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Stingless bee honeys (SBHs) from Australian and Malaysian species were analysed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the presence of pyrrolizidine alkaloids (PAs) and the corresponding N-oxides (PANOs) due to the potential for such hepatotoxic alkaloids to contaminate honey as a result of bees foraging on plants containing these alkaloids. Low levels of alkaloids were found in these SBHs when assessed against certified PA standards in targeted analysis. However, certain isomers were identified using untargeted analysis in a subset of honeys of Heterotrigona itama which resulted in the identification of a PA weed species (Ageratum conyzoides) near the hives. The evaluation of this weed provided a PA profile matching that of the SBH of H. itama produced nearby, and included supinine, supinine N-oxide (or isomers) and acetylated derivatives. These PAs lacking a hydroxyl group at C7 are thought to be less hepatoxic. However, high levels were also observed in SBH (and in A. conyzoides) of a potentially more toxic diester PA corresponding to an echimidine isomer. Intermedine, the C7 hydroxy equivalent of supinine, was also observed. Species differences in nectar collection were evident as the same alkaloids were not identified in SBH of G. thoracica from the same location. This study highlights that not all PAs and PANOs are identified using available standards in targeted analyses and confirms the need for producers of all types of honey to be aware of nearby potential PA sources, particularly weeds. [ABSTRACT FROM AUTHOR]

Within the last decades, in the EU, there has been an increasing interest in toxic plant alkaloids as food contaminants, especially after the continuous and growing consumption of plant‐based foods compared with food of animal origin. In this regard, the once neglected presence of these tropane alkaloids (TAs) and pyrrolizidine alkaloids (PAs) has recently been reconsidered by the European Food Safety Authority, highlighting the lack of data and the need to develop risk assessment strategies. For this reason, the emphasis has been placed on detecting their occurrence in food through the development of accurate and sensitive analytical methods to achieve the determination of these compounds. The present study aims to elaborate and validate an analytical method based on QuEChERS sample preparation approach, exploiting the UHPLC coupled to the HRMS to simultaneously identify and quantify 21 PAs and two TAs in cereals and spices. For TAs, the obtained limit of detection (LOD) is 0.1 μg·kg−1 and the limit of quantification (LOQ) is 0.4 μg·kg−1, while for PAs, the LODs values ranging between 0.2 to 0.3 μg·kg−1 and the LOQ, between 0.4 and 0.8 μg·kg−1, ensuring compliance with the recently established European Regulations. Several commercial samples were analysed to further verify the applicability of this comprehensive analytical approach. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are noted for their hepatotoxic, genotoxic, and carcinogenic effects in animals and humans following metabolic activation in the liver. In this study, herbal supplements sold in Ghana for sexual improvement were analysed for the presence of 64 PAs using LC-MS/MS analysis. Up to 17 different PAs were identified in 19 out of the 37 samples analysed. The sum of PAs in samples ranged from 5 to 3204 μg kg−1. Since the PA content in the herbal medicinal preparations was generally lower than in honey samples, their presence was mainly attributed to cross-contamination. The observed levels would result in estimated daily intakes from 0.01 to 12 μg per day or 0.0002 to 0.2 μg kg−1 bw day−1 for a person weighing 70 kg. The margins of exposure ranged from 1200 to 1,400,000 with eight samples showing values below 10,000, thus indicating a health concern. [ABSTRACT FROM AUTHOR]

Comfrey (Symphytum officinale L.) has a long tradition of use in the treatment of musculoskeletal disorders. However, due to hepatotoxic pyrrolizidine alkaloids (PAs), the EMA restricts the use of comfrey root (CR) to external use only and for short periods of time. Recent studies indicate a low permeability of PAs across the skin, calling into question the safety of topical application of products containing comfrey preparations. The aim of our work was to develop and validate an HPLC method enabling the separation of isomeric PAs from comfrey and, on this basis, to assess the potential toxicity of CR and comfrey leaf (CL) obtained from various Polish sources. The qualitative and quantitative analysis of PAs via HPLC-MS/MS was performed in MRM mode. The results obtained confirmed a lower content of PAs in CL than in CR and showed a wide variation in the composition of PAs in CR, with a much more stable profile of PAs in CL. Factor analysis confirmed that CRs and CLs differ in PA content, which is influenced by the growth conditions and geographical origin. The determined concentrations of PAs prove that in some CRs available on the Polish herbal market, the content of PAs may exceed the daily dose considered safe. [ABSTRACT FROM AUTHOR]

• Presence of pyrrolizidine alkaloids (PAs) are of global concern in herbal teas. • First study to show direct transfer of PAs from weed residues via soil to rooibos plants. • Rooibos plants mainly accumulated the PA senecivirnine-N-oxide in field and pot trial. • Weeds with highest PAs in Niewoudtville rooibos fields were Spergula arvensis and Senecio species. • Rooibos plants in fields contained on average 20 ug kg−1 total PAs. Pyrrolizidine alkaloids (PAs) contamination of rooibos tea products has prompted global concern amongst distributors. Lateral transfer of PAs from weeds as a result of co-harvesting has been previously demonstrated. However, it is not currently clear whether there can be a direct transfer of PAs from decomposing weed material to rooibos tea via the soil. Furthermore, a large number of weed species occur in rooibos tea plantations in the Nieuwoudtville region, Northern Cape Province, South Africa and it is not fully known which species produce PAs. Therefore, the objectives of this study were, firstly, to quantify and identify PAs in rooibos tea and dominant weed species occurring in rooibos plantations in the Nieuwoudtville region using ultra performance liquid chromatography mass spectrometry, and secondly, to determine whether PAs from weed materials can be transferred to rooibos plants via the soil in a pot trial. amongst the 19 weed species identified in the plantations in spring, Spergula arvensis, Senecio abruptus, Senecio arenarius and Senecio burchellii were found to contain substantial contents of PAs (> 1000 µg kg−1). Rooibos tea at the sites was found to contain average total PA concentrations of 20 µg kg−1, dominated by senecivirnine-N-oxide (76%). In the pot trial, only rooibos plants where S. burchellii material was applied contained measurable PA levels in the form of senecivirnine-N-oxide (4.25 μg kg−1, SE 2.53 μg kg−1). However, the dominant PAs occurring in S. burchellii material applied in the pot trial were senecivirnine (30.1%), senecionine-N-oxide (25.4%), and retrorsine-N-oxide (23.5%). This indicates that senecivirnine-N-oxide was the most absorbable PA taken up the rooibos plants or was the one of the primary metabolic products from adsorbed PAs in rooibos. These findings also help to explain why rooibos plants at the field sites contained predominantly senecivirnine-N-oxide. This is the first study to demonstrate the direct transfer of PAs from PA-containing weed residues to rooibos tea by the soil. [ABSTRACT FROM AUTHOR]

Background: Farfarae Flos (FF) is a frequently used traditional herbal medicine with outstanding antitussive actions. The adulteration of FF decoction pieces is common. Objective: This study aimed to study the effect of adulteration on the safety and quality of FF decoction pieces. Methods: The proportion of impurities was conducted by cone quartering method. A simple and accurate ultra-highperformance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was established to simultaneous determinate three pyrrolizidine alkaloids (PAs) as endogenous toxic compounds in FF. The traditional medicinal parts (flower bud), impurities (pedicel and rhizome) and unselected samples were determined respectively. The values of estimated daily intake (EDI) and margin of exposure (MOE) were used for risk assessment. Results: Twenty batches of samples were collected from different habitats, and the proportion of impurities ranged from 17.51% to 41.27%. Pedicel and rhizome were the main impurities, accounting for more than 87.40% of the total impurities. The content of PAs in impurities was significantly higher. The EDI value range was 5.34 to 16.59 lg/kg bw/day, which was much higher than the standard safety value of 7.00X10-3μg/kg bw/day. The MOE values ranges for life long time and shorter exposure were 14.29 to 44.37 and 371.53 to 1153.63, respectively, indicating that at least 80% of the samples had safety risks. Correlation analysis showed that the proportion of adulterated impurities had significant correlation with the values of EDI and MOE. Conclusions: Adulteration of non medicinal parts may significantly increase the risk of medications of FF decoction pieces. Highlights: This study provides an efficient methodology reference for the control of PAs and a basis for adulteration to affect the safety and quality of FF decoction pieces. [ABSTRACT FROM AUTHOR]

Background: Studies on pyrrolizidine alkaloid (PA) contamination in honey produced in China are scarce. Previously reported HPLC-MS/MSmethods for the determination of PAs in honey often suffer from insufficient separation and uncertainties in PA isomers. Objective: To develop and validate an Ultra-HPLC (UHPLC)-MS/MS method for baseline separation of PA isomers towards precise determination of 32 PAs in Chinese wild honey. Methods: PAs were extracted from honey samples and separated on an ACQUITY BEH C18 (2.1mm 100mm, 1.7 lm) column with (A) 0.1% formic acid aqueous solution containing 5mM ammonium acetate and (B) methanol as mobile phase. The column temperature was maintained at 30 °C, and flow rate was 0.3 mL/min. Detection was performed by tandem mass spectrometry. The total run time was reduced to 18 min. Results: Thirty-one of 32 PAs were baseline separated efficiently within 18 min. The LOD and LOQ were 0.06-0.25 lg/kg and 0.22-0.82 lg/kg, respectively, except for that of clivorine, for which LOD and LOQ were 2.03 and 6.78 lg/kg, respectively. The average recoveries ranged between 66.3 and 95.1% and the average RSDs were 3.2 to 8%. The established method was used to analyze PAs in 22 types of Chinese wild honey, and the predominant PAs found in these honey samples were intermedine and lycopsamine. Conclusion: A high-throughput method for the determination of isomeric PAs in honey was developed and validated. Five of the 22 types of Chinese wild honey were contaminated with PAs concentrations of 2.2-207.0 lg/kg. Highlights: A new method capable of monitoring more PAs and providing better separation than previously reported protocols for the determination of multiclass PAs in honey is established. [ABSTRACT FROM AUTHOR]

Summary: Two analytical methods for the quantification of pyrrolizidine (PAs) and tropane alkaloids (TAs) in honey bee‐collected pollen, teas and herbal infusions have been in‐house validated using the UHPLC‐MS/MS technique. These methods showed good linearity, good accuracy and robustness. The methods have been applied to a preliminary investigation of forty‐seven pollen samples and thirty‐three packs of tea‐bags in light of Commission Regulations (EU) 2020/2040 and 2021/1408 regarding maximum limits of PAs and TAs in foodstuffs, respectively. About half of the pollen samples contained searched PAs but in concentrations significantly lower than the maximum limit established; two samples were contaminated with TAs. One third of teas or herbal infusions contained PAs or TAs, one sample exceeded the EU limits for PAs and one for TAs. Despite the final consumer's health does not seem threatened, these results recommend accurate analytical methods and broader investigations for PAs and TAs in foods. [ABSTRACT FROM AUTHOR]

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid–liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected. [ABSTRACT FROM AUTHOR]

1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid–liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) constitute a class of phytotoxin which demonstrates strong hepatotoxicity. In China, many plants containing PAs are used as traditional medicines or medicinal preparations, which could harm human health and safety. Xiaoyao Tablet (XYT) is an antidepressant drug registered in the European Union (EU), Compound Danshen Dropping Pills (CDDP) is a commonly used drug for coronary heart disease, and phase III clinical study is ongoing in the United States. The purpose of this study is to provide data to support the use of Chinese medicine preparations internationally and to establish analytical methods for 32 PAs in XYT and CDDP. The extraction parameters that were optimized include solid-phase extraction (SPE) cartridge, extraction method, and extraction solvent. Then ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-traptandem mass spectrometry (UPLC-MS/MS) was developed to effectively and efficiently quantify the 32 PAs of the XYT and CDDP. The analytical methods for XYT and CDDP were verified respectively. For XYT, the analytical method for 32 PAs was linear, and the correlation coefficient r was greater than 0.994; the recovery (REC%) at 10–2000 μg/kg was 73.3%–118.5%, and the relative standard deviation (RSD%) was 2.1%–15.4%. The CDDP REC% was 71.8%–112.0%, and the RSD% was 2.0%–17.1%. This study provides technical and data support for the registration of Chinese patented medicines in the EU, controls quality and ensures safety, and is committed to the internationalization and standardization of Chinese patented medicines. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are produced by various plant species and have been detected as contaminants in food and feed. Monitoring programmes should include PAs that are present in relevant matrices and that exhibit a high toxic potential. The aim of the present study was to use a bioassay-directed analysis approach to identify relevant PAs not yet included in monitoring programmes. To that end, extracts of Heliotropium europaeum and H. popovii were prepared and analysed with LC–MS/MS for the presence of 35 PAs included in monitoring programmes, as well as for genotoxic activity in the HepaRG/γH2AX assay. Europine, heliotrine and lasiocarpine were found to be the most abundant PAs. The extracts showed a higher γH2AX activity than related artificial mixtures of quantified known PAs, which might point to the presence of unknown toxic PAs. The H. europaeum extract was fractionated and γH2AX activities of individual fractions were determined. Fractions were further analysed applying LC–Orbitrap-MS analysis and Compound Discoverer software, identifying various candidate PAs responsible for the non-explained genotoxic activity. Altogether, the results obtained show that bioassay-directed analysis allows identification of candidate PAs that can be included in monitoring programmes. [ABSTRACT FROM AUTHOR]

Plants produce many secondary metabolites showing considerable inter‐ and intraspecific diversity of concentration and composition as a strategy to cope with environmental stresses. The evolution of plant defenses against herbivores and pathogens can be unraveled by understanding the mechanisms underlying chemical diversity. Pyrrolizidine alkaloids are a class of secondary metabolites with high diversity. We performed a qualitative and quantitative analysis of 80 pyrrolizidine alkaloids with liquid chromatography‐tandem mass spectrometry of leaves from 17 Jacobaea species including one to three populations per species with 4–10 individuals per population grown under controlled conditions in a climate chamber. We observed large inter‐ and intraspecific variation in pyrrolizidine alkaloid concentration and composition, which were both species‐specific. Furthermore, we sequenced 11 plastid and three nuclear regions to reconstruct the phylogeny of the 17 Jacobaea species. Ancestral state reconstruction at the species level showed mainly random distributions of individual pyrrolizidine alkaloids. We found little evidence for phylogenetic signals, as nine out of 80 pyrrolizidine alkaloids showed a significant phylogenetic signal for Pagel's λ statistics only, whereas no significance was detected for Blomberg's K measure. We speculate that this high pyrrolizidine alkaloid diversity is the result of the upregulation and downregulation of specific pyrrolizidine alkaloids depending on ecological needs rather than gains and losses of particular pyrrolizidine alkaloid biosynthesis genes during evolution. [ABSTRACT FROM AUTHOR]

[Display omitted] • Analysis of pyrrolizidine alkaloids (PAs) in ginseng samples was developed. • The solvent extraction and purification process were systemically optimized. • Single adsorbent nano-TiO 2 was applied instead of conventional adsorbent combination. • This method gave rise to satisfactory purification effect and recoveries for all PAs. • PAs were detected in the ginseng samples at concentration of 13.3 to 174 μg/kg. Pyrrolizidine alkaloids (PAs) are a group of naturally occurring toxins, which have been widely found in herbs. However, the complexity of the herbs made the accurate and simple determination of PAs challenging. Herein, the present work described the development of a simple and effective QuEChERS method for simultaneous determination of 18 PAs in various ginseng samples. The extraction and purification processes were systematically optimized and validated coupled to LC-MS/MS analysis. After intensive screening, a single adsorbent nano-TiO 2 was employed as a cleanup adsorbent during the dispersive solid phase extraction process to remove the matrix interferences and retain the target PAs in the sample solution, achieving the simultaneous determination of 18 PAs. The validation results displayed that the PAs had good linearity, sensitivity and satisfactory accuracy by the developed method. The method LODs and LOQs of PAs ranged from 0.05-1.49 μg/kg and 0.17–4.95 µg/kg, respectively. The average recoveries were 70.1 %-111 % with RSD less than 13 % under different spiked concentrations. Compared to the conventional QuEChERS method, this method gave rise to better purification effect and recoveries under the condition of only using one adsorbent nano-TiO 2 , which obvious improved its simplicity and versatility. Furthermore, this method was used to analyze the real ginseng samples and PAs were detected with concentrations ranging from 1.02 to 139 μg/kg, which further verified the feasibility of the developed method in the PAs analysis from the traditional herbal medicines. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, and their 1,2-unsaturated derivatives, which contain the retronecine, heliotridine, or otonecine type of the necine base, have raised concern due to their ability to form hepatotoxic intermediates and exhibit serious toxic effects. Several hundred individual pyrrolizidine alkaloids and their N-oxides have been identified mostly using liquid chromatography coupled with mass spectrometry, although the number of available reference standards is limited. In this work, characteristic fragment ions and their abundance in the mass spectra of different PAs were used to reveal typical fragmentation patterns for various classes of PAs that can be further employed to distinguish monoesters (retronecine, heliotridine type), open chain diesters and macrocyclic diesters, and corresponding N-oxides. Fragment ions at m/z 120 and 138 were found in all types of PAs with a different relative abundance. Additional observation of fragment ions at m/z 94 and 156 was found to be typical for monoester PAs esterified at position C9 of the necin base, whereas fragment ions at m/z 111 and 172 were characteristic for monoester N-oxides. Fragment ions at m/z 180 and 220 were found to be typical for open chain diesters with esterification at C7 with acetic and angelic acid, respectively, whereas fragment ions at m/z 214 and 254 were characteristic for the respective N-oxides. For the 3'-acetyl PA monoester or open chain diester derivatives, characteristic fragment ions were observed after loss of the acetyl moiety ([M+H]+-60), whereas for macrocyclic diesters and their N-oxides, fragment ions due to the neutral loss of CO were found ([M+H]+-28). [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are considered as the major constituents that cause hepatoxicity in Senecio plants. PAs can be found in about 3%–5% of the world's flowering plants. Nowadays, the identification method of PAs by separation and preparation was too slow and lacked effective power. A rapid method to identify PAs in plants must be developed. Based on the fragmentation regularity, the hepatoxic PAs and nonhepatoxic PAs were characterized by liquid chromatography-mass spectrometry (LC-MS). The detailed structures of PAs in five Senecio plants were identified based on tandem mass spectrometry (MS/MS) spectrum and chemical research information. In the present study, some new fragmentation regularities of PAs have been found, such as product ions at m/z 122, m/z 140 and m/z 124, m/z 142, which have been discovered as the characteristic fragments of lactone and mono-esterase type of saturated PAs, respectively. Moreover, two product ions at m/z 120 and m/z 138 have been reported as the characteristic fragments of unsaturated PAs. Some of them were found in Senecio species for the first time, and some of them may be new nature product or even new compound. Finally, we classified these plants into five categories based on PAs which were identified in the present study; the result corresponded with the classification by morphology. In addition, we have found some constituents that have odd molecular weight number only in Senecio species but not in Ligularia species; the detailed structures of these non-PAs constituents need penetrating study. LC-MS was rapid and sensitive method for detecting and identifying PAs in plants. Pyrrolizidine alkaloids were the toxiferous constituent of Senecio plants. In this study, we found that PAs can be used as the characteristic constituent of Senecio species. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids are toxins having hepatotoxic and carcinogenic effects on human health. A ultra high performance liquid chromatography tandem mass spectrometry technique was developed for the first time for the simultaneous determination of eight pyrrolizidine alkaloids, including four diastereoisomers (intermedine, lycopsamine, rinderine, and echinatine) and their respective N‐oxide forms, in different parts of Eupatorium lindleyanum. The risk assessment method for pyrrolizidine alkaloids in Eupatorium lindleyanum was explored using the margin of exposure strategy for the first time based on a real‐life exposure scenario. Differences were found in all eight pyrrolizidine alkaloids in various parts of Eupatorium lindleyanum. Besides, the total levels of pyrrolizidine alkaloids in Eupatorium lindleyanum followed the order of root > flower > stem > leaf. Moreover, the risk assessment data revealed that the deleterious effects on human health were unlikely at exposure times of less than 200, 37, and 12 days during the lifetimes of Eupatorium lindleyanum leaves, stems, and flowers, respectively. This study reported both the contents of and risk associated with Eupatorium lindleyanum pyrrolizidine alkaloids. The comprehensive application of the novel ultra high performance liquid chromatography tandem mass spectrometry technique alongside the risk assessment approach provided a scientific basis for quality evaluation and rational utilization of toxic pyrrolizidine alkaloids in Eupatorium lindleyanum to improve public health safety. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids and their derived oxides are toxins naturally produced by plants when they are exposed to stress factors. In particular, the unsaturated pyrrolizidine alkaloids exhibiting a double bond between the 1,2-positions have caused great interest in recent years since they have pneumotoxic, genotoxic and carcinogenic effects upon ingestion. In this review, focus on their chemical structure justifying their toxicity is provided as well as insight on the recently adopted EU regulatory framework. In addition, to reveal current trends and highlight the research effort in the field, an all-time bibliometric analysis was performed for the first time. It was found that tea, infusions, honey, spices, and cereals are the most common foodstuffs in which pyrrolizidine alkaloids have been detected. In terms of pre-analytical steps, the application of efficient sample preparation is necessary to detect pyrrolizidine alkaloids in complex food matrices. Solvent extraction followed by a clean-up stage, e.g., solid-phase extraction was the most applied option. Focusing on the analytical methods, liquid chromatography combined with various mass spectrometry detectors has been the golden standard in the field. Gas chromatography methods were also applied, but the need for analyte derivatisation has hindered their application. All in all, this review provides an overview on the analysis of pyrrolizidine alkaloids and their derived oxides, comprehensively discussing the up-to-date advances and highlighting the analytical challenges to be faced. [Display omitted] • Tea, infusions, honey and cereals are contaminated with pyrrolizidine alkaloids. • Efficient sample preparation methods are needed in complex food matrices. • Solvent extraction followed by a clean-up stage is the most applied option. • Liquid chromatography-mass spectrometry is the most useful analytical technique. • Gas chromatography required derivatization that hindered its application. [ABSTRACT FROM AUTHOR]

[Display omitted] • LC-MS/MS method proposed for food safety control in cereal-based food. • Simple and fast sample preparation. • Method succesfully validated in acordance with EU regulations. Alkaloids are naturally occurring compounds containing basic nitrogen atoms. They are biosynthesized mainly by plants but also by some fungi species. Many alkaloids are toxic to humans and animals, and they have been classified as food contaminants. Among them, ergot, tropane, and pyrrolizidine alkaloids have maximum levels in foods, established by the Commission Regulation (EU) 2023/915. In this study, an analytical method was successfully developed and validated for the simultaneous determination of 42 ergot, tropane, and pyrrolizidine alkaloids and their N-oxides in cereal-based food. The method includes QuEChERS-based extraction followed by liquid chromatography coupled with tandem mass spectrometry. The proposed method was validated providing recoveries ranging from 71 to 119 %, intra- and inter-day precision lower than 19 %, and limits of quantification between 0.5 and 1.0 µg kg−1. Finally, the analysis of reference materials coming from FAPAS proficiency tests demonstrated the suitability for purpose of the methodology (z-scores < 2). Nine cereal-based products samples were analyzed of which ergot alkaloids were detected in two of them, while one sample showed the presence of three pyrrolizidine alkaloids. [ABSTRACT FROM AUTHOR]

Bee pollen may be contaminated with pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs), which are mainly detected by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), even though the use of fast near-infrared (NIR) spectroscopy is an ongoing alternative. Therefore, the main challenge of this study was to assess the feasibility of both a lab-stationary (Foss) and a portable (Polispec) NIR spectrometer in 60 dehydrated bee pollen samples. After an ANOVA-feature selection of the most informative NIR spectral data, canonical discriminant analysis (CDA) was performed to distinguish three quantitative PA/PANO classes (µg/kg): < LOQ (0.4), low; 0.4–400, moderate; > 400, high. According to the LC–MS/MS analysis, 77% of the samples were contaminated with PAs/PANOs and the sum content of the 17 target analytes was higher than 400 µg/kg in 28% of the samples. CDA was carried out on a pool of 18 (Foss) and 22 (Polispec) selected spectral variables and allowed accurate classification of samples from the low class as confirmed by the high values of Matthews correlation coefficient (≥ 0.91) for both NIR spectrometers. Leave-one-out cross-validation highlighted precise recognition of samples characterised by a high PA/PANO content with a low misclassification rate (0.02) as false negatives. The most informative wavelengths were within the < 1000, 1000–1660 and > 2400 nm regions for Foss and > 1500 nm for Polispec that could be associated with cyclic amines, and epoxide chemical structures of PAs/PANOs. In sum, both lab-stationary and portable NIR systems are reliable and fast techniques for detecting PA/PANO contamination in bee pollen. [ABSTRACT FROM AUTHOR]

Senecio scandens as a commonly used traditional Chinese medicine that is used alone or in combination with other herbs in preparations such as QianBai BiYan tablets has attracted much attention because of its hepatotoxic pyrrolizidine alkaloids. Nowadays, most studies for pyrrolizidine alkaloids are only performed on herbs or a preparation, however, production of preparations is a dynamic process, control of toxic impurities for raw materials, or finished products cannot monitor the production process dynamically. Thus, in this study, qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from S. scandens to its preparations was carried out with HPLC-MS/MS for the first time, which was more comprehensive and dynamic than the previous single-layer analysis. First, the species of pyrrolizidine alkaloids in S. scandens were analyzed, and the characteristic fragmentation rules of pyrrolizidine alkaloids containing common parent nucleus were found, which can be used to identify these components rapidly in the future. Then, a quantitative method for S. scandens to QianBai BiYan tablets and other nine S. scandens-containing preparations was established, and after the medication safety speculation, all of them met the relevant safety requirements. After that, in order to ensure the stability and controllable of drug quality, the limit of pyrrolizidine alkaloids in preparations was determined according to the safe dosage that is stipulated to be the same as raw materials. Finally, the factors causing the content change of pyrrolizidine alkaloids in S. scandens from different source were studies, which can provide theoretical basis for selecting suitable raw materials for production. [ABSTRACT FROM AUTHOR]

Background & Aims: Patients with non‐alcoholic fatty liver disease (NAFLD) exhibit higher levels of plasma 1‐deoxysphingolipids than healthy individuals. The aim of this study was to investigate the role of farnesoid X receptor (FXR) in 1‐deoxysphingolipid de novo synthesis and degradation. Methods: Mice were fed with a high‐fat diet (HFD) to induce obesity and NAFLD, and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Sphingolipid patterns from NAFLD patients and mouse models were assessed by liquid chromatography‐mass spectrometry. The molecular mechanism underlying the effect of FXR activation on sphingolipid metabolism was studied in Huh7 cells and primary cultured hepatocytes, as well as in a 1‐deoxysphinganine‐treated mouse model. Results: 1‐deoxysphingolipids were increased in both NAFLD patients and mouse models. FXR activation by OCA protected the liver against oxidative stress, apoptosis, and reduced 1‐deoxysphingolipid levels, both in a HFD‐induced mouse model of obesity and in 1‐deoxysphinganine‐treated mice. In vitro, FXR activation lowered intracellular 1‐deoxysphingolipid levels by inducing Cyp4f‐mediated degradation, but not by inhibiting de novo synthesis, thereby protecting hepatocytes against doxSA‐induced cytotoxicity, mitochondrial damage, and apoptosis. Overexpression of Cyp4f13 in cells was sufficient to ameliorate doxSA‐induced cytotoxicity. Treatment with the Cyp4f pan‐inhibitor HET0016 or FXR knock‐down fully abolished the protective effect of OCA, indicating that OCA‐mediated 1‐deoxysphingolipid degradation is FXR and Cyp4f dependent. Conclusions: Our study identifies FXR‐Cyp4f as a novel regulatory pathway for 1‐deoxysphingolipid metabolism. FXR activation represents a promising therapeutic strategy for patients with metabolic syndrome and NAFLD. [ABSTRACT FROM AUTHOR]

Gynura procumbens is commonly consumed as a vegetable and has been approved as an ingredient for food and dietary supplements in China. However, Gynura species are known to contain toxic pyrrolizidine alkaloids (PAs) and the PA profiles in G. procumbens are not known. This study was to extract and enrich PA from G. procumbens or health care products using PCX solid-phase extraction (SPE) cartridges identify the main PAs in the herb and to develop an liquid chromatography–mass spectrometry (LC-MS/MS) method for assaying the PA contents in the plant and its derived products. Upon using multiple reaction monitoring (MRM) acquisition together with comparison to the characteristics of mass fragmentations and retention times of reference standards, 11 PAs were identified as the main PAs in the plant. After clean-up and enrichment with PCX solid-phase extraction (SPE) cartridges, which resulted in better PA recoveries than either C18 or SCX cartridges, the LC-MS/MS method was subjected to validation in terms of linearity, repeatability, limits of quantification (LOQ) and recovery. The validated method was applied to quantify PAs in 12 plant samples and 7 commercial finished products. The total amounts of targeted PAs were found to vary from 15.6 to 848 μg/kg in the herbs and from 9.9 μg/kg to 33.9 mg/kg in the commercial products. The present work was the first to demonstrate that G. procumbens contained PAs in the herb and its derived products and the PA contents might exceed the daily dose limits in food and herbal medicinal products proposed by the European Medicines Agency (i.e. 0.35 μg PA per day for 50 kg adult). [ABSTRACT FROM AUTHOR]

• pyrrolizidine alkaloids using differential mobility spectrometry (vDMS-MS) at low pressure. • LC-DMS-SIM/MS (vDMS) with a short LC column in trap/elute mode. • LC-vDMS-SIM/MS versus LC-MRM/MS and SFC-MRM/MS assays. • Combination of supercritical fluid chromatography with vDMS-MS. The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry – Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15–100% MeOH and ACN), flow rate (8–100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO 2 /MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS. [ABSTRACT FROM AUTHOR]

Foods derived from lupin are attracting consumers and producers for their nutritional value and low cost of production. Lupin products may pose a health risk to consumers due to the presence of toxic alkaloid compounds. To monitor alkaloids in lupin seeds, we developed and validated a rapid LC-MS/MS method for the determination of five quinolizidine alkaloids and one indole alkaloid in lupin seeds. The final method involves extraction by water/acetonitrile, ultrasonication, separation using NaCl and MgSO 4 , alkalinisation by NaOH and analysis using HPLC-MS/MS. The separation was achieved in 6 min using HPLC directly coupled to MS/MS on a 6500 + QTRAP in ESI-positive mode. The method showed acceptable recovery for all tested compounds in soybean grain at a wide range from 0.55 (LOQ) to 55 mg/kg, with a mean recovery of 93% for the total alkaloid content (SD <2.5%). The method was tested on a commercial lupin flour sample yielding an alkaloid concentration of 130 mg/kg (SD 3.6%). Alkaloid profiles were assessed across different cultivars of five Lupinus species (Lupinus angustifolius, L. cosentinii, L. albus, L. luteus, and L. mutabilis). Using the method presented, toxic alkaloid levels can be monitored in lupin-based products to ensure their safety for human consumption. The method was deployed to measure the alkaloid content across different seeds of narrow-leaf lupin from the same farm, revealing that the alkaloid content varied widely from seed to seed from the same farm. • Development of a rapid and sensitive LC-MS/MS method for alkaloid analysis in Lupinus species and lupin-based products. • Validation of method, demonstrating suitability for routine alkaloid analysis. • The variability of alkaloids between Lupinus species is demonstrated. • Different seeds from the same farm and cultivar have different levels of alkaloids. [ABSTRACT FROM AUTHOR]

Two sample preparation methods were developed (graphitised carbon and C18 solid phase extraction clean-up) and validated in house using liquid chromatography and tandem mass spectrometry (MS/MS) for the determination of 30 pyrrolizidine alkaloids (PAs) in salads, herbs, tea, herbals teas as well as tea infusion and ice-tea beverages. Total PAs concentration of samples purchased on the Belgian market varied greatly with matrix type ranging from

Pyrrolizidine alkaloids (PA) from borage (Borago officinalis) consumed as herb and tea, may pose a food safety risk. Therefore, the European Union (EU) set maximum levels of PA in borage, among other foodstuffs, which are applicable since July 1st, 2022. Here, a comprehensive LC-MS/MS based profiling of PA and their N -oxides (PANO) in B. officinalis leaves is presented. Based on these results a targeted, quantitative LC-MS/MS method for the determination of individual PA/PANO present in borage was developed. Chromatographic separation was achieved for all PA/PANO detected in B. officinalis. An easy and fast extraction procedure was developed using a design of experiments approach (DOE). The most efficient extraction was achieved using 0.2% formic acid in 10% methanol at a temperature of 47.5 °C for 60 min. The final method was validated and showed good overall accuracy (recoveries 85–121%) and precision (RDS ≤11%). The method was applied to B. officinalis leave material, demonstrating its suitability for the intended purpose. In these borage samples, the acetylated forms, which are not regulated by EU, were among the quantitatively most relevant PA. [Display omitted] • Detailed profiling of pyrrolizidine alkaloids in borage by UHPLC-QToF analysis. • Validated method for quantitation of 18 pyrrolizidine alkaloids found in borage. • Extraction of pyrrolizidine alkaloids optimized by design of experiment approach. • Presence of acid increased pyrrolizidine alkaloid extraction from borage. • High percentage of pyrrolizidine alkaloids in borage not covered by EU regulations. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are natural toxins present in over 6000 plants. Humans are exposed to PAs through the intake of PA-containing plants and/or PA-contaminated foodstuffs, such as honey. PA contamination in honey has been extensively studied in many countries, but rarely investigated in China. The present study investigated PA contamination in retail honey purchased in four representative regions (Hong Kong, Macau, Beijing, Shanghai) in China. Among 120 honey samples tested, 58% of them contained PAs with the concentration range over 0.04–288.07 μg/kg. The prevalence and content of PAs in honeys purchased in Hong Kong and Macau were significantly higher than those detected in Beijing and Shanghai. Lycopsamine and echimidine were determined as predominant PAs in the contaminated honeys, and their potential botanic sources including plants of Echium and Senecio genera were identified by pollen analysis. Moreover, clivorine, which has not been reported previously in PA-contaminated honey, was found with a high prevalence (34%) in PA-contaminated honeys in China. Our study represented an integrated report on PA contamination in retail honey in China. The data provided information useful for the global assessment of human risk posed by consumption of PA-contaminated honey. [ABSTRACT FROM AUTHOR]

There is a vast amount of information about the nutritional and medicinal properties of honey as a result of its numerous benefits. However, honeys have been found to be contaminated with hepatotoxic and carcinogenic pyrrolizidine alkaloids (PAs) on account of bees foraging on PA-containing plants. This study deals with the analysis of PAs in tropical honeys emanating from different agro-ecological zones of Ghana in order to assess its potential health risk. PAs of 48 honey samples were analysed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results show that a total of 85% of the honeys from various agro-ecological zones were PA positive including all honeys from supermarkets. The highest concentration of PAs was 2639 μg kg−1, while the average PA concentration of the samples was 283 μg kg−1. The study also foundChromolaena odoratapollens in majority of the honeys, thus indicating the plant as major source of PA contamination of honeys in the tropical regions. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Several PA quantitative methodologies were developed based on a limited number of certified standards, including time consuming solid phase extraction (SPE) purification steps. Herein, we shed light on the variability of PA in herbal extracts and propose a quantification methodology based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. Particularly in the focus of the investigation were Alkanna spp. (Boraginaceae), which possess a wide range of pharmaceutical properties. In addition, a comparative PA screening of crude and SPE enriched extracts was performed and PA-containing plants from Fabaceae and Compositae families were included to demonstrate universal applicability. In total, 105 PA were identified using HRMSe experiments, specific MS/MS fragmentation PA patterns, a customized in-house library and literature data. Among them, 18 glycosidic PA derivatives were reported for the first time in literature. Using a hierarchical clustering approach, PA distribution in herbal extracts was shown to be family-dependent and significantly different among species. This was further supported by the results of the total PA concentrations, obtained using a retronecine/heliotridine/internal standard-based targeted UHPLC-HRMS quantification method, which varied from 8.64 ± 0.08–3096.28 ± 273.72 μg RE/g extract dry weight in shoots extracts of Alkanna spp. and leaves extracts of Crotalaria retusa L. respectively. Worth mentioning is that the procedure allowed to quantify PA in Alkanna spp. If the procedure based on 35 specific PA recommended by European regulations had been used, results would have been equal to zero for the four species since none were observed in Alkanna spp. Finally, by combining the RE results with the corresponding dereplication results, a customized correction factor for each extract (ranging from 2.12 to 2.48) was assessed leading to a more accurate estimate of the PA content regardless of the molecular weight of each PA. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation the real PA content due to limited availabilty of authentic reference compounds in botanical extracts used in phytomedicines or food supplements/cosmetics. [Display omitted] • Profiling of pyrrolizidine alkaloids (PA) using a retronecine-based untargeted metabolomics approach. • Eighteen new glycosidic pyrrolizidine alkaloids annotated. • Quantification of PA directly from crude botanical extracts expressed as total Retronecine equivalents. • A correction factor considering the dereplication step for more accurate total Retronecine equivalents. [ABSTRACT FROM AUTHOR]

B Correction to: Analytical and Bioanalytical Chemistry (2022) 414:8107-8124 b https://doi.org/10.1007/s00216-022-04344-5 The original article contained a mistake. Instead of "After washing with 10 mL 2% aqueous formic acid and 10 mL of methanol, the analytes were eluted with 6 mL of ammoniated methanol (5%) into a glass vial" it should have been "After washing with 10 mL water and 10 mL of methanol, the analytes were eluted with 6 mL of ammoniated methanol (5%) into a glass vial". Correction to: A sensitive LC-MS/MS method for isomer separation and quantitative determination of 51 pyrrolizidine alkaloids and two tropane alkaloids in cow's milk. [Extracted from the article]

A fast and simple method was developed to determine pyrrolizidine alkaloids (PAs) in honey using liquid chromatography tandem mass spectrometry (LC- MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was carried out by simply diluting with water, without the need of any additional clean-up steps. A full validation of the method was performed according to Commission Decision 2002/657/EC. The method was linear in the 050 μg kg −1 range and presented satisfactory intra-day and inter-day precision with relative standard deviations of 1.45–10.2% and 1.60–1–0.2%, respectively. The measurement uncertainty, limit of detection (LOD) (0.1–1.0 μg kg −1 ) and limit of quantification (LOQ) (0.2–1.5 μg kg −1 ) were also calculated. The proposed method was applied to analyse eight PAs, namely, senecionine, senecionine-N-oxide, echimidine, intermedine, lycopsamine, retrorsine, monocrotaline and retrorsine-N-oxide, in 92 commercial honey samples from Brazil. At least three PAs were detected in 99.1% of the samples. PAs were not detectable (

