Determination of ergot alkaloids from grains with UPLC‐MS/MS

A simple and rapid method for determining six ergot alkaloids and four of their respective epimers was developed for rye and wheat. The analytes were extracted from the sample matrix with ACN/ammonium carbonate solution. The extract was purified with a commercial push‐through SPE column (Mycosep^® 150 Ergot). After concentration and filtration steps, the final separation of the analytes was achieved with ultra‐performance LC‐MS/MS. The chromatographic separation of the ergot alkaloids was achieved in 4.5 min. The method performance proved satisfactory in the preliminary validation. The calculated LOQs were low ranging from 0.01 to 1.0 μg/kg for wheat and from 0.01 to 10.0 μg/kg for rye. At the concentration levels of 10, 50 and 200 μg/kg, the recoveries were between 80 and 120% in most cases and the within‐day repeatability (expressed as RSD) ranged between 1.3 and 13.9%. Despite the cleanup of the samples, some matrix effect was observed in the MS, highlighting the necessity of using matrix‐assisted standards. This is the first article to describe the application of the push‐through columns and ultra‐performance LC in the analysis of ergot alkaloids.     

Formyl migration product of chanoclavine-I aldehyde in the presence of the old yellow enzyme FgaOx3 from Aspergillus fumigatus: a NMR structure elucidation

A previous study showed that together with the festuclavine synthase FgaFS, the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalyzed the conversion of chanoclavine-I aldehyde to festuclavine in the biosynthesis of ergot alkaloids. In the absence of FgaFS, a mixture containing two compounds with a ratio of 7:3 was detected in the enzyme assay of FgaOx3. NMR experiments including (DQF)-COSY, HSQC, HMBC and NOESY identified their structures as E/Z isomers of N-methyl-N-[(5R,10R)-10-(2-oxo-propyl)-2,4,5,10-tetrahydrobenzo[cd]indol-5-yl]formamide and proved the migration of the formyl group at C-8 in chanoclavine I-aldehyde to N-6 in the identified products.     

Stereoselective total syntheses of (±)-chanoclavine I and (±)-isochanoclavine I by an intramolecular nitrone-olefin/cycloaddition. Preliminary communication

The racemic alkaloids chanoclavine I ( 1 ) and isochanoclavine I ( 2 ) have been synthesized stereoselectively from indole-4-carbaldehyde ( 3 ) by a sequence of 11 operations in overall yields of 14% and 2.4%, respectively. The key step 6 → 8 (Scheme 2) involves a transient nitrone 7 which undergoes a regio- and stereoselective intramolecular cycloaddition to a 1,2-disubstituted olefinic bond.     

Pergolide mesyl­ate form II

A new polymorph of pergolide mesyl­ate or 8β‐[(methyl­sul­fan­yl)­methyl]‐6‐propyl­ergoline methane­sulfonate, C₁₉H₂₇N₂S⁺·CH₃SO₃⁻, is reported. Pergolide mesyl­ate form II crystallizes in the trigonal system, which is unique for ergot derivatives. Although the hydrogen‐bond system in form II differs completely from that in form I, the conformation of the pergolide moiety in various related structures is very similar.     

Catalytic Asymmetric Reactions of 4‐Substituted Indoles with Nitroethene: A Direct Entry to Ergot Alkaloid Structures

A domino Friedel–Crafts/nitro‐Michael reaction between 4‐substituted indoles and nitroethene is presented. The reaction is catalyzed by BINOL‐derived phosphoric acid catalysts, and delivers the corresponding 3,4‐ring‐fused indoles with very good results in terms of yields and diastereo‐ and enantioselectivities. The tricyclic benzo[cd]indole products bear a nitro group at the right position to serve as precursors of ergot alkaloids, as demonstrated by the formal synthesis of 6,7‐secoagroclavine from one of the adducts. DFT calculations suggest that the outcome of the reaction stems from the preferential evolution of a key nitronic acid intermediate through a nucleophilic addition pathway, rather than to the expected “quenching” through protonation.     

Low field chemical shifts in 13carbon NMR spectra of 1,3-dehydroadamantane compounds possessing “inverted carbon atoms”

1,3-Dehydroadamantanes, which possess “inverted carbon atoms” in a cyclopropyl moiety, display low field ¹³C-NMR chemical shifts for carbon atoms situated across from the cyclopropyl group. This long range effect also shows in 2,4-dehydroadamantane and is therefore not due to the presence of especially strained inverted carbons.     

