Determination of Veratrum alkaloid contents in three Veratrum species by HPLC-MS/MS.

INTRODUCTION: Veratrum alkaloids have gained attention due to their toxic effects and potential pharmaceutical applications, particularly in cancer and cardiology. Over 200 alkaloids are found in species of the Veratrum genus. The alkaloid composition and concentrations can greatly vary in plants depending on factors like species, plant part, location, season, weather, or nutrients. OBJECTIVE: This study aims an analytical approach to analyze and quantify Veratrum alkaloids in different plant parts of Veratrum species. The purpose is to contribute essential alkaloid concentration data for future research on the pharmacological and toxicological aspects of Veratrum alkaloids. METHODS: This study focuses on five Veratrum alkaloids (cevadine, jervine, protoveratrine A, veratramine, and veratridine) in three Veratrum species (Veratrum album L., Veratrum californicum Durand, and Veratrum nigrum L.) collected from four German botanical gardens (Dresden, Leipzig, Marburg, and Schellerhau). A liquid-liquid extraction method and a sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method operating in multiple reaction monitoring (MRM) mode were applied for the alkaloid determination. RESULTS: Quantification revealed varying alkaloid concentrations among plant parts and Veratrum species in the µg/g to mg/g range. Protoveratrine A exhibited the highest content, while veratramine concentrations were generally lower. Especially in fruit, roots and rootstock of Veratrum album L. alkaloid concentrations were significant high. CONCLUSION: The developed HPLC-MS/MS method successfully determined Veratrum alkaloid concentrations in plant samples. The study contributes valuable data on Veratrum alkaloid distribution in different species and plant parts, crucial for understanding their potential medicinal and toxicological significance.

Point-of-Care Assessment of Direct Oral Anticoagulation in Acute Ischemic Stroke: Protocol for a Prospective Observational Diagnostic Accuracy Study.

BACKGROUND: Treatment of ischemic stroke with recombinant tissue plasminogen activator for intravenous thrombolysis (IVT) must be delivered within a narrow time window after symptom onset. This effective hyperacute treatment can be administered after ruling out active anticoagulation with direct oral anticoagulants (DOACs). Whenever this is impractical, e.g., due to aphasia, plasmatic DOAC levels are measured with a consequent delay in the IVT decision-making process ranging from 30 to 60 minutes of time. This study will test the hypothesis that hyperacute point-of-care assessment of clotting time in the patient's whole blood has sufficient diagnostic accuracy to determine immediately whether stroke patients are pretreated with DOAC. METHODS AND DESIGN: This will be a prospective single-center diagnostic accuracy study in 1,850 consecutive acute ischemic stroke patients at a tertiary stroke center in Saxony, Germany. Presence of active anticoagulation with DOAC will be determined by point-of-care quantification of clotting time via whole blood viscoelastic testing (ClotPro) using Russell venom viper and ecarin assay compared with high-performance liquid chromatography-tandem mass spectrometry as the reference standard. DISCUSSION: Viscoelastic point-of-care assessment of clotting time in whole blood might improve swift delivery of time-sensitive hyperacute treatment with IVT in stroke patients.

Development and validation of an analytical method for the determination of direct oral anticoagulants (DOAC) and the direct thrombin-inhibitor argatroban by HPLC-MS/MS.

Since direct oral anticoagulants (DOAC) are administered frequently to an elderly, co-morbid population, medical emergencies including trauma, acute bleeding or organ failure are not uncommon. In these situations, the type, dosage or the time of last intake of anticoagulants is often unknown and single substance analysis by functional tests is only possible if the substance contained in the sample is known. A reliable and validated toxicology screen of DOAC and argatroban would be helpful inform not only attending physicians in the emergency department but also law enforcement and courts of justice. After precipitation with acetone, HPLC separation was achieved on a Phenomenex Luna Pentafluorophenyl Colum using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis MRM Scans (MS/MS) with positive ionization were chosen. The method was validated for blank serum as the matrix of choice. Limits of detection are between 0.5 and 1.0 ng/mL, limits of quantification are between 1.9 and 3.6 ng/mL and recoveries are above 60%. The applicability of the method was demonstrated by the determination of DOAC in body fluids from forensic cases and in therapeutic drug monitoring. The rapid simultaneous detection and quantification of apixaban, argatroban, dabigatran etexilate, dabigatran, edoxaban and rivaroxaban in body fluids by HPLC-MS/MS closes an important gap in emergency toxicology.

Simultaneous determination of selected catechins and pyrogallol in deer intoxications by HPLC-MS/MS.

A selective and sensitive analytical method for the determination of selected catechins (catechin, epicatechin, gallocatechin, epigallocatechin) and pyrogallol in biological matrices by HPLC-MS/MS was developed. The utilized sample preparation technique was a two-stage liquid-liquid extraction using ethyl acetate. The HPLC-system was equipped with a Phenomenex Luna Pentafluorophenyl Column (150 × 2 mm, 5 µm) and operated with an acetonitrile-water gradient as a mobile phase system. Detection was performed with a 3200 Q Trap mass spectrometer. For analysis the mass spectrometer was used in the MRM-mode with negative ionization. The method validation was performed with serum as matrix. The selectivity of the method as well as the linearity of calibration was successfully proven for all analytes. The limits of quantification were between 5.3 and 11.2 ng/mL and the recovery rates were above 50 % for all analytes. Results from the samples of three deer poisoning cases demonstrated that the developed HPLC-MS/MS method is applicable to real biological samples.

Red Arils of Taxus baccata L.-A New Source of Valuable Fatty Acids and Nutrients.

The aim of this study, focused on the nutritional value of wild berries, was to determine the contents of macronutrients, profiles of fatty (FAs) and amino acids (AAs), and the contents of selected elements in red arils (RA) of Taxus baccata L., grown in diverse locations in Poland. Protein (1.79-3.80 g/100 g) and carbohydrate (18.43-19.30 g/100 g) contents of RAs were higher than in many cultivated berries. RAs proved to be a source of lipids (1.39-3.55 g/100 g). Ten out of 18 AAs detected in RAs, mostly branched-chain AAs, were essential AAs (EAAs). The EAAs/total AAs ratio approximating were found in animal foods. Lipids of RA contained seven PUFAs, including those from n-3 family (19.20-28.20 g/100 g FA). Polymethylene-interrupted FAs (PMI-FAs), pinolenic 18:3Δ5,9,12; sciadonic 20:3Δ5,11,14, and juniperonic 20:4Δ5,11,14,17, known as unique for seeds of gymnosperms, were found in RAs. RAs may represent a novel dietary source of valuable n-3 PUFAs and the unique PMI-FAs. The established composition of RAs suggests it to become a new source of functional foods, dietary supplements, and valuable ingredients. Because of the tendency to accumulate toxic metals, RAs may be regarded as a valuable indicator of environmental contamination. Thus, the levels of toxic trace elements (Al, Ni, Cd) have to be determined before collecting fruits from natural habitats.

Effects of regional anesthesia techniques on local anesthetic plasma levels and complications in carotid surgery: a randomized controlled pilot trial.

BACKGROUND: The ultrasound guided intermediate cervical plexus block with perivascular infiltration of the internal carotid artery (PVB) is a new technique for regional anesthesia in carotid endarterectomy (CEA). We conducted a pilot study investigating the effects of deep cervical block (DCB), intermediate cervical block alone (ICB) and PVB on perioperative complications in patients undergoing elective CEA. We hypothesized, that the ropivacaine plasma concentration is higher in patients receiving DCB compared to PVB and ICB. METHODS: In a randomized controlled pilot study thirty patients scheduled for elective CEA were randomly assigned into three groups: DCB receiving 20 mL ropivacaine 0.5% (n = 10), ICB receiving 20 mL ropivacaine 0.5% (n = 10) and PVB receiving 20 mL ropivacaine 0.5% and 10 mL ropivacaine 0,3% (n = 10). As primary outcome, plasma levels of ropivacaine were measured with high performance liquid chromatography before, 5, 10, 20, 60, and 180 min after the injection of ropivacaine. Secondary outcomes were vascular and neurological complications as well as patients' and surgeons' satisfaction. All analyses were performed on an intention-to-treat basis. Statistical significance was accepted at p < 0.05. RESULTS: No conversion to general anesthesia was necessary and we observed no signs of local anesthetic intoxication or accidental vascular puncture. Plasma concentration of ropivacaine was significantly higher in the DCB group compared to PVB and ICB (p < 0.001) and in the PVB group compared to ICB (p = 0.008). Surgeons' satisfaction was higher in the PVB group compared to ICB (p = 0.003) and patients' satisfaction was higher in the PVB group compared to ICB (p = 0.010) and DCB group (p = 0.029). Phrenic nerve paralysis was observed frequently in the DCB group (p < 0.05). None of these patients with hemi-diaphragmatic paralysis showed signs of respiratory distress. CONCLUSION: The ultrasound guided PVB is a safe and effective technique for CEA which is associated with lower plasma levels of local anesthetic than the standard DCB. Considering the low rate of complications in all types of regional anesthesia for CEA, larger randomized controlled trials are warranted to assess potential side effects among the blocks. TRIAL REGISTRATION: The trial was registered at German Clinical Trials Register (DRKS) on 04/05/2019 (DRKS00016705, retrospectively registered).

[Poisonous plants-a new approach for assessing the risk of poisoning in small children]. / Risiko Pflanze ­ Ein neuer Ansatz zur Einschätzung des Vergiftungsrisikos für Kleinkinder.

BACKGROUND: Plant poisoning in small children (from 0.5 to <6 years of age) is the third most frequent cause for phone contact with a poison center. For prevention of poisonings, a list of poisonous plants that should not be planted close to playgrounds or other places frequently visited by children was published in 2000 by the Bundesanzeiger. This list has been reevaluated and updated by the "Toxicity of Plants" working group of the Committee of the Assessment of Intoxications at the Federal Institute of Risk Assessment (BfR). MATERIALS AND METHODS: Relevant plants were taken from a recent publication. A literature search was conducted in PubMed concerning all plant poisonings in children and the toxic ingredients of plants. Also, monographs and the database POISINDEX were integrated in the evaluation. A classification was made for plants that after oral, dermal, or ocular contact of small quantities could cause severe, moderate, mild, or no intoxications in small children. RESULTS: Based on data of exposure and potentially toxic ingredients of the involved plants, a risk assessment was executed, which diverges from other publications because it concerns the actual basic risk of an intoxication. In total, 251 plants were reevaluated. For 11 plants, there was a high risk, for 32 a moderate, for 115 a mild, and for 93 plants no risk of intoxication could be determined. CONCLUSION: The new assessment of evaluating a toxicity risk for small children on the basis of exposure data and including the toxicity of ingredients allows for a more realistic assessment of the risk of poisoning with outdoor plants. In this way, infant exposure carrying a high risk of intoxication can be identified.

[Accidental intoxication by outdoor and garden plants : Data from two German poison centres]. / Akzidentelle Vergiftungen mit Gartenpflanzen und Pflanzen in der freien Natur : Daten aus zwei deutschen Giftinformationszentren.

BACKGROUND AND OBJECTIVES: Accidental exposure of children to plants occurs often and results in numerous calls to poison centres. The aim of this study was to identify outdoor plants that led to moderate or severe poisoning after accidental exposure and to identify patterns of paediatric plant exposures. MATERIALS AND METHODS: Human exposure data on accidental exposures provided by two German poison centres were retrospectively evaluated regarding the number and the routes of exposure. Special attention was turned to the kind and severity of symptoms. Based on these data a modified Litovitz factor was calculated. RESULTS: Out of 42,344 confirmed exposures to 227 plant species, 39,346 (93%) were asymptomatic, 2415 (5.7%) experienced minor, 580 (1.3%) moderate and 3 (0.007%) severe symptoms. Twenty-six plant genera were responsible for 70% of all exposures. Only eight of these plants (Arum spec., Laburnum anagyroides, Narcissus spec., Phaseolus vulgaris/coccineus, Prunus laurocerasus, Sambucus spec., Taxus baccata, Thuja spec.) led to at least moderate symptoms. Accidental exposure of children aged 0.5-5 years was mainly by oral ingestion (98%) and involved mostly fruits (60%). CONCLUSIONS: Exposure data collected by poison centres are very useful for hazard identification of outdoor plants. The data give a comprehensive overview of observed symptoms, which offers valuable instruments for use in clinical practice.

Grayanotoxin I Intoxication in Pet Pigs.

Contaminated honey is a common cause of grayanotoxin intoxication in humans. Intoxication of animals, especially cattle, is usually due to ingestion of plants of the Ericaceae family, such as Rhododendron. Here, we report the ingestion of Pieris japonica as the cause of grayanotoxin I intoxication in 2 miniature pigs that were kept as pets. The pigs showed sudden onset of pale oral mucosa, tachycardia, tachypnea, hypersalivation, tremor, and ataxia that progressed to lateral recumbency. The pathological examination of one pig revealed no specific indications for intoxication except for the finding of plant material of Pieris japonica in the intestine. Grayanotoxin I was identified in the ingested plant, gastric content, blood, liver, bile, kidney, urine, lung, and skeletal muscle via HPLC-MS/MS. Grayanotoxin I should be considered as a differential etiological diagnosis in pigs with unspecific signs and discovery of ingested plant material as the only indication in the pathologic examination.

Taxus ingredients in the red arils of Taxus baccata L. determined by HPLC-MS/MS.

INTRODUCTION: Taxus baccata L. is an evergreen conifer whose plant parts are cardiotoxic. Only the red arils of the berries are described as non-toxic and taxane-free. OBJECTIVE: Extraction and HPLC-MS/MS methods were developed for the investigation of the Taxus compounds 3,5-dimethoxyphenol, 10-deacetylbaccatin III, baccatin III, cephalomannine, taxol A and taxinine M in the red arils of the yew berries. METHODOLOGY: A liquid-liquid extraction method for the red arils of the fruits from three yews were developed. An accurate (ESI+) HPLC-MS/MS method was performed for the simultaneous detection and determination of the target compounds in multiple reaction monitoring (MRM) mode. RESULTS: All Taxus agents obtained were detected in the red arils. Highest concentrations were determined for baccatin III and 10-deacetylbaccatin III. CONCLUSION: The developed quantitative method is reliable and selective and was successfully applied for quantification of selected Taxus ingredients in red arils of Taxus baccata. It was disproved that the red arils of the berries do not contain the selected Taxus compounds.

Simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC-MS.

A selective and sensitive analytical method for the simultaneous determination of cucurbitacin B, E, I and E-glucoside in plant material and body fluids by HPLC-MS was developed. After liquid-liquid extraction with dichlormethane, separation was achieved on a Phenomenex Luna Pentafluorophenyl Column (150mm×2mm, 5µm) using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis Q1 Scans with negative ionisation were chosen. The method was validated for serum as the matrix of choice. Limits of detection are in the picogram range, limits of quantification are between 0.05 and 0.42ng/mL, recoveries are above 50%. The assay was linear in the calibration range from 1.0 to 50ng/mL for cucurbitacin E and from 0.10 to 50ng/mL for the cucurbitacins B, I and E-glucoside. The applicability of the method was demonstrated by the determination of cucurbitacins in zucchini plant material and body fluids from intoxication cases.

Investigations into distribution of lidocaine in human autopsy material.