• Novel nano-LC-MS method for determining & screening pyrrolizidine alkaloids in food. • Post-column solvent for stabilising nanoflow electrospray during aqueous gradient. • C 6 H 8 N+ fragment ion is selective for detecting pyrrolizidine alkaloids in food. • Occurrence of pyrrolizidine alkaloids in food from the Latvian market was at low levels. A method for the determination of pyrrolizidine alkaloids in tea, honey, herbal tinctures, and milk samples was developed by employing nano-LC-MS with high-resolution Orbitrap mass spectrometry. Quantitation was performed using the available analytical standards, and a MS2 target ion screening approach was developed using fragment ions that were specific for pyrrolizidine alkaloids under collision-induced dissociation. Proof of concept was delivered for the screening approach, proposing that the C 6 H 8 N+ fragment ion is a highly selective fragment ion for the detection of potential pyrrolizidine alkaloids. The elaborated quantitation was applied for the occurrence study of pyrrolizidine alkaloids in food products available on the Latvian market, including samples of tea (n = 15), honey (n = 40), herbal tinctures (n = 15), and milk (n = 10). The median LOQ over all analytes was 0.33 µg kg-1 in honey, 3.6 µg kg-1 in tea, 3.3 µg kg-1 in herbal tinctures, and 0.32 µg kg-1 in milk. The herbal tinctures samples and milk samples did not contain pyrrolizidine alkaloids above LOQ values. Analytes were detected in 33% of honey and 47% of tea samples. Most common were echimidine, intermedine, and enchinatine N -oxide. Pyrrolizidine alkaloids in tea samples were mainly N -oxides, with the highest total concentration being 215 µg kg-1 among the samples, exceeding the maximum limit of 200 µg kg-1 set by Commission Regulation (EU) 2020/2040. In honey samples, lycopsamine-type alkaloids were detected most frequently, with the highest total concentration equal to 74 µg kg-1. Advantages of the developed nano-LC-MS methods included increased sensitivity in comparison with conventional flow LC-MS, low solvent consumption typical with nano-LC and the novel use of a selective common target ion for detection and discovery of potential pyrrolizidine alkaloids using high resolution mass spectrometry. [ABSTRACT FROM AUTHOR]

Rationale We report the electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis of a pyrrolizidine alkaloid extract containing both retrorsine [C18H25NO6] and its N-oxide [C18H25NO7] and N-hydroxyl [C18H26NO7] derivatives measured with a QqTOFMS hybrid instrument. Methods A solution of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives was directly infused into an electrospray ionization-quadrupole-time-of-flight (ESI-QTOF) mass spectrometer and product ion scans of the protonated molecules of each species were acquired. Labile protons of each compound were deuterated and computational energy calculations of the proposed structures of the product ions were used to determine the fragmentation pathways of retrorsine and its N-oxide and N-hydroxyl derivatives. Results ESI-MS of the pyrrolizidine alkaloid extract containing retrorsine and its N-oxide and N-hydroxyl derivatives afforded the protonated retrorsine [M1 + H]+ at m/z 352.1760 and the protonated retrorsine N-oxide [M2 + H]+ at m/z 368.1631 in addition to the formation of the unexpected protonated N-hydroxyl radical [M3 + H]+ • at m/z 369.1686. CID-MS/MS of this series of protonated molecules allowed the evaluation of their gas-phase fragmentations and the establishment of their fragmentation pathways. It was also found that several product ions could be assigned to different structures. Deuterium exchange and computational energy calculations allowed us to determine the most probable structures for the characterized product ions. Conclusions To our knowledge, the identification of the protonated retrorsine N-hydroxyl radical [M3 + H]+ • is reported for the first time. In addition, the MS/MS results can be used for the identification of retrorsine and its N-oxide and N-hydroxyl derivatives in different complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

• Food control of pyrrolizidine alkaloids in aromatic herbs. • Mesostructured silicas as dSPE clean-up sorbents in miniaturized QuEChERS. • Cost-effective and environmentally friendly microextraction method. • Lasiocarpine, europine N -oxide and senecivernine N -oxide main sample contaminants. • Reinforcement of horizontal natural transfer of pyrrolidine alkaloids. This work proposes the miniaturization and modification of the QuEChERS strategy using different large pore mesostructured silicas, non-modified and modified with amino groups (NH 2), as dispersive clean-up sorbents for multi-component extraction of 21 pyrrolizidine alkaloids from different aromatic herbs, combined with ultra-high performance liquid chromatography coupled to tandem mass spectrometry analysis. The procedure was miniaturized by reducing the amounts of sample (0.2 g), solvents (2 mL), clean-up sorbents (25 mg sorbent + 150 mg MgSO 4) and partitioning salts (0.65 g) employed. Best results were achieved using mesostructured silicas (LP-MS-NH 2) than conventional PSA. The method was validated (overall recoveries 73–105%) and applied to the analysis of 17 samples. All the samples were contaminated with PAs (average concentration 262 µg/Kg). Thyme and basil samples were the most contaminated, whereas rosemary was the least. Lasiocarpine, senecivernine N -oxide and europine N -oxide were the main PAs that contributed to their contamination. [ABSTRACT FROM AUTHOR]

• SALLE is suitable for efficient and fast extraction of PAs/PANOs from beehive products. • SALLE procedure allows the simultaneous determination of PAs and PANOs. • Pyrrolizidine alkaloids were determined by UHPLC-MS/MS. • Proposed procedure shows negligible matrix effect for all analyzed. • The validated method was applied to the analysis of honey and pollen samples. The application of salting out assisted liquid-liquid extraction for the rapid and simple simultaneous determination of pyrrolizidine alkaloids and related N-oxide in honey and pollen was evaluated. The determination of PAs and PANOs was obtained by ultra high performance liquid chromatography coupled with tandem mass spectrometry. The critical parameters (pH of the aqueous solution, extraction solvent, type and amount of salting-out agents) affecting SALLE performance were carefully studied and optimized using a chemometric approach. Under the optimal experimental conditions, the developed procedure showed excellent extraction efficiency (82–104 %) and recoveries (82–113 %) for all target analytes, except for Intermedine N-oxide, at three concentration levels (0.25, 2.5 and 25 μg kg−1 and 2.5, 25 and 250 μg kg-1 for honey and pollen samples, respectively), and satisfactory intra-day (<10) and inter-day (<13) precisions. The proposed method showed negligible matrix effect for both studied matrices and method quantification limits at very low levels (0.1−0.2 and 1−2 μg kg-1 for honey and pollen, respectively). The method was successfully applied to the analysis of honey (n = 71) and pollen (n = 6) samples. The 97 % and the 67 % of analyzed honey and pollen samples, respectively, turned out to be contaminated by at least one PA. Echimidine, echimidine N-oxide, intermedine and intermedine N-oxide significantly contributed to the contamination of the samples. [ABSTRACT FROM AUTHOR]

The UHPLC–QToF-MS analysis of pyrrolizidine alkaloids (PAs) from various parts of 37 botanicals and 7 products was performed. A separation by LC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. MS–MS detection was used because of its high selectivity, and ability to provide structural information. Free base and N -oxides were observed by this method. PAs were analyzed and detected in plants from three different families, viz ., Asteraceae , Boraginaceae and Fabaceae . The Asteraceae family was found to contain senecionine and lycopsamine type PAs. The Boraginaceae family contained lycopsamine and heliotrine type PAs and the Fabaceae family contained senecionine and monocrotaline type PAs. These PAs may serve as important markers for the detection of these plant materials in food and dietary supplements. PAs were identified in 44 samples by comparing their retention times, accurate mass and mass fragmentation patterns with those of 25 reference standards. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with serious hepatic disease in humans and animals. In this study, rapid and sensitive analytical method was developed for the determination of 9 toxic PAs in popularly high lipid foodstuffs by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). PAs in lipid foodstuff were effectively purified by freezing lipid precipitation (FLP) and strong cation exchange (SCX)-solid-phase extraction (SPE). Especially, FLP could easily remove the large amounts of triacylglycerols in the lipid sample extract and effectively combine with SPE cleanup. During the FLP procedure, over 77% of the lipids in the foodstuff extracts were rapidly eliminated without any significant loss of the PAs with over 81% recovery. The elimination efficiency of lipids by FLP was tested with LC-atmospheric chemical ionization (APCI)-MS. For further purification, SCX-SPE cartridge could successfully purify PAs from the remaining interfering substances by the variation pH with 5% NH 4 OH in methanol. For precise quantification and confirmation of PAs in complicate sample matrices, appropriate transition ions in LC-MS/MS-multiple-ion reaction monitoring (MRM) mode were selected on the basis of MS/MS fragmentation pathways of PAs. The established analytical method was validated in terms of the linearity, limits of detection (LOD), and quantification (LOQ), precision, and accuracy. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation < 11.06%). Overall limits of detection and quantitation of PAs were approximately 0.06–0.60 ng/mL at a signal-to-noise ratio (S/N) of 3 and were about 0.20–1.99 ng/mL at a S/N of 10 for all foodstuffs. The established method was successfully applied for the monitoring of toxic PAs in several types of high lipid foodstuffs such as soybeans, seed oil, milk, and margarine. [ABSTRACT FROM AUTHOR]