Ergot alkaloids in Norwegian wild grasses: a mass spectrometric approach

Ergot alkaloids are mycotoxins which are produced among fungi in the family Clavicipitaceae. Poisoning with ergot alkaloids is an important veterinary problem in animal husbandry and has recently also been recognised in wild animals. While the poisoning syndrome observed in domestic animals such as cattle, horses and sheep is usually caused by endophyte-infected grass, the recently observed ergotism among Norwegian cervids is probably due to infection of wild grasses with Claviceps. Mass spectrometry is today the method of choice for the rapid qualitative and quantitative determination of many natural compounds. This study uses tandem quadrupole mass spectrometry as well as ion trap mass spectrometry in connection with electrospray(+) ionisation for the quantification, screening and fragmentation of ergot alkaloids in extracts from Claviceps sclerotia that had been picked from wild grasses from several locations in Norway. Ergotamine, ergovaline, ergonovine and ergocryptine were available as standards and were quantified in the extracts, while ergocrystine, ergocornine, ergonine/ergosine, lysergic acid and lysergol were identified on the basis of their molecular weights and semi-quantified. Ergocrystine dominated the alkaloid spectrum of most extracts. Levels of the quantified alkaloids were in the range 0.2-9300 microg/g. Several unknown ergot alkaloids were found in the extracts. MS(n) experiments identified some as simple lysergic acid amide derivatives, while othes are probably related to ergocrystine and ergocryptine by dehydration, dehydrogenation and/or amino acid substitution at R(1) of the peptide moiety.     

Synergistic Effects of Metals in a Promising RuII−PtII Assembly for a Combined Anticancer Approach: Theoretical Exploration of the Photophysical Properties

Ru^II?Pt^II complexes are a class of bioactive molecules of interest as anticancer agents that combine a light‐absorbing chromophore with a cisplatin‐like unit. The results of a DFT and TDDFT investigation of a Ru^II complex and its conjugate with a cis‐PtCl₂ moiety reveal that a synergistic effect of the metals makes the assembly a promising multitarget anticancer drug. Inspection of type I and type II photoreactions and spin–orbit coupling computations reveals that the cis‐PtCl₂ moiety improves the photophysical properties of the Ru^II chromophore, ensuring efficient singlet oxygen generation and making the assembly suitable for photodynamic therapy. At the same time, the Ru^II chromophore promotes a new alternative activation mechanism of the Pt^II ligand via a triplet metal‐to‐ligand charge transfer (³M LCT) state, before reaching the biological target. The importance of the supramolecular architecture is accurately derived, opening interesting new perspectives on the use of bimetallic Ru^II?Pt^II assemblies in a combined anticancer approach.     

The synthesis of alkaloids of the ergotoxin group. 70. Section on ergot alkaloids

The syntheses of the alkaloids of the ergotoxine-group i.e. ergocristine, α- and β-ergokryptine, and ergocornine, are described. Using starting material with known stereochemistry these syntheses allowed to determine the absolute configurations also at C-2′ and C-12′ in the peptide part, which could not be derived from analytical data. All ergot alkaloids of the peptide type possess the same stereochemical structure.     

Quantitative determination of bioactive alkaloids lysergol and chanoclavine in Ipomoea muricata by reversed-phase high-performance liquid chromatography

A rapid, simple, sensitive, gradient and reproducible, reverse‐phase high‐performance liquid chromatographic method was developed for the quantitative estimation of bioactive alkaloids, lysergol and chanoclavine in the seeds of Ipomoea muricata. The clavine alkaloid, lysergol, is a bioenhancer for the drugs and nutrients. The samples were analyzed by reverse‐phase chromatography on a Waters spherisorb ODS2 column (250 × 4.6 mm, i.d., 10 µm) using binary gradient elution with acetonitrile and 0.01 m phosphate buffer (NaH₂PO₄) containing 0.1% glacial acetic acid at a flow rate of 0.8 mL/min, a column temperature of 25 °C and UV detection at λ 254 nm. The limits of detection (LOD) and quantitation (LOQ) were 0.035 and 0.106 µg/mL for lysergol and 0.039 and 0.118 µg/mL for chanoclavine, respectively. Standard curves were linear in the range of 2–10 µg/mL (r > 99) for both analytes. Good results were achieved with respect to repeatability (RSD < 2%) and recovery (99.20–102.0). The method was validated for linearity, accuracy repeatability, LOQ and LOD. The method is simple, accurate and precise, and may be recommended for routine quality control analysis of I. muricata seed extracts containing these two clavine alkaloids (1, 2) as bioactive principles of the herb. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC–MS–MS

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS–MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 μg kg⁻¹ depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 μg kg⁻¹ were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 μg kg⁻¹ the expanded measurement uncertainty ranged from ±0.56 to ±1.49 μg kg⁻¹, at a concentration level of 100 μg kg⁻¹ the expanded measurement uncertainty ranged from ±8.9 to ±20 μg kg⁻¹. Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Étude par chromatographie liquide à haute pression des alcaloïdes de l''ergot

High-performance liquid chromatography of ergot alkaloid.

A method for the analysis of ergot alkaloids has been developed by high-performance liquid chromatography. A silica column was used for preparation of ergotinine and ergotoxine from ergot. Separation of the individual ergot alkaloids was successfully accomplished on a column of Hitachi Gel No. 3011-O (porous polystyrene modified by hydroxymethyl) with an eluent of n-hexane—ethanol—triethylamine (70:30:0.5, v/v). The method was applied to the analysis of -ergocryptinine, ergocorninine, ergocristinine, -ergocryptine, ergocornine and ergocristine in ergot and dihydroergocryptine ( + β), dihydroergocornine and dihydroergocristine in pharmaceutical tablets.     