With screening methods in the legal medicine drugs were often detected in autopsy material. In this study the antiarrhythmic and the local anesthetic drug lidocaine could be proved in fifty-one cases and determined in different autopsy materials. For the first time the comparison of so many distribution patterns of lidocaine in human compartments was possible. A liquid-liquid extraction procedure, a standard addition method and LC/MS/MS were used for analytics. The measured concentrations in blood were in the therapeutic range or lower. The time between lidocaine application and death was given in twenty-nine cases. These data were very helpful to estimate and interpret the distribution process of lidocaine between application and death. This time exerted a crucial influence on the distribution of lidocaine in the compartments. Most of the intravenous applicated lidocaine was found in heart blood after a very short time of distribution. Afterwards the highest concentrations were measured in brain. Later the highest concentration was found in the kidney samples or in urine. If the time between lidocaine application and death is known, the results of this study can be used to deepen the knowledge of its pharmacokinetics. If this time is unknown, the circumstances and the causes of death can be better explained.

Illegal drug-related deaths in East Germany between 1995 and 2004.

The retrospective analysis determines changes between the deaths caused by illegal drugs in East Germany between 1995 and 2004 with specific regard to the number and manner of deaths, common intoxicants, concomitant drug use, the age and gender of the victims as well as the places of death. The data was collected by the institutes of forensic medicine in the German federal states of Saxony, Thuringia, Brandenburg, Mecklenburg West-Pomerania, Saxony-Anhalt and the State Offices of Criminal Investigation of these federal states. A comparison of these two different sources of data is also made. 510 drug-related deaths occurred in East Germany between 1995 and 2004. This was equivalent to a death rate of 0.4 per 100,000 inhabitants and represented 3% of all drug-related deaths throughout Germany. The average age of the victims was 24 years and male accounted for 85% of all the fatalities. Opiates, especially heroin, caused the majority of deaths (55%). Comparison of the two sources revealed that approximately half of the drug-related deaths were accounted for by national statistics. The analysis reveals an increase of drug-related deaths in East Germany after reunification but no relevant difference between the five East German states.

Theophylline induces changes in the electro-olfactogram of the mouse.

Options for the treatment of hyposmia are limited;available therapies do not provide a long-lasting effect.A recent study suggests that an unspecific phosphodiesterase inhibitor (PDE-I) increases olfactory sensitivity due to interaction with the signal transduction in the olfactory epithelium. The aim of the present study was to investigate whether theophylline, an unspecific PDE-I, evokes changes in the electro-olfactogram (EOG) which would support the hypothesis of a drug-related impact on signal transduction.In addition, the uptake of topically administered theophylline in the olfactory epithelium should be investigated. EOG was obtained in 29 samples of supravital mouse olfactory epithelia. Olfactory stimulation (phenylethyl alcohol, PEA and hydrogen sulfide, H2S) was performed using an air dilution olfactometer. Theophylline concentration in the olfactory epithelium of five samples was measured by means of high pressure liquid chromatography. Administration of theophylline resulted in a tendency towards smaller EOG amplitudes (p = 0.055), being reduced by 13 and 25% in response to stimulation with PEA or H2S,respectively. In comparison to the application of Ringer's solution, theophylline resulted in a significant (p = 0.031)decrease of the EOG amplitude. Latency was not significantly(p = 0.10) influenced by drug administration. The theophylline concentration in the olfactory epithelium ranged from 0.21 to 1.53 microg/mg. Theophylline seems to be taken up into the olfactory epithelium of supravital mice and to interact with the olfactory signal transduction.

Simultaneous determination of three anticonvulsants using hydrophilic interaction LC-MS.

A specific and automated method was developed to quantify the anticonvulsants gabapentin, pregabalin and vigabatrin simultaneously in human serum. Samples were prepared with a protein precipitation. The hydrophilic interaction chromatography (HILIC) with a mobile phase gradient was used to divide off ions of the matrix and for separation of the analytes. Four different HILIC-columns and two different column temperatures were tested. The Tosoh-Amid column gave the best results: single small peaks. The anticonvulsants were detected in the multiple reaction monitoring mode (MRM) with ESI-MS-MS. Using a volume of 100 microL biological sample the lowest point of the standard curve, i.e. the lower LOQs were 312 ng/mL. The described HILIC-MS-MS method is suitable for therapeutic drug monitoring and for clinical and pharmcokinetical investigations of the anticonvulsives.

