Analysis Of Pyrrolizidine Alkaloids Research Articles

LC–MS/MS analysis of pyrrolizidine alkaloids in bee bread and commercial pollen from Brazil

LC–MS/MS analysis of pyrrolizidine alkaloids in bee bread and commercial pollen from Brazil

Analysis of Pyrrolizidine Alkaloids in Stingless Bee Honey and Identification of a Botanical Source as Ageratum conyzoides.

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.

Analysis of pyrrolizidine alkaloids in Eupatorium fortunei Turcz. and their in vitro neurotoxicity

This study was to analyze the pyrrolizidine alkaloids (PAs) in Eupatorium fortunei herbs and its derived finished products with a view to evaluating their effects on the proliferation and oligodendrogenesis of neural progenitor cells (NPCs). Using a LC-MS/MS method with 32 PAs reference standards, 8 PAs including intermedine, intermedine N-oxide, lycopsamine, lycopsamine N-oxide, retronecine, seneciphylline and senkirkine and 7-acetylintermedine N-oxide were identified with intermedine N-oxide and lycopsamine N-oxide being most abundant. The total PA amounts were found to vary from 0.18 to 61.81 μg/g in 30 batches of herbs and from 0.86 to 36.96 μg/g in 4 commercial finished products, respectively. Risk assessments indicated that the short-term intake seemed unlikely lead to acute toxic effects but the chronic use warranted cautions. Using NPCs derived from mouse induced pluripotent stem cells as an in vitro testing model, intermedine, intermedine N-oxide and lycopsamine N-oxide appeared to decrease cell viability at 30 μM whereas intermedine N-oxide inhibited oligodendrogenesis of NPCs at 10 μM. The present results suggested that the PAs in the majority of E. fortunei herbs and the derived products not only resulted in their exposure far exceeding the acceptable intake limit (i. e. 1.0 μg PA per day for adults) in herbal medicinal products recommended by the European Medicines Agency but also induced neurotoxicity to NPCs in vitro.

Analysis of Pyrrolizidine Alkaloids in Queensland Honey: Using Low Temperature Chromatography to Resolve Stereoisomers and Identify Botanical Sources by UHPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples.

Quantitative analysis of pyrrolizidine alkaloids in Gynura procumbens by liquid chromatography–tandem quadrupole mass spectrometry after enrichment by PCX solid-phase extraction

ABSTRACTGynura 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).

Quantitative and qualitative analysis of pyrrolizidine alkaloids in liqueurs, elixirs and herbal juices

Pyrrolizidine alkaloids (PAs and corresponding N-oxides (PANOs)) are known to have adverse health effects. Their toxic effects on liver cells are especially well-documented. In addition, potential carcinogenic and mutagenic effects in chronic exposure via food and/or herbal medicines have been a subject of vivid discussion in the last decade. Liqueurs and elixirs are traditionally used alcoholic extracts made from parts of plants and herbs. PA cross-contamination of the final products seems likely. Hence, this study aims to detect and quantify the PAs in such products in the light of a possible PA-contamination. The PA content was determined in the form of a single sum parameter using HPLC-ESI-MS/MS and a stable isotope-labeled internal standard. Overall, 56 products available at German pharmacies, drugstores, or internet shops were analyzed, comprising in total 38 samples of liqueurs (mainly bitters), 12 samples of plant elixirs and six different herbal juices. The results showed that 9 out of 38 liqueurs were PA-positive (24%). The total amount of PAs ranged from non-detectable to 9.5 μg/kg. Seven out of ten elixirs were PA-positive (70%) with a maximum PA-content of 3121 μg/kg. Four out of six plant juices were PA-positive (67%) with an average of 4.4 μg/kg (PA-positive samples only).The results and potential risks are discussed in the light of recommended portions for daily consumption or daily doses, in association with the detected PA amounts for individual products and product classes.

Characterization and screening of Pyrrolizidine Alkaloids and N-oxides from botanicals and dietary supplements using UHPLC-high resolution mass spectrometry

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, sensitivity 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.

Development of a fast isocratic LC-MS/MS method for the high-throughput analysis of pyrrolizidine alkaloids in Australian honey

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−1 and 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−1 for adults and 0.204 µg kg−1 bw day−1 for 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.

The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods

Pyrrolizidine alkaloids (PAs) serve an important function in plant defence.Objective - To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC-NPD) and liquid chromatography tandem mass spectrometry (LC-MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC-NPD, reduction is required to transform PA N-oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. The lowest PA concentration measured with GC-NPD was approximately 0.03 mg/g and with LC-MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC-NPD. With the LC-MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC-NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC-MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC-NPD analysis with sulfuric acid extraction and PA N-oxide reduction with zinc and purification. In terms of sensitivity and discrimination, formic acid extraction in combination with LC-MS/MS detection is the method of choice for analysing PAs (both free and N-oxides forms) in plant material.

A New High Performance Liquid Chromatography Method for the Simultaneous Quantitative Analysis of Pyrrolizidine Alkaloids and their N-Oxides in Plant Material

A New High Performance Liquid Chromatography Method for the Simultaneous Quantitative Analysis of Pyrrolizidine Alkaloids and their N-Oxides in Plant Material

Analysis of pyrrolizidine alkaloids: a competitive enzyme-linked immunoassay (ELISA) for the quantitative determination of some toxic pyrrolizidine alkaloids.

A highly sensitive and specific competitive enzyme-linked immunoassay (ELISA) for the determination of retrorsine, senecionine, and integerrimine in the nanogram range has been developed. Pyrrolizidine alkaloids were detected in Senecio rupestris. No cross-reactivity was seen with either retrosine N-oxide, otonecine alkaloids, or other macrocyclic pyrrolizidine alkaloids.