Application of UHPLC-QqQ-MS/MS Method for Quantification of Beta-Adrenergic Blocking Agents (ß-Blockers) in Human Postmortem Specimens.

Betablockers are one of the most frequently used medications in cardiology. They can lead to fatal drops in blood pressure and heart rhythm disturbances. Death is functional, and poisoning with this group of drugs can be difficult to detect. The liquid-liquid extraction (LLE) method developed using ethyl acetate at pH 9 successfully identified 18 ß-blockers in human blood. The method's limit of quantification (LOQ) was in the range of 0.1 to 0.5 ng/mL. No carryover of substances between samples was detected, and no interfering ion current signals were observed in the biological samples at the retention times of the compounds or internal standards. All compounds had a coefficient of determination (R2) above 0.995. Intraday and interday precision (RSD%) and accuracy (RE%) for low and high QC levels were within 1.7-12.3% and -14.4 to 14.1%, respectively. Very good recovery (80.0-119.6%) and matrix effect (±20.0%) values were achieved for all compounds. In addition, fragmentation spectra were collected for all the examined substances, and high-resolution spectra were presented for landiolol and metipranolol, because they are not available in commercial HRMS spectra databases. The developed method was applied in authentic postmortem samples.

The stability of cyanide in human biological samples. A systematic review, meta-analysis and determination of cyanide (GC-QqQ-MS/MS) in an authentic casework 7 years after fatal intoxication.

A 30 year old man was found with no signs of life in front of the house. The cyanide concentration in blood and urine was determined five years after the man's death. What is more, a stability study was conducted for 730 days in an authentic casework blood sample. Sample preparation procedure included precipitation with methanol:water mixture, solid phase extraction (SPE) and derivatization with the use of PFB-Br (pentafluorobenzyl bromide). The sample was analyzed using GC-QqQ-MS/MS (gas chromatopraphy coupled with tandem mass spectrometry) isotope dilution method. Separation was done using a SH-RXI-5MS column (30 m x 0.25 mm, 0.25 µm). Detection of PFB-CN and PFB-13CN was achieved using a triple-quadrupole mass spectrometer with an electron ionization (EI) ion source in multiple reaction monitoring (MRM) mode. After 5 years from the man's death, cyanide concentration was: 1900 ng/mL in blood and 500 ng/mL in urine. Stability study performed in an authentic blood sample 6 and 7 years after the man's death revealed cyanide concentrations of 1898.2 ng/mL and 1618.7 ng/mL, respectively. While spectrophotometric and colorimetric methods recorded both decrease and increase in cyanide concentration over time, newer chromatographic methods mainly indicate a decrease. The studies presented in this paper seem to confirm this trend. However, in order to interpretate the results of cyanide concentration in biological material reliably, more research is still necessary.

Novel Technique for Simultaneous Ethylene Glycol and Its Metabolites Determination in Human Whole Blood and Urine Samples Using GC-QqQ-MS/MS.

Toxicological analyses often necessitate the identification of compounds belonging to diverse functional groups. For GC-MS analyses, derivatization of compounds belonging to different functional groups can pose a challenge and requires the development of comprehensive methods of analysis. One example could be ethylene glycol, whose widespread use is related to possible unintentional or suicidal intoxications. This fact clearly indicates the need to develop sensitive methods for the determination of ethylene glycol and its metabolites in biological material, as only such complex analysis allows for proper toxicological expertise. A simultaneous GC-QqQ-MS/MS method for the determination of ethylene glycol together with its metabolites, glyoxal and glycolic acid, as well as the detection of glyoxylic acid and oxalic acid, was developed and fully validated. A novel approach for simultaneous derivatization of substances from different groups (alcohols, aldehydes, and carboxylic acids) was established. Sample preparation included the addition of three internal standards (BHB-d4, ethylene glycol-d4 and methylglyoxal), precipitation with acetonitrile and subsequent derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), as well as pentafluorophenylhydrazine (PFPH). Detection was carried out with the use of triple quadrupole mass spectrometer. The ionization method was electron impact, and quantitative analysis was carried out in multiple reaction monitoring mode. The lower limit of quantification was 1 µg/mL, 0.1 µg/mL, and 500 µg/mL for ethylene glycol, glyoxal, and glycolic acid, respectively. The presented method was applied in three authentic postmortem cases of ethylene glycol intoxication.

Subinhibitory concentrations of antibiotics affect development and parameters of Helicobacter pylori biofilm.

Introduction: Helicobacter pylori causes chronic gastric diseases in nearly 50% of people around the world. It is suggested that biofilm formation has a pronounced effect on the dynamic resistance spread and recurrence of these infections. Methods: To mimic the scenario of therapeutic ineffectiveness, we investigated the impact of sub-minimal inhibitory concentrations (sub-MICs) of antibiotics on the development and parameters of biofilms produced by clinical H. pylori strains. Results: We observed that constant exposure of planktonic forms to metronidazole or levofloxacin stimulated the speed of autoaggregation and the amount of extracellular matrix, resulting in increased dimensions of the developed biofilms. Contrary to this, continuous exposure to clarithromycin negatively affected a number of biofilm-related reactions and led to the biofilm-weakening effect. Through assessing the membrane fatty acid profiles of antibiotic-exposed cells, we confirmed that metronidazole and levofloxacin induced a biofilm-like phenotype, while clarithromycin kept bacteria in a planktonic form. Discussion: Our results suggest that sub-MICs of antibiotics affect the biochemical and biophysical properties of the developing biofilm of H. pylori strains and may impact the effectiveness of antibiotic treatment.

New approach for barbiturates, phenytoin, methyprylon and glutethimide determination and fragmentation (UHPLC-MS/MS).

Barbiturates which are old pharmaceutical drugs are still widely used in medical treatment of epilepsy and for general anesthesia. To date, more than 2500 different barbituric acid analogs have been synthesized, and 50 of them were introduced into medical use over the last century. Due to their highly addictive properties, pharmaceuticals containing barbiturates are under strict control in many countries. However, by considering the worldwide problem with new psychoactive substances (NPS) the introduction of new designer barbiturate analogs into the dark market might serve a serious public health problem in the near future. For this reason there is an increasing need for application methods for barbiturates monitoring in biological samples. The UHPLC-QqQ-MS/MS method for determination of 15 barbiturates, phenytoin, methyprylon and glutethimide was developed and fully validated. The biological sample volume was reduced to only 50 µL. A simple LLE (pH 3 with ethyl acetate) was successfully applied. The lower LOQ was 10 ng/mL. The method enables differentiation of structural isomers: hexobarbital and cyclobarbital; as well as amobarbital and pentobarbital. Chromatographic separation was achieved with the use of the alkaline mobile phase (pH 9) and Acquity UPLC BEH C18 column. Furthermore, the novel fragmentation mechanism of barbiturates was proposed, which may have a great impact in identification of novel barbiturates analogs introduced to illegal marketplaces. The presented technique has a great potential to be applied in forensic, clinical and veterinary toxicological laboratories, as was evidenced by the positive results of international proficiency tests.

Nitrites: An Old Poison or a Current Hazard? Epidemiology of Intoxications Covering the Last 100 Years and Evaluation of Analytical Methods.

In recent times, there has been a concerning and noteworthy rise in the global use of sodium nitrite for suicidal purposes. This is facilitated either through the employment of specialized "suicide kits" or by acquiring sodium nