diterpene that can be found in pines, blocking resin canals when they are pierced by pathogens. Saponins are triterpenoids that are covalently attached to one or more sugar moieties. Carotenoids are an important group of tetraterpenes, functioning as pigments. As for polyterpenes, a well-known compound is rubber, containing over 1500 isopentenyl units.

An important diterpene is paclitaxel, a high molecular weight compound that can be extracted from the bark of the yew tree, also known as Taxus brevifolia [23]. It is used for treatment of various forms of cancer. During cell division, specifically mitosis, microtubules play an important role to form the mitotic spindle. These microtubules are in an equilibrium with tubulin polymers and heterodimers. Paclitaxel inhibits

Table 2: Classification of terpenoids, based on the number of isoprenoid units in their structures. Reproduced from ref. [20]

Isoprene units n

Carbon atoms n

Name Example Structure of example

1 5 Hemiterpenes Isoprene 2 10 Monoterpenes Pulegone 3 15 Sesquiterpenes Polygodial 4 20 Diterpenes Paclitaxel 5 25 Sesterterpenes 6 30 Triterpenes β-Amyrin 8 40 Tetraterpenes β-Catorene 9-30000 >40 Polyterpenes Rubber

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depolymerization of tubulin, preventing the required (dis)assembly and causing disruption of normal microtubular dynamics, finally resulting in cell death. Because of this specific effect, paclitaxel is applied for treatment of prostate, lung, breast and ovarian cancer [24]. One of the difficulties of paclitaxel involves the extraction and/or synthesis of the compound. To obtain 1 kg of extract of paclitaxel, around 3,000 trees are required for extraction. Extraction generally involves washing of the plant material, followed by extraction with an organic solvent such as methanol or acetone. Clean-up includes steps such as salting-out followed by a chromatographic purification. Synthesis has been tested and achieved, obtaining yields varying from 0.07 up to 2.7%. Because of these low yields, the synthesis is too expensive and too complex to be applied in industrial use.

2.3 Saponins

One of the unique features of saponins, is that they are able to foam. Their name is derived from the Latin “sapo”, meaning “soap”. Therefore, a similar structure to soap can be expected. Saponins have an amphiphilic structure, consisting of a hydrophilic sugar part and a hydrophobic part [25]. The sugar part can consist of up to three sugar chains (straight or branched), being mostly L-arabinose, fucose, galactose, glucose, D-glucuronic acid, L-rhamnose or D-xylose. An example of the structure of a saponin is shown in Figure 5.

Figure 5: Example of a steroidic saponin, reproduced from ref. [26]

The hydrophobic part, here also called the aglycone, genin or sapogenin, can have a steroid or triterpenoid structure [27]. Triterpenoid saponins have 30 carbon atoms, where steroidal saponins have 27 carbon atoms with either a six-membered ring spirostane or a five-membered ring furostane skeleton. The triterpenoid saponins are mostly found in the class of Magnoliopsida, while the steroidal saponins are common in the class of Liliopsida. The composition of saponins depends on different factors, such as the genetic background of the plant and environmental factors.

Saponins are produced in the leaves of plants and stored in the roots, functioning as antimicrobial phytoprotectants [27]. For instance, the saponin avenacin A-1 is stored in the epidermal cell layer of a plant’s root, functioning as a chemical barrier to microbes. The mechanism of synthesis of saponins is quite unique. In general, the production of plant toxins is stimulated upon (a)biotic stress. However, some saponins are produced regardless of external signals. Precursors of saponins are already present in the plant. After pathogen infection, chemical processes alter the precursors, resulting in an increased amount of saponins. Various properties have been ascribed to saponins, including their potency to permeate the membrane, to affect protein digestion and the uptake of minerals and vitamins [5].

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2.4 Alkaloids

Compounds in the alkaloid group all contain a heterocyclic ring, with a nitrogen atom in the ring [28]. They originate from lysine, tryptophan or tyrosine and with over 12,000 different alkaloids, they can be found in about 20% of the different plant species [29]. Alkaloids are commonly present as salts of organic acids, including lactic and oxalic acid. They can also exist freely in nature, such as the weak basic alkaloid nicotine. Furthermore, they can occur as glycosides with sugars such as glucose and rhamnose. Because the various alkaloids have various biological properties, they are used as stimulants, narcotics, and pharmaceuticals. Alkaloids that are known for their pharmacological, but also addictive effects, include morphine and codeine. Alkaloids may have beneficial pharmacological effects at lower doses but may be poisonous at high doses. Because of their possible effect as toxins, alkaloids are involved in the defence mechanism of plants, especially against mammals. Some examples of various alkaloids are shown in Figure 6.

Caffeine is a purine alkaloid that can be found in the seeds and leaves of the families Malvaceae and Theaceae [30]. Caffeine may have various effects in the body. By acting as an antagonist on the adenosine receptors, caffeine indirectly affects the release of serotonin, dopamine, acetylcholine, gamma-aminobutyric acid (GABA) and norepinephrine, explaining its behavioural effect [31]. These effects include affecting sleep, memory, and learning. Furthermore, caffeine inhibits phosphodiesterase enzymes, causing a cardio stimulatory effect such as an increased rate of heartbeat.

Figure 6: Various structures of alkaloids, including caffeine, morphine, nicotine, and cocaine, reproduced from ref. [29]

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2.4.1 Pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are alkaloids that have a great structural diversity [32]. PAs are derived from ornithine and occur rarely as pyrrolizidine base, also known as the free form. Instead, they commonly occur as esters (mono-, di- or macrocyclic diesters) or as tertiary bases or as pyrrolizidine alkaloids N-oxides (PANOs). Their origin comes from necine bases (amino alcohols) or necic acids (mono- or dicarboxylic aliphatic acids), with examples of structures depicted in Figure 7.

Pyrrolizidine alkaloids that occur in food, are mostly esters of 1-hydroxymethyl-1, 2-dehydropyrrolizidine [33]. PAs can be found in eggs, vegetables, milk, and honey, but also in seeds from plants. PAs can be found in the leaves of Borago officinalis that is used as spice in salads and soup, whereas the leaves or roots of

Symphytum officinale can be used in herbal tea. In the leaves up to 1800 mg PAs can be found per kg, while

the roots can contain up to 2900 mg PAs per kg. PAs may be transferred from Senecio jacobaea by bees to honey, where up to 3.9 mg PAs per kg honey were found. Furthermore, research has shown that when cows are fed with PA containing ragwort, it may be transferred to milk [34].

PAs are absorbed from the gastrointestinal tract after oral ingestion [32]. Most of the PAs are excreted in urine and faeces and some may pass the placenta. There are three main pathways involved in the metabolism of PAs. One involves hydrolysis of PAs resulting in necines and necic acids, a second one involves N-oxidation resulting in the formation of PANOs and the third involves oxidation leading to the formation of pyrrolic esters or dehydropyrrolizidine alkaloids (DHPA). The metabolic pathways are summed in Figure 8. DHPAs are able to form DNA adducts, potentially resulting in damage mainly in the hepatocyte. This may result in hepatic sinusoidal obstruction syndrome. The syndrome may cause an enlarged liver and may possibly end in death [33]. Furthermore, long-term exposure to PAs may also affect the lungs by resulting in pulmonary hypertension or affecting the cardiovascular system by resulting in cardiac right ventricular hypertrophy. EFSA already concluded in 2010 that DHPAs may act as genotoxic carcinogens in humans, and toddlers and children that consume large amounts of honey may be at possible risk [35]. As a result, the EFSA Panel on Contaminants in the Food Chain concluded that there is a possible concern for the human health related to exposure to PAs and a list of PAs to be monitored in food products was proposed [36].

Figure 7: Structure of a pyrrolizidine alkaloid (PA) and its different forms,

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Figure 8: Metabolic pathways of PAs. Figure reproduced and adapted from ref. [32]

2.4.2 Tropane alkaloids

The tropane alkaloids are tertiary or quaternary (N-oxides) bases having a tropane structure that originates from L-ornithine and/or L-arginine and contain one or more hydroxyl groups at position 3 [37]. Mostly, tropane alkaloids can be divided into main groups based on the substituent on position 3. More specifically, when the hydroxyl group is in axial position, the group is referred to as 3α-hydroxytropanes, also known as tropine. On the contrary, when the hydroxyl group is in equatorial position, the group is referred to as 3β-hydroxytropanes, also known as pseudotropine. The structure of the tropane skeleton, tropine and pseudo-tropine with corresponding biosynthetic pathways are shown in Figure 9. Tropane alkaloids can be found in various plants, including Proteaceae, Erythroxylaceae, Rhizophoraceae and Solanaceae. Although present in all parts of the plants, the highest concentrations will be in the roots and seeds [38]. In addition, the concentration can vary for each species, season, or location of the plant. The plants may produce berries that can be very toxic: less than three berries of henbane or nightshade, can have lethal effects in children [1]. These berries contain scopolamine and hyoscyamine. The racemic form of hyoscyamine is known as atropine, known for its property to dilate the pupil. It is a muscarinic acetylcholine receptor antagonist, also used for treatments of poisoning with e.g. nerve gas. Scopolamine can be used to prevent motion sickness or during labour as a sedative, when combined with morphine. In general, tropane alkaloids act as competitive antagonists of acetylcholine by blocking its binding to muscarinic receptors [37]. Additional effects can be observed on the central nervous system when a tertiary amine is present. For instance, cocaine (structure

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shown in Figure 9) acts as a reuptake inhibitor of serotonin-norepinephrine-dopamine. Although cocaine has already been used for a long time by South American indigenous people, nowadays it is mostly used for its euphoric and energetic effects, being highly addictive as well.

Figure 9: Structure and numbering of tropane skeleton (a) and biosynthetic pathways of tropane alkaloids, forming α-tropine and pseudo-tropine (b). Reproduced and adapted from ref. [37]

2.5 Cyanogenic glycosides

Cyanogenic glycosides are a large and diverse group of compounds that are able to release hydrogen cyanide (HCN), causing toxic effects [39]. Glycosylation of cyanohydrin results in the formation of a cyanogenic glycoside. With oximes and cyanohydrins as intermediates, they are derived from different amino acids such as aromatic, aliphatic and nonproteinogenic amino acids. Through hydrolysis of cyanogenic glycosides, the intermediate cyanohydrin is formed and when it dissociates, HCN is formed. This process is called cyanogenesis. The enzymes that catalyse the hydrolysis and the cyanogenic glycosides are stored in different tissues or in separate compartment within cells to prevent accidental autotoxicity. Certain events, such as chewing herbivores or another physical process such as freezing, can cause the two components to get into contact, initiating the cyanogenesis. The production of HCN is what causes the potential toxicity of cyanogenic glycosides. The acute dose of cyanide is around 1 mg/kg body weight and can have lethal effects

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[40]. Cyanide acts very quick, starting with dermal absorption or absorption via the respiratory system [41]. According to Egekeze et al., clinical signs of toxicity can occur within 25 minutes, leading to death in about 1 hour, after dermal absorption of 10% NaCN. However, another greater part is metabolized into thiocyanate, which is less toxic and excreted in the urine during a few days. As a result, animals are able to ingest lower doses of cyanogenic glycosides for a longer period of time, without any toxic effects. This depends on different factors, such as the size of the animal and the ability to metabolize cyanide. Once absorbed, cyanide forms a complex with cytochrome oxidase (the cytochrome oxidase-CN complex). The electron transfer to oxygen is blocked and therefore the chain of cellular respiration. Oxyhemoglobin is not able to release its oxygen, which results in bright red blood, full of oxygen, unable to be utilized in the cells. The first sign of intoxication can be an increase in the rate and depth of the breathing, followed by slower breathing, ending with respiratory arrest.

Cyanogenic glycosides are found in many different plants. For instance, the cyanogenic glycoside Linamarin is found in the cassava (Manihot esculenta), but also in the family of Rosaceae, such as peaches and bitter almonds [40]. The great variety of cyanogenic glycosides is caused by different aspects. For instance, cyanogenic glycosides are derived from different amino acids. Secondly, one or more hydroxylations modify the structures. Third, there is the structural diversity of the sugar moiety. For instance, the cyanohydrin is always linked through a β-glucosidic linkage to D-glucose. For cyanogenic diglycosides, the second sugar moiety may be a D-glucose such as amygdalin (prunasin-6’ -glucoside) or linustatin (linamarin-6’ -glucoside), being attached by a β-1,6 linkage. However, other variations in linkages may occur, such as β-1.2, β-1.3 and β-1.4 linkages. Other sugar moieties may also be attached, such as xylose and arabinose. In Figure 10, some examples of structures of cyanogenic glycosides are shown.

Figure 10: Structures of different cyanogenic glycosides, showing the structural diversity. Reproduced from ref. [39]

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Page 21 of 103

3 Methods for analysis of plant toxins

The aim of the research was to obtain an overview of the methods that are currently available for analysing plant toxins in feed and food, that can be applied in the feed and food industry for monitoring. The search for literature started broad, focusing on determining which plant toxins should be excluded from this thesis. All keywords used for searching were tracked and the articles related to the thesis were processed in Excel. For each article, the year of publication, keywords, plant toxins, matrices, sample pre-treatment and other specifics were described. Articles used for background information were processed as well. This resulted in more than two-hundred articles that were found. To narrow it down, the articles were selected based on their year of publication, plant toxins, sample pre-treatment, separation, and detection methods. Furthermore, some articles lacked a clear description of the methods applied and were excluded as well. This resulted in approximately seventy articles and methods, that will be further discussed in this chapter. The aim of this chapter is to give an overview of the articles that were selected and of the methods that have been described. In chapter four, the methods are discussed more deeply, and a critical view is taken.

3.1 Sample pre-treatment

3.1.1 Introduction

Different matrices and analytes of interest require different sample pre-treatments. During this research, the focus was on feed and food. This makes the spectrum very broad, where each matrix and compound (group) of interest requires a specific sample pre-treatment. The sample pre-treatments discussed here include steps for extracting the compounds from the matrix, as well as possible clean-up steps. The different methods for sample pre-treatment that are discussed, include solid-liquid extraction (SLE), strong cation exchange (SCX), solid-phase extraction (SPE), liquid-liquid extraction (LLE), QuEChERS and microwave-assisted extraction methods. For each different kind of method, various aspects are shown to give an overview. This chapter serves the information required for the discussion that is followed in chapter four.

3.1.2 Solid-liquid extraction

Considering feed and food, many matrices will be solid. For these matrices, quite often a solid-liquid extraction is applied. Simply this means adding a liquid to a solid sample and the analytes of interest are extracted into the liquid. This has been applied for many different matrices and plant toxins. For instance, ethanol and water have been used to extract amygdalin, a cyanogenic glycoside, from seeds [42, 43]. Cyanogenic glycosides were also extracted with water and methanol from seeds [44]. An aqueous extraction for PAs in medicinal tea is applied by Bosi et al. [45], whereas a static extraction is used to extract tropane alkaloids from buckwheat [46]. Coumarins have been extracted from plant material using methanol [47]. An overview of solid-liquid extractions applied, is given in Table 3. Here solid-liquid extraction is interpreted as adding a solvent or a mixture of solvents that are at least miscible, to a solid sample.

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Table 3: Selected articles that apply solid-liquid extraction (SLE) to extract plant toxins from solid matrices. Abbreviations include: formic acid (FA), methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), acetic acid (AA), capillary electrophoresis (CE), electro-chemiluminescence (ECL), ultrasound-assisted extraction (UAE), accelerated solvent extraction (ASE), pressurized liquid extraction (PLE), (ultra) high-performance liquid chromatography ((U)HPLC), photodiode array (PDA), mass spectrometry (MS), electrospray ionization (ESI), nuclear magnetic resonance (NMR), gas chromatography (GC), high resolution mass spectrometry (HRMS), time of flight (TOF), integrated pulsed amperometric detection (IPAD), fluorescence detection (FLD), molecularly imprinted solid-phase extraction (MISPE), magnetic molecularly imprinted polymers (MMIPs), supercritical fluid chromatography (SFC)

Ref. Matrix Compound Extraction Time (min)

Clean-up Recovery (%) Separation & detection

Avula et al., 2016 [48] Plant Coumarins SLE with MeOH, sonication 30 None 97-104 UHPLC-PDA-MS & HPLC-TOF-MS Bodi et al., 2014 [49] Tea (honey) PAs SLE with 0.05 M H2SO4, ultra sonication 15 SPE 72-122 HPLC-ESI-MS/MS Bolarinwa et al., 2014a

[42]

Seeds Cyanogenic glycoside

SLE with EtOH under reflux 100 None 98 HPLC-DAD Bolarinwa et al., 2015

[43]

Food Cyanogenic glycoside

SLE with EtOH under reflux 100 None >98 HPLC-DAD Bosi et al., 2013 [45] Plant PAs SLE with boiling H2O, followed by column

chromatography and then lyophilization

Not mentioned

Column chromatography

Not mentioned NMR & HPLC-HRMS Caligiani et al., 2011

[46]

Plant Tropane alkaloids Static extraction with CH2Cl2 1440 None 89-110 GC-MS Chen et al., 2012 [50] Fruit Coumarins Reflux extraction with MeOH 60 None 95-105 HPLC-MS/MS Cirlini et al., 2018 [51] Food Tropane alkaloids SLE with extraction solvent: MeOH:H2O

(3:2, v/v) with 0.2% ACN and 0.2% FA, pH 3.2; shaken

90 None 78-103 UHPLC-MS/MS

Cirlini et al., 2019 [52] Tea Tropane alkaloids SLE with ACN:H2O (3:2, v/v) with 0.2% FA, shaken

90 None 83-107 UHPLC-MS/MS

De Nijs et al., 2014 [53] Feed PAs SLE with 2% FA in water on a rotary tumbler 60 SPE Not mentioned LC-MS/MS Dresler et al., 2018 [54] Plant Coumarins SLE in ultrasonic bath with 80% MeOH 30 None 96-109 CE-DAD Dugrand-Judek et al.,

2015 [9]

Fruit Coumarins SLE with MeOH:H2O (80/20), overhead shaken

60 None Not mentioned UPLC-MS Fiorito et al., 2019 [55] Seeds Coumarins Ultrasound-assisted extraction with EtOH 1 None 96-100 UHPLC-UV/VIS Gómez-Caravaca et al.,

2011 [56]

Plant Saponins SLE in ultrasonic bath with MeOH:H2O with AA

20 None Not mentioned HPLC-DAD-ESI-TOF-MS

Guo et al., 2015 [57] Plant Tropane alkaloids SLE with MeOH in ultrasonic bath 30 None 98-107 CE-ECL Hroboňová et al., 2018

[58]

Plant Coumarins SLE with MeOH, stirred and mechanical shaken

60 None 88.6-95 HPLC-DAD

Huybrechts et al., 2015 [59]

Food, feed PAs Solid samples: SLE with extraction solvent and hexane1_{, overhead shaken}

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Kubrak et al., 2015 [60] Plant Coumarins SLE with MeOH, ultrasonic extraction 30 None 92-97 CE-DAD Lee et al., 2017 [61] Plant Saponins Reflux extraction of sample powder with 80%

EtOH

120 None 97.73-102.57 LC-IPAD Li et al., 2014 [62] Plant Coumarins SLE with MeOH in ultrasonic bath 60 None Not mentioned Off-line

2D-HPLC-ESI/MS Li et al., 2019 [63] Citrus juice (food) Coumarins After centrifugation, SLE was performed with

ACN (three times) on the residual solid material

Not mentioned

SPE 93-101 LC-DAD-FLD

Ling et al., 2016 [64] Plant Saponins SLE with MeOH in ultrasonic bath 30 None Not mentioned HPLC-ESI-QTOF-MS/MS

Llorent-Martinez et al., 2015 [65]

Plant Saponins Ultrasound-assisted solvent extraction with MeOH, 35 Hz, 200 W (60 min)

60 None Not mentioned HPLC-ESI-MSn Machyňáková et al.,

2017 [66]

Food Coumarins SLE with water, stirring 60 MMIP-SPE 71.4-90.3 HPLC-DAD Machyňáková et al.,

2017 [67]

Food Coumarins SLE with water, stirring 60 On-line MISPE 87.7-112.2 On-line MISPE-HPLC-DAD

Marín-Sáez et al., 2017 [68]

Food Tropane alkaloids SLE with MeOH/AA (0.5%) (2/1 v/v), rotary shaker

30 SPE mixed-mode 60-109 LC-MS (Orbitrap) Marín-Sáez et al.,

2019a [69]

Food Tropane alkaloids SLE with MeOH:H2O (2:1 v/v) (0.5% AA), rotary shaker

30 None 75-101 LC-MS (Orbitrap) Marín-Sáez et al.,

2019b [70]

Food Tropane alkaloids SLE with MeOH:H2O (2:1 v/v) (0.5% AA), rotary shaker

30 SPE mixed-mode 66-98 LC-MS Mehari et al., 2016 [71] Food Alkaloids SLE with water, shaken 30 Precipitation 98.6-103.2 HPLC-DAD Mudge et al., 2015 [72] Plant (honey) PAs SLE with 80% MeOH, shaken 60 None 84.6-108.2 HPLC-MS Mulder et al., 2016 [73] Food Tropane alkaloids SLE with MeOH/H2O/FA (75/25/0.4, v/v/v),

rotary tumbler

30 SPE mixed-mode 61-105 LC-MS/MS Mulder et al., 2018 [74] Food

(animal-derived)

PAs SLE with H2O (0.2% FA) and hexane, rotary tumbler

30 SPE 56-107 LC-MS/MS

Mulder et al., 2018 [74] Food supplements (oily plant-derived)

PAs SLE with 0.05 M H2SO4 in MeOH, overhead shaken

15 SPE mixed-mode Supp. Data not found

LC-MS/MS

Mulder et al., 2018 [74] Tea PAs SLE with water (ISO 3103) 5 SPE 72-1222 45-983

LC-MS/MS Muzashvili et al., 2014

[75]

Plant (seeds) Cyanogenic glycosides

ASE with 80% MeOH Not

mentioned

SPE Not mentioned UPLC-MS/MS Pfeifer et al., 2016 [47] Plant Coumarins SLE with MeOH, sonication 20 None 96.5-104.2 SFC-PDA Qiu et al., 2018 [76] Food Coumarins PLE with diatomaceous earth (1:1, w/w) and

80% EtOH

10 None 96.2-104.3 LC-DAD Reim et al., 2015 [77] Food Saponins SLE with MeOH (water bath 4 h)

Extra precipitation step for 20 h

240 SPE 90 HPTLC-MS &

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Romera-Torres et al., 2018 [78]

Tea Tropane alkaloids SLE with MeOH/H2O/FA (75:25:0.4, v/v/v), rotary agitator

30 SPE mixed-mode 75-128 LC-MS Orbitrap Romera-Torres et al.,

2018 [79]

Feed Tropane alkaloids PLE with Hydromatrix and MeOH/H2O/FA (75/25/0.4, v/v/v)

10 SPE mixed-mode 70-109 LC-HRMS Senica et al., 2016 [80] Seeds Cyanogenic

glycosides

SLE with MeOH/H2O (70/30 v/v) 30 None Not mentioned HPLC-DAD & HPLC-MS Senica et al., 2017 [81] Plant Cyanogenic

glycosides

SLE with MeOH/H2O (70/30 v/v) 30 None Not mentioned HPLC-DAD & HPLC-MS Senica et al., 2019 [44] Seeds Cyanogenic

glycosides

SLE with MeOH/H2O (70/30 v/v) 30 None Not mentioned HPLC-MS Shimshoni et al., 2015

[82]

Tea PAs and tropane alkaloids

SLE with MeOH/acetic acid (2/1), shaken 30 None 70-110 LC-MS/MS Tine et al., 2017 [83] Plant Coumarins SLE with MeOH under magnetic stirring 2880 None Not mentioned LC-MS/MS Urban et al., 2019 [84] Food PAs, tropane

alkaloids

SLE with ACN/H2O (80/20, v/v), overhead shaken

30 None 91-104 2D-LC-MS/MS

Wang et al., 2013 [85] Plant Coumarins SLE with EtOH/H2O (70:30, v/v), ultrasonic extraction 30 SPE 56.95-105.47 HPLC-UV Waszkowiak et al., 2015 [86] Seeds Cyanogenic glucosides

Aqueous SLE: H2O, stirred Ethanolic SLE: 60% EtOH, shaken

60 None Not mentioned GC-FID Winderl et al., 2016

[87]

Fruit Coumarins SLE with MeOH, sonication 15 None 97.2-103.6 SFC-PDA Xu et al., 2015 [88] Fruit Coumarins SLE with ethyl acetate, ultrasonic cleaner 30 None Not mentioned UPLC-PDA Yoon et al., 2015 [89] Food PAs SLE with MeOH4_{or chloroform-methanol,}

ultrasonic bath

40 SPE mixed-mode 89-109 LC-ESI-MS/MS & LC-APCI-MS Zhao et al., 2019 [90] Seeds Cyanogenic

glycosides

Ultrasound-assisted SLE with MeOH/H2O (75:25, v/v) 5

16 Hydrolysis 92.3-102.5 UPLC/ESI-HRMS Zhang et al., 2016 [91] Plant Saponins SLE with 70% MeOH under heat reflux 120 Column

chromatography

98.53-102.82 LC-CAD-ESI-MS Zheng et al., 2010 [92] Plant Coumarins SLE with 75% MeOH in ultrasonic bath 30 None 92.1-105.6 HPLC-ESI-MS/MS

1_{: extraction solvent consisted of 20 g NaCl, 800 mL H2O, filled up to 1000 mL with 37% HCl.} 2_{: fennel, mixed herbal, and rooibos tea.}

3_{: chamomile and black tea.}

4_{: Milk and soybean powder extracted with chloroform:MeOH, margarine and seed oil extracted with MeOH. Followed by precipitation of lipids and extracting PAs again.} 5_{: final chosen sample preparation: aqueous MeOH ultrasound-assisted extraction, followed by alkaline ultrasound-assisted extraction (0.08 M NaOH/MeOH). Finally,} hydrolysis performed with 0.02 M NaOH.

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3.1.3 Liquid-liquid extraction

For liquid matrices, a liquid-liquid extraction (LLE) can be applied. However, from the selected articles, an LLE procedure was only performed by Huybrechts et al. [59]. Therefore, LLE will not be further discussed except for this article. Huybrechts et al. focused on extracting PAs from food and feed, including milk and honey samples. To extract from milk, HCl and hexane were added, and extraction took place in a water bath (60 °C, 60 min). HCl was added to denaturate the proteins, removing an extra clean-up step. In case of honey, an extraction solution was added consisting of an aqueous solution of NaCl and HCl. Their research shows a confirmation of the fact that PAs can be carried over from feed into milk, since PAs were detected in nine out of 63 milk samples. As for honey, fifteen out of sixteen samples contained one or more PAs, with one even containing all sixteen PAs of interest. It is possible that LLE is not further applied, because most feed and food samples that were analysed, were solid. Perhaps the selected articles did not include sufficient liquid matrices, or little research is carried out for liquid matrices and plant toxins. Since plant toxins originate from plants, they may require some kind of transfer (such as carry-over to milk) to end up in liquid matrices.

3.1.4 QuEChERS

QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged and Safe [93]. The procedure utilizes a low volume of organic solvent and addition of salts and solid-phase extraction (SPE) packing sorbents. Initially, a hydrophilic solvent such as acetone or acetonitrile (ACN) is added to the sample, allowing analytes of interest to transfer to the solvent (step 2, see Figure 11). Next, salts are added (step 3), resulting in a separation of the organic solvent from water related to the sample. Simultaneously, this step promotes extraction of the analytes of interest into the organic solvent. Then, the internal standard is added (step 4) and the sample is shaken and centrifuged. To an aliquot of the organic phase, SPE packing sorbents are added in order to remove interfering compounds (step 5). Additionally, the pH can be adjusted (step 6) and finally the sample is analysed (step 7). Official methods already exist [94] and have been applied for the analysis of pesticides in fruit and vegetables, extending to other matrices such as fish products and meat. For analysis of plant toxins, QuEChERS was applied eight times to matrices such as feed, food, tea, and seeds. An overview of these articles is given in Table 4.

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Table 4: Overview of selected articles that have applied QuEChERS for extraction and analysis of plant toxins. Described are the matrices, plant toxins, extraction specifics, recovery and separation and detection. Abbreviations include acetonitrile (ACN), formic acid (FA), matrix solid phase dispersion (MSPD), graphitized carbon black (GCB), primary secondary amine (PSA) and ethylenediaminetetraacetic acid (EDTA).

Ref. Matrix Compound Extraction Recovery (%) Separation & detection

Bolechová et al., 2015 [96] Feed PAs ACN and H2O (0.1% FA); addition of MgSO4 and NaCl, organic phase analysed 72-94 at 5 µg/kg 80-98 at 100 µg/kg

UPLC-MS/MS Chen et al., 2017 [97] Food Tropane

alkaloids

H2O and ACN (1% FA); addition of NaSO4 and ammonium acetate, supernatant (ACN) PSA and GCB added and finally analysed

75-92 UHPLC-MS/MS Dzuman et al., 2015 [98] Food, tea PAs H2O (0.2% FA) and ACN; addition of MgSO4 and NaCl. Organic layer:

Bondesil-C18 and MgSO4, extract further analysed

70-120 HPLC-HRMS/MS Jandrić et al., 2011 [99] Seeds Tropane

alkaloids

Addition of 0.5% FA in ACN/H2O (75:25, v/v), followed by addition of MgSO4, NaCl, sodium citrate dihydrate and sodium hydrogen citrate sesquihydrate. To soybean and linseed, extra MSPD C18 material was added.

61-111 LC-MS/MS

León et al., 2016 [100] Feed Alkaloids H2O and ACN (1% AA), addition of MgSO4, NaCl, sodium citrate, disodium citrate sesquihydrate and ceramic homogenisers; organic phase analysed

80-120 UHPLC-HRMS Martinello et al., 2017 [101] Honey Tropane

alkaloids

ACN and ceramic homogenizer. Addition of Q-sep QuEChERS extraction salts1_, supernatant purified with purification Q-sep dSPE2_{, purified supernatant analysed}

92.3-114.8 LC-HRMS Melough et al., 2017 [102] Food Coumarins ACN; addition of QuEChERS powder (MgSO4/NaOAc) 95 UPLC-MS Zheng et al., 2019 [103] Food (porcine

muscle, egg, milk)

Tropane alkaloids

EDTA and ACN (0.5% trifluoroacetic acid); QuEChERS reagent added3_{, to} supernatant C18 sorbent and MgSO4 added.

73-104 LC-MS/MS

1_{: magnesium sulphate 4 g, trisodium citrate dehydrate 1 g, disodium hydrogen citrate sesquihydrate 0.5 g and sodium chloride 1 g} 2_{: PSA 0.15 g and magnesium sulphate 0.9 g}

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Page 27 of 103

3.1.5 Microwave-assisted extraction

Microwaves are a form of electromagnetic radiation in the frequency range of 0.1 to 100 cm-1_{, corresponding} to 0.3 to 300 GHz [104]. Microwave heating differs from conventional heating, since conventional heating transfers thermal energy through conduction and convection, while in microwave heating ionic conduction and dipole rotation transfer electromagnetic energy into heat. As a result, only compounds that contain a dipole can absorb microwaves. Applying a closed-vessel system allows higher temperatures and higher pressures to be achieved, reducing the required time for extraction [105]. Furthermore, less solvent can be applied and a high through-put can be obtained (e.g. up to twelve samples per hour for closed-vessel systems) [104].

Although only applied twice, microwave-assisted extraction will still be discussed. A recent study focused on the optimization of a microwave-assisted extraction (MAE) to extract saponins, phenolics and flavonoids from fenugreek seeds [106]. A two-level factorial design was used, considering the extraction time, ethanol concentration, microwave power, ratio of feed-to-solvent and extraction temperature. The highest yield was obtained for 600 W at 70 °C for 3 min, using 1:10 g/mL and 60% solvent concentration. The optimization showed that increasing the temperature, microwave power and the irradiation time, increased the yield. However, an increase in the ethanol concentration or the feed-to-solvent ratio decreased the yield. In fact, the ethanol concentration was the most significant factor, with a contribution of 26.03%. When the solvent concentration is increased, more time and energy is required for the compounds to be absorbed by the solvent. This explains the negative effect.

Microwave-assisted extraction was also applied by Carro et al. for extraction of terpenoids from plant leaves [107]. Compared to steam distillation (SD) and ultrasound-assisted extraction (UAE), MAE provided the highest number of compounds to be identified (18 terpenoids), while UAE resulted in 14 detected compounds and SD in only 11. SD gave a higher deviation and resulted in about ten-fold lower concentrations in its extract compared to UAE, whereas MAE gave the highest concentrations. Based on these results, MAE was considered the best extraction method. Since MAE was only applied few times, it is only briefly discussed in chapter four.

3.1.6 Solid-phase extraction

Solid-phase extraction (SPE) is a commonly used extraction technique that can also be used as clean-up step. In SPE, separation or removal of an analyte from a mixture of compounds is achieved by selective partitioning of the compounds between a solid phase (the sorbent) and a liquid phase (the solvent). This principle is similar to separation achieved in liquid chromatography (LC), however the columns in SPE contain larger particles, reducing their efficiency compared to regular LC columns and allowing compounds to be trapped and eluted. Since interferences may remain on the cartridge, SPE columns are more used as consumables and only used once. Larger particles result in lower backpressure, allowing faster sample pre-treatment because pressure or a vacuum can be applied.

In general, an SPE procedure involves six steps. The first step is sample pre-treatment, e.g. diluting the sample. Secondly, the SPE column is conditioned. This will clean and solvate the sorbent and allow removal of air channels and interferences. Third, the column is equilibrated, removing the solvent that was applied in the previous step, normalizing the sorbent to match the sample condition. Furthermore, equilibration will promote the optimal environment for retention. Fourth, the sample is applied or loaded. In the fifth step, interfering compounds that have no interaction with the column are eluted. This is also called the wash step. In the sixth step, the analyte of interest is eluted by applying a solvent strong enough to overcome the interaction between the analyte and the column. One of the advantages of SPE includes the availability of many different stationary phases, such as polymeric stationary phases, strong cation exchange (SCX) and mixed-mode stationary phases. The latter two will be discussed more deeply later in this subsection.

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During this research, SPE was applied in seven articles, where Mulder et al. used two different SPE procedures for two different matrices [74]. In Table 5 an overview is given of the matrices, plant toxins, and several steps from the SPE procedure.

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Page 29 of 103 Table 5: Overview of different articles in which solid-phase extraction (SPE) was applied as clean-up step. The various matrices and compounds are described, as well

as specifics for the SPE procedure. Finally, the separation and detection are briefly mentioned. Abbreviations include methanol (MeOH), double-distilled water (ddH2O),

formic acid (FA), acetonitrile (ACN), diode array detection (DAD), fluorescence detection (FLD), high-performance thin-layer chromatography (HPTLC) and matrix-assisted laser desorption/ionization (MALDI).

Ref. Matrix Compound Column Pre-condition Wash Elution Recovery (%) Separation & detection

Bodi et al., 2014 [49]

Tea PAs Discovery

DSC-C18

MeOH and H2O H2O, then dry MeOH (2.5% ammonia) 72-1221 45-982

HPLC-ESI-MS/MS De Nijs et al., 2014

[53]

Feed PAs Strata-X SPE MeOH and H2O 1% FA and 1% ammonia, then dry

MeOH Not mentioned LC-MS/MS Wang et al., 2013

[85]

Plant Coumarins Florisil Hexane MeOH 56.95-105.47 HPLC-UV

Li et al., 2019 [63] Citrus juice Coumarins C18 (1 cc, Sep-Pak plus)

MeOH and H2O 75:25 H2O:ACN Ethyl acetate 93-101 HPLC-DAD-FLD & HPLC-MS/MS Mulder et al., 2018

[74]

(Herbal) teas and supplements

PAs Discovery DSC-C18

MeOH and H2O H2O and dry MeOH or MeOH (2.5% ammonia) 72-1221 45-982 LC-MS/MS Mulder et al., 2018 [74] Food (animal-derived)

PAs StrataX MeOH and 0.1% ammonia 0.1% ammonia and dry MeOH 56-107 LC-MS/MS Muzashvili et al., 2014 [75] Plant Cyanogenic glycosides

C18 Sep-Pak H2O H2O 20% (v/v) MeOH Not mentioned UPLC-MS/MS Reim et al., 2015

[77]

Food Saponins Chromabond C18

MeOH

Equilibrate: ddH2O

ddH2O:MeOH (95:5, v/v)

Fractionation: five steps MeOH:H2O gradient, alkaline pH by adding 17% ammonia

90 HPTLC-MS & MALDI-TOF-MS

1_{: fennel, mixed herbal, and rooibos tea.} 2_{: chamomile and black tea.}

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Mixed-mode solid-phase extraction

Mixed-mode cartridges have mixed sorbents of C8 or C18 with either a weak/strong anion or weak/strong cation exchanger. The mixed-mode solid-phase extraction (SPE) allows more robust procedures to be performed. The columns are designed to provide multiple interactions, resulting in cleaner extracts. Furthermore, the columns have the ability to fractionate complex mixtures. For instance, acids and neutral compounds can be eluted with a neutral pH solvent, while basic compounds can be eluted at a high pH or with an ionic strength solvent. Three examples of mixed-mode sorbents are shown in Table 6, including the Strata-X-C sorbent from Phenomenex, the MCX sorbent from Oasis and the Bond Elut Plexa PCX sorbent.

Table 6: Overview of three different mixed-mode columns with their stationary phases. Reproduced from ref. [108] (Strata-X-C, technical note TN-0026), [109] (Oasis MCX, webpage from Waters), [110] (Bond Elut Plexa PCX structure)

Strata-X-C Oasis MCX Bond Elut Plexa

Mixed-mode SPE was applied nine times and the specifics of the SPE procedures are shown in Table 7. The results and specifics will be further discussed in chapter four.

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Page 31 of 103 Table 7: Overview of articles that applied mixed-mode SPE for extraction and/or clean-up of plant toxins. The matrices and plant toxins are described, as well as specifics of the mixed-mode SPE procedure such as the columns and elution solvents. Finally, the recovery, separation and detection are described. Abbreviations include methanol (MeOH), acetic acid (AA) and formic acid (FA).

Ref. Matrix Compound Column Pre-condition Wash Elution Recovery (%) Separation & detection

Griffin et al., 2013 [111]

Honey PAs Strata-X-C MeOH and H2O (0.05% FA)

After loading, dry. Wash: H2O (0.05% FA)

and MeOH MeOH (0.1% ammoniated) 87 LC-MS/MS Griffin et al., 2015 [112]

Honey PAs Strata-X-C MeOH and H2O H2O (0.1% FA) and MeOH MeOH (3% ammonia) 75-97 LC-MS/MS Marín-Sáez et al., 2017 [68] Food Tropane alkaloids

Strata-X-C MeOH and MeOH:H2O (1% AA) (2:1, v/v)

MeOH:H2O (1% AA) (2:1, v/v)

MeOH (2.5% NH4OH) 60-109 LC-MS (Orbitrap)

Mulder et al., 2016 [73]

Food and tea Tropane alkaloids OASIS MCX or StrataX MeOH Equilibrate: MeOH/H2O/FA (75/25/1, v/v/v) MeOH/H2O/FA (75/25/1) and then dried

MeOH (0.5% ammonia) 61-105 LC-MS/MS Mulder et al., 2018 [74] Food supplements (oily plant-derived)

PAs PCX (Bond Elut Plexa)

MeOH and MeOH (0.05 M H2SO4)

MeOH and dry Two steps with MeOH (2.5% ammonia)

Supp. Data not found LC-M/MS Romera-Torres et al., 2018 [78] Tea Tropane alkaloids

Strata-X-C column MeOH Equilibration: MeOH/H2O/FA MeOH/H2O/FA Dry MeOH with 3% ammonia solution (25%) 75-128 LC-MS Orbitrap Romera-Torres et al., 2018 [79] Feed Tropane alkaloids

Strata-X-C column MeOH Equilibration: MeOH/H2O/FA MeOH/H2O/FA Dry MeOH with 3% ammonia solution (25%) 70-109 LC-HRMS Wang et al., 2019 [113]

Honey PAs Biotage Evolute Express CX column

Not applied. 0.05 M H2SO4 and MeOH MeOH (2.5% NH4OH) 79.2-104.4 HPLC-ESI-QTOF-MS

Yoon et al., 2015 [89]

Food PAs Strata-X-C column MeOH and H2O (0.1% FA) H2O (0.05% FA) and MeOH

Dry

MeOH (5% NH4OH) 89-109 LC-ESI-MS/MS & LC-APCI-MS

The analysis of plant toxins in the feed and food industry

MSc Chemistry

Analytical Sciences

Literature Thesis

by

Naomi Dam

11811374

August 2020, 12 EC

Supervisor/Examiner:

Examiner:

Dr. Jeroen Kool

Dr. Rob Haselberg

Abstract

Abbreviations

Table of contents

1 Introduction

2 Theoretical background

2.1 Phenolics

2.1.1 Flavonoids and phenolic acids

2.1.2 Coumarins

2.2 Terpenoids and terpenes

2.3 Saponins

2.4 Alkaloids

2.4.1 Pyrrolizidine alkaloids

2.4.2 Tropane alkaloids

2.5 Cyanogenic glycosides

3 Methods for analysis of plant toxins

3.1 Sample pre-treatment

3.1.1 Introduction

3.1.2 Solid-liquid extraction

3.1.3 Liquid-liquid extraction

3.1.4 QuEChERS

3.1.5 Microwave-assisted extraction

3.1.6 Solid-phase extraction