Pediatric plant exposures in Germany, 1998-2004.

INTRODUCTION: Each year, 80,000 to 100,000 calls to the Poison Information Centres (PIC) concern pediatric exposures in Germany. Plant exposures are the fourth most common category, accounting for 22% of pediatric exposures. METHODS: Information on plant exposures in children (0-14 years) was collected from annual reports of German PIC. The severity of pediatric plant exposures was classified using the number of ingestions and a calculated hazard factor. RESULTS: A total of 58,641 cases involving 248 different plant genera were reported from 1998 to 2004. Most plant exposures were not associated with clinical effects at time of call, but 9.6% of cases had noticeable effects, including 0.4% classified as moderate and major effect. CONCLUSIONS: The majority of plant genera have low hazard factors. Most severe poisoning (highest hazard factors and exposures) in children involved Brugmansia, Laburnum, Phaseolus, and Thuja.

Simultaneous determination of thirteen plant alkaloids in a human specimen by SPE and HPLC.

A new screening method for the simultaneous determination of thirteen plant alkaloids (aconitine, anabasine, atropine, brucine, colchicine, cotinine, cytisine, harmine, ibogaine, nicotine, scopolamine, strychnine, yohimbine) in a human specimen was developed based on solid-phase extraction and reversed-phase liquid chromatography with photodiode array detection. The validated method enables selective identification as well as accurate and sensitive quantification. The analysis of forensic and clinical samples emphasizes the applicability for intoxications and drug abuse, as well as for compliance control.

Production of drinking water from raw water containing cyanobacteria--pilot plant studies for assessing the risk of microcystin breakthrough.

Toxins in cyanobacteria are a recognized risk in the treatment of drinking-water treatment. Cyanotoxins can occur in two modifications: cell bound and dissolved in water. The process of toxin release may occur naturally, but it also may be induced through the processes of drinking-water treatment. Both causes of release are relevant to the safety of drinking water. This study investigated cyanotoxin release and elimination through different treatment trains in systematic pilot-scale studies with water from the Weida Reservoir, in Thuringia, Germany. The Weida Reservoir is a dimictic mesoeutrophic reservoir typical for a number of mountainous areas in Europe, with Planktothrix rubescens as the dominant phytoplankton species, and shows a characteristic seasonal pattern of population development and microcystin occurrence. To assess the risk of microcystin breakthrough, the pilot-scale results as well as results of laboratory-scale experiments were used for developing a kinetic model of toxin release in relation to elimination. By calculating removal efficiency of total microcystins (cell bound and dissolved) for different treatment trains, raw water quality was related to the quality targets for finished water, and breakthrough risks could be calculated for given treatment trains and varying cyanobacterial population densities in the reservoir.

Highly active palladium/activated carbon catalysts for Heck reactions: correlation of activity, catalyst properties, and Pd leaching.

A variety of palladium on activated carbon catalysts differing in Pd dispersion, Pd distribution, Pd oxidation state, and water content were tested in Heck reactions of aryl bromides with olefins. The optimization of the catalyst (structure-activity relationship) and reaction conditions (temperature, solvent, base, and Pd loading) allowed Pd/C catalysts with very high activity for Heck reactions of unactivated bromobenzene (turnover number (TON) approximately 18000, turnover frequency (TOF) up to 9000, Pd concentrations down to 0.005 mol %) to be developed. High Pd dispersion, low degree of reduction, sufficient content of water, and uniform Pd impregnation are criteria for the most active system. The catalysts combine high activity and selectivity under ambient conditions (air and moisture), easy separation (filtration), and quantitative recovery of palladium. Determination of Pd in solution after and during the reaction, and catalyst characterization before and after the reaction (transmission electron microscopy (TEM), X-ray diffraction (XRD)), indicate dissolution/reprecipitation of palladium during the reaction. The Pd concentration in solution is highest at the beginning of the reaction and is a minimum (< 1 ppm) at the end of the reaction. Palladium leaching correlates significantly with the reaction parameters.