Honey samples originating from Australia were purchased and analysed for targeted pyrrolizidine alkaloids (PAs) using a new and rapid isocratic LC-MS/MS method. This isocratic method was developed from, and is comparable with, a gradient elution method and resulted in no loss of sensitivity or reduction in chromatographic peak shape. Isocratic elution allows for significantly shorter run times (6 min), eliminates the requirement for column equilibration periods and, thus, has the advantage of facilitating a high-throughput analysis which is particularly important for regulatory testing laboratories. In excess of two hundred injections are possible, with this new isocratic methodology, within a 24-h period which is more than 50% improvement on all previously published methodologies. Good linear calibrations were obtained for all 10 PAs and four PA N-oxides (PANOs) in spiked honey samples (3.57–357.14 µg l−1;R2 ≥ 0.9987). Acceptable inter-day repeatability was achieved for the target analytes in honey with % RSD values (n= 4) less than 7.4%. Limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked PAs and PANOs samples; giving an average LOD of 1.6 µg kg−1and LOQ of 5.4 µg kg−1. This method was successfully applied to Australian and New Zealand honey samples sourced from supermarkets in Australia. Analysis showed that 41 of the 59 honey samples were contaminated by PAs with the mean total sum of PAs being 153 µg kg−1. Echimidine and lycopsamine were predominant and found in 76% and 88%, respectively, of the positive samples. The average daily exposure, based on the results presented in this study, were 0.051 µg kg−1 bw day−1for adults and 0.204 µg kg−1 bw day−1for children. These results are a cause for concern when compared with the proposed European Food Safety Authority (EFSA), Committee on Toxicity (COT) and Bundesinstitut für Risikobewertung (BfR – Federal Institute of Risk Assessment Germany) maximum daily PA intake limit of 0.007 µg kg−1 bw day−1. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) suchas retrorsine are common foodcontaminants that are known to be bioactivated by cytochrome P450enzymes to putative hepatotoxic, genotoxic, and carcinogenic metabolitesknown as dehydropyrrolizidine alkaloids (DHPs). We compared how bothelectrochemical (EC) and human liver microsomal (HLM) oxidation ofretrorsine could produce short-lived intermediate metabolites; wealso characterized a toxicologically important metabolite, (3H-pyrrolizin-7-yl)methanol. The EC cell was coupled onlineor offline to a liquid chromatograph/mass spectrometer (LC/MS), whereasthe HLM oxidation was performed in 100 mM potassium phosphate (pH7.4) in the presence of NADPH at 37 °C. The EC cell oxidationof retrorsine produced 12 metabolites, including dehydroretrorsine(m/z350, [M + H+]),which was degraded to a new reactive metabolite at m/z136 ([M + H+]). The molecular structureof this small metabolite was determined using high-resolution massspectrometry and NMR spectroscopy followed by chemical synthesis.In addition, we also identified another minor but reactive metaboliteat m/z136, an isomer of (3H-pyrrolizin-7-yl)methanol. Both (3H-pyrrolizin-7-yl)methanoland its minor isomer were also observed after HLM oxidation of retrorsineand other hepatotoxic PAs such as lasiocarpine and senkirkin. In thepresence of reduced glutathione (GSH), each isomer formed identicalGSH conjugates at m/z441 and m/z730 in the negative ESI-MS. Because(3H-pyrrolizine-7-yl)methanol) and its minor isomersubsequently reacted with GSH, it is concluded that (3H-pyrrolizin-7-yl)methanol may be a common toxic metabolite arisingfrom PAs. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are a large class of natural compounds amongst which the esterified 1,2-unsaturated necine base is toxic for humans and livestock. In the present study, a method was developed and validated for the screening and quantification of nine PAs and one PA N-oxide in teas ( Camellia sinensis (L.) O. Kuntze) and herbal teas (camomile, fennel, linden, mint, rooibos, verbena). Samples were analysed by HPLC on a RP-column, packed with sub-2 μm core-shell particles, and quantified using tandem mass spectrometry operating in the positive electrospray ionisation mode. These PAs and some of their isomers were detected in a majority of the analysed beverages (50/70 samples). In 24 samples, PA concentrations were above the limit of quantification and the sum of the nine targeted PAs was between 0.021 and 0.954 μg per cup of tea. Thus, in some cases, total concentrations exceed the maximum daily intake recommended by the German Federal Institute for Risk Assessment and the UK's Committee On Toxicity (i.e. 0.007 μg kg bw). [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Consumers often believe that "natural food" is harmless, however naturally occurring toxins in food represent a health risk to humans. Honey as a natural, nutritious sweetener, is one of the most commonly consumed foods throughout the world. However, food safety concerns for honey arise when honeybees collect nectar from poisonous plants such as Rhododendron sp., Coriaria arborea, and Tripterygium wilfordii Hook F. Such honey contains natural plant toxins. Humans may develop intoxication symptoms after consuming toxic honey; in some cases, it can be fatal. As a result, toxic honey poses an often-ignored threat to public health. Typical plant toxins such as grayanotoxins, triptolides, tutin and pyrrolizidine alkaloids, have been identified in toxic honey. Although different toxic honeys elicit similar symptoms, such as vomiting, nausea, and dizziness, the mechanism of toxicity may be different. Thus, it is necessary to determine the exact toxicity mechanism of different toxins to further develop effective antidotes and cures. Another important challenge is preventing toxic honey from entering the food chain. Liquid chromatography-mass spectrometry has a wide range of applications in the detection of different toxins due to its accuracy and simplicity. More methods, however, are urgently needed to detect multiple plant-derived toxins in honey and its derivatives. Developing uniform international standards for toxin detection during quarantine using advanced techniques is critical for preventing human consumption of toxic honey. [Display omitted] • Honey contaminated with plant toxins is a potential risk to human health. • Typical plant toxins were identified from honey and comprehensively reviewed. • Toxicological mechanisms by toxic honey-derived toxins were reviewed. • Recent advances in detections of these toxins were explored. • Uniform international standards for toxin detection are warranted. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) are natural plant toxins, which are widely distributed in various types of teas. The source of PAs contamination in tea is probably related to weeds in tea gardens. The method of simultaneously detecting PAs/PANOs in teas and weeds has not been reported. A sensitive method was developed for detecting 14 PAs/PANOs simultaneously in teas and weeds using ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Sample preparation was optimized step by step. Sulfuric acid at 0.1 M was used to extract the target compounds. Large capacity PCX column was used to enrich target compounds and remove complex matrices effectively, following by the elution solvent with methanol containing 0.5% NH 4 OH. The method showed good linearity for all 14 analytes with regression coefficients (R2) > 0.99. Satisfactory recoveries of target compounds spiked at three concentrations (10, 50, 100 μg/kg) ranged from 68.6% to 110.2%, while intra-day and inter-day precision were below 18.5%. Limits of detection (LODs) and limits of quantification (LOQs) of 14 PAs/PANOs in different matrices were in the range of 0.001–0.4 μg/kg and 1–5 μg/kg, respectively. This method was firstly applied to monitor the occurrence of PAs/PANOs in different kinds of teas from China and weed samples collected in the local tea gardens. Seven PAs/PANOs (>LOQs) were detected in 15.32% of tea samples and maximum total PAs/PANOs content was 151.33 μg/kg. Pollution levels and patterns of PAs/PANOs were different significantly in various types of teas. Based on the occurrence of PAs/PANOs in teas and weeds, weeds in tea garden were likely to be the source of PAs/PANOs in teas and the most relevant contaminating species of tea samples could be Crassocephalum crepidioides (Benth.) S. Moore. • 14 PAs/PANOs were determined in teas and weeds using UHPLC-MS/MS. • Targets were extracted using 0.1M H 2 SO 4 and complex matrices were purified by SPE. • Weeds in tea garden were likely to be source of PAs in teas. • The most relevant contaminating species of teas was found. [ABSTRACT FROM AUTHOR]

Abstract: Pyrrolizidine alkaloids (PAs) are toxins biosynthesised by plants and they are known to be present in approximately 6000 plant species, about 3% of all flowering plants. PAs are probably the most widely distributed natural toxins and represent a potential risk to human health, since poisoning caused by these toxins is associated with acute and chronic liver damage and may lead to death. One of the most common sources of PAs exposure in humans is honey consumption. We have developed a quick and easy method to quantify nine different PAs (echimidine, heliotrine, intermedine, lycopsamine, lasiocarpine, retrorsine, seneciphylline, senecionine, senkirkine) in honey based on QuEChERS sample extraction and ultra fast liquid chromatography coupled with mass spectrometry detection. We performed a validation study of the method and it resulted in good precision and accuracy, high recoveries, and good linear calibrations. The limit of detection ranged from 0.021 to 1.39 μg Kg−1 and the limit of quantification from 0.081 to 4.35 μg Kg−1. This new approach was applied to the quantification of PAs in retail honeys purchased in local supermarkets, classified by their country of origin: Italian honeys, blends of honey of European countries and blends of honey of European and non-European countries. The concentrations detected ranged from 1 to 169 μg PAs/kg−1 with higher concentrations in blends of European and non-European honeys. This study reveals that many samples tested would exceed the tolerable daily intake suggested for these substances and they could be a hazard to human health. [Copyright &y& Elsevier]

• 17-PAs/PANOs profile in 775 honey was investigated by a validated LC-MS/MS method. • Local honey analysed does not pose a double risk to health compared to large retail. • PAs/PANOs level in honey aid local beekeepers to select the safest production area. • S-type PAs/PANOs characterized mainly the local honey. • Machine learning applied to LC-MS/MS data could be a tool to trace honey's origin. A validated method in liquid chromatography tandem mass spectrometry was applied to assess the contamination profile of 17 pyrrolizidine alkaloids and their N-oxide metabolites (PAs/PANOs) in 775 honey samples of different origins, including from local beekeepers (579, LB), large retail and online stores (Italian (LRI) and foreign (LRF) products). Fifty-five percent of the honey samples contained the 17 monitored compounds at below the limit of quantification. The LB samples were characterised by a total mean concentration of PAs/PANOs at 6.7 and 3.3 times lower than those of the LRF and LRI samples, respectively. The PA echimidine characterised mainly honey samples from the large retail stores (75.8 % and 72.5 % of LRI- and LRF-contaminated samples, respectively). The LB samples showed a senecionine-type contamination profile, providing beekeepers clues to the plants responsible for contamination. The results highlighted that the consumption of the local honey samples analysed does not represent a double risk for human health compared to the consumption of products from large retail stores. Careful attention must be paid to honey imported from areas where lycopsamine-type and senecionine-type PAs/PANOs-producing plants are invasive. A machine learning approach could predict the origin of the sample given its contaminant profile at an accuracy of 0.74, which is useful for detecting fraud in the declared honey origin. [ABSTRACT FROM AUTHOR]

Abstract: Pyrrolizidine alkaloids (PAs) are known secondary plant metabolites which can cause hepatotoxicity in both humans and livestock. PAs can be consumed through the use of plants for food, medicinal purposes and as contaminants of agricultural crops and food. PA contaminated grain has posed the largest health risk, although any PA contamination in our food chain should be recognised as a potential health threat. For this purpose, retail honeys were tested by LC–MS/MS. The method allows for specific identification of toxic retronecine and otonecine-type PAs by comparison to reference compounds via a spectral library. In total, 50 honey samples were matched to the reference spectra within a set of tolerance parameters. Accurate data analysis and quick detection of positive samples was possible. Positive samples contained an average PA concentration of 1260μgkg−1 of honey. Good linear calibrations were obtained (R 2 >0.991). LOD and LOQ ranged from 0.0134 to 0.0305 and 0.0446 to 0.1018μgmL−1, respectively. [Copyright &y& Elsevier]

OBJECTIVE: Hepatic sinusoidal obstruction syndrome (HSOS) induced by a Chinese medicinal herb Tusanqi is increasingly being reported in recent years. The aim of the study was to investigate the possibility of using blood pyrrole-protein adducts test as a confirmatory diagnostic method. METHODS: Patients with HSOS according to international diagnostic criteria associated with Tusanqi from January 2006 to August 2010 in Zhongshan Hospital Fudan University were included and clinical features were collected. Pyrrole-protein adducts in blood sample were determined with ultra performance liquid chromatography-mass spectrometry (UPLC-MS) while pyrrolizidine alkaloids (PAs) in available herbal preparations were analyzed by high performance liquid chromatography-ultraviolet (HPLC-UV). RESULTS: Five patients (age 41-72 years, median age 54 years, all women) were included. Ascites (5/5), jaundice (5/5) and hepatomegaly (4/5) were common manifestations. The imaging features were diffused, patchy hepatic enhancement, periportal edema and ascites. Pathology ascertained that blood flow was obstructive at the site of sinusoid. PAs ( Seneionine and seneciphylline) were identified in all the three available herbal preparations ingested by the HSOS patients. Pyrrole-protein adducts were unequivocally found in all the five blood samples. Two patients recovered, two developed chronic illness and one died due to liver failure and hepatic encephalopathy. CONCLUSIONS: The detection of blood pyrrole-protein adducts using a UPLC-MS approach is a specific, reliable, unambiguous and confirmatory test for HSOS induced by PA, and should be used together with the conventional HSOS clinical diagnostic criteria for the definitive diagnosis of PA-induced HSOS. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are a class of natural toxins with hepatotoxicity, genotoxicity and carcinogenicity. They are endogenous and adulterated toxic components widely found in food and herbal products. In this study, a sensitive and efficient ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to detect the PAs in 386 kinds of Chinese herbal medicines recorded in the Chinese Pharmacopoeia (2020). The estimated daily intake (EDI) of 0.007 μg/kg body weight (bw)/day was adopted as the safety baseline. The margin of exposure (MOE) approach was applied to evaluate the chronic exposure risk for the genotoxic and carcinogenic potential of PAs. Results showed that PAs was detected in 271 out of 386 samples with a content of 0.1–25,567.4 μg/kg, and there were 20 samples with EDI values above the baseline, 0.007 μg/kg bw/day. Beyond that, the MOE values for 10 out of 271 positive samples were below 10,000. Considering the actual situation, Haber's rule was used to assume two weeks exposure every year during lifetime, and still the MOE values for four out of 271 positive samples were under 10,000, indicating these products may have potential health risk. The developed method was successfully applied to detect the PAs-containing Chinese herbal medicines. This study provides convincing data that can support risk management actions in China and a meaningful reference for the rational and safe use of Chinese herbal medicines. [ABSTRACT FROM AUTHOR]

Senecio coronatus (known as izonkozonko and ubulibazi in Zulu) is commonly used in traditional medicine in South Africa as purification purgative and enemas for infants during weaning. We show for the first time that this species does not contain pyrrolizidine alkaloids and that reported cases of fatal hepatic sinusoidal obstruction syndrome in infants were caused by wrongly identified Senecio species containing large amounts of retrorsine-N-oxide. A validated ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection and quantitation of pyrrolizidine alkaloids is described. [ABSTRACT FROM AUTHOR]