Capillary zone electrophoresis separation of enantiomers of lisuride

Lisuride is an ergot alkaloid derivative with dopaminergic activity. It is used for treatment of Parkinsonism and some other diseases associated with high level of prolactine. Lisuride is a chiral compound derived from natural ergot alkaloids. A new capillary zone electrophoresis (CZE) method capable of separating the enantiomers of lisuride was developed. Using the optimized conditions (acidic electrolyte with the addition of gamma-cyclodextrin (gamma-CD)) as low as 0.02% of undesirable L-lisuride can be detected. Selected method characteristics, i.e., linearity (0-20 mg/l), precision (2.0% at 5 mg/I), and accuracy (101 +/- 4% at 5 mg/l) were evaluated. The optimized method was applied for the analysis of real batches of Lisuride hydrogenmaleate and Lisuride base manufactured by IVAX Pharmaceuticals. It was found that they contain less than 0.02% of undesirable L-enantiomer.     

Some new information on the formation of substituted 4‐amino‐1‐substituted phenyl‐1H‐pyrazoles from β‐enaminones and diazonium tetrafluoroborates

Upon reaction of 2‐methyl‐, 3‐ethoxycarbonyl, and 4‐ethoxycarbonylbenzenediazonium tetrafluoroborate with 1‐cyclopropyl‐3‐phenylaminohex‐2‐en‐1‐one 3‐cyclopropylcarbonyl‐1‐(substituted phenyl)‐5‐ethyl‐4‐phenylamino‐1H‐pyrazoles are formed. On the other hand, the reaction of 1‐cyclopropyl‐3‐phenylaminohex‐2‐en‐1‐one and 5‐methylaminohept‐4‐en‐3‐one with sterically more demanding 2‐ethoxycarbonylbenzenediazonium tetrafluoroborate does not give the corresponding pyrazoles but the probable intermediates on the route to the pyrazoles: 1‐cyclopropyl‐3‐phenyliminohexane‐1,2,4‐trione 2,4‐bis(2‐ethoxycarbonylphenylhydrazone) and 3‐methyliminoheptane‐2,4,5‐trione 2,4‐bis(2‐ethoxycarbonylphenylhydrazone), respectively. All the compounds were identified on the basis of ¹H‐ and ¹³C‐NMR spectra. The structure of 1‐cyclopropyl‐3‐phenyliminohexane‐1,2,4‐trione 2,4‐bis(2‐ethoxycarbonylphenylhydrazone) was confirmed by means of ¹⁵N‐NMR spectra and X‐ray. The bis(2‐ethoxycarbonylphenylhydrazones) were found to show atropoisomerism due to a hindered rotation around the bond between the carbons of imino group and the hydrazono group next to carbonyl. In the case of the crystalline cyclopropyl derivative, the unit cell was found out to contain two molecules of opposite chirality. J. Heterocyclic Chem., (2011).     

Identification of ergot alkaloids with a photochemical reaction detector in liquid chromatography

An on-line photochemical reaction detector is described for the identification of ergot alkaloids by high-performance liquid chromatography. The fluorescence signal of alkaloids decreases within about 20 sec of irradiation and disappears selectively from complex chromatograms. The application of this principle to urine samples is described.     

Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha-ergocryptine, and ergocristine are extracted from wheat with methanol-0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 microg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovine and 20-400 ng/g for ergotamine, ergocornine, alpha-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of <5 and <20 ng/g, respectively, for each ergot alkaloid tested.     

Carbophilic 3‐Component Cascades: Access to Complex Bioactive Cyclopropyl Diindolylmethanes

Naturally occurring indole‐3‐carbinol and 3,3‐diindolylmethane show bioactivity in a number of disparate disease areas, including cancer, prompting substantial synthetic analogue activity. We describe a new approach to highly functionalised derivatives that starts from allene gas and proceeds via the combination of a three‐component Pd⁰‐catalysed cascade with a one‐pot, three‐component carbophilic Pt^II cascade linked to a stereoselective acid‐catalysed Mannich–Michael reaction that generates complex cyclopropyl diindolylmethanes which show selective activity against prostate cancer cell lines.     

Analysis of ergot alkaloids by high-performance liquid chromatography : I. Clavinces and simple derivatives of lysergic acid

A method of high-performance liquid chromatography has been developed for the separation and quantitative analysis of a mixture of ergot alkaloids on MicroPak NH₂ columns using isocratic and gradient elution. The mobile phase is diethyl ether-ethanol. Ultraviolet detection is employed at various wavelengths, and the ergot alkaloids are determined using the method of internal normalization.     

Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α‐Synuclein‐Induced Cytotoxicity in Baker′s Yeast

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans‐splicing in combination with a highly efficient split‐intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI‐I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α‐synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI‐I in a ratio of 1:5×10⁴. This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide‐based libraries in eukaryotic cells.