An integrated targeted and untargeted approach for the analysis of ergot alkaloids in cereals using UHPLC - hybrid quadrupole time-of-flight mass spectrometry

An ultra-high performance liquid chromatography hybrid quadrupole time of flight (Q-TOF) mass spectrometry (MS) method is described for the simultaneous quantitative determination of common ergot alkaloids and the screening, detection and identification of unexpected (less studied or novel) members of this class of toxic fungal secondary metabolites. The employed analytical strategy involves an untargeted data acquisition (consisting of full scan TOF MS survey and information dependent acquisition MS/MS scans) and the processing of data using both targeted and untargeted approaches. Method performance characteristics for the quantitative analysis of 6 common ergot alkaloids i.e. ergometrine, ergosine, ergotamine, ergocornine, ergocristine, ergokryptine and their corresponding epimers in rye were comparable to those previously reported for triple-quadrupole (QqQ) MS/MS. The method limits of quantification (LOQ) were in the range from 3 to 19 mu g/kg, and good linearity was observed for the different ergot alkaloids in the range from LOQ to 1000 mu g/kg. Furthermore, the method demonstrated good precision (relative standard deviations at 50 mu g/kg not higher than 14.6 and 16.2% for the intra-day and inter-day precision, respectively), and the trueness values at different concentration levels were all between 89 and 115%. The method was applied for the analysis of a set of 17 rye samples and demonstrated the presence of these ergot alkaloids in the range from <LOQ to 2,811 mu g/kg. Further mining of the same data based on a 'non-targeted peak finding' algorithm and the use of full MS and MS/MS accurate mass data allowed the detection and identification of 19 ergot alkaloids that are commonly not included in most analytical methods using QqQ instruments. Some of these alkaloids are reported for the first time in naturally contaminated samples

Identification of “Kratom” (Mitragyna speciosa) Alkaloids in Commercially Available Products

“Kratom” is the common name for the botanical mitragyna speciosa. It is a tree native to Southeast Asia in which leaves contain the psychoactive alkaloids mitragynine and 7-hydroxymitragynine. Kratom is often ingested as teas, chewed, or smoked. It acts as a stimulant in small doses and as an opioid in large doses. Overdoses can result in vomiting, seizures, and death. Recently the Drug Enforcement Agency (DEA) placed Kratom on Schedule 1, but, due to public outcry, it was almost immediately removed. Eleven kratom based products were obtained from various tobacco shops, “headshops” and via the internet including: Choice brand capsule and powder, Krave brand capsule, Lucky brand powder, King Kratom and Purple Haze e-liquids with 0 mg nicotine and 12 mg nicotine, Mojo brand capsule, O.P.M.S Liquid Kratom concentrate, and a K. Kratom chocolate bar. These products and methanol extracted samples were analyzed for psychoactive alkaloids and other components using an AccuTOF Direct Analysis in Real Time Mass Spectrometry (DART-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). The psychoactive alkaloids mitragynine and 7-hydroxymitragynine along with four other alkaloids, corynantheidine, corynoxine, paynantheine, and speciofoline, were identified in all 11 products. Unregulated commercial products made from kratom contain psychoactive alkaloids such as mitragynine and 7-hydroxymitragynine. They are readily available in a variety of forms and may be prone to abuse.https://scholarscompass.vcu.edu/uresposters/1246/thumbnail.jp

Determination of pyrrolizidine alkaloids in dietary sources using a spectrophotometric method

Pyrrolizidine alkaloids (PAs) are a class of toxic compounds found in the composition of more than 6000 plants. People can be exposed to PAs by consuming phytotherapeutic products, food from crops contaminated with seeds of some species with high content of PAs, and/ or contaminated animal products like bee products. For this reason we developed and validated a method for quantitative determination of PAs, from the most frequently contaminated food sources, honey and flour. Colorimetric Ehrlich reagent method was used with standard addition (1mg/kg senecionine). The extraction solvent was methanol 50% acidified with citric acid to pH 2-3, as this solvent can be used for alkaloids and N-oxides. We found that, in extracting the alkaloid only once from the dietary sources, the percent of recovery is low (52.5% for honey, and 45.75% for flour). Using successive extractions, three times with the same solvent, the senecionine retrieval percentage increased to 86.0% for honey and 76.0% for flour. The method was validated using the following parameters: selectivity, linearity (0,25- 20 mg/ mL senecionine), accuracy (average recovery 93.5 - 107.93%) and precision (RSD 3,26-4.55%.). The calculated limit of quantification (0.174 mg/ mL) makes this method applicable for determining Pas occurring at toxic levels for consumers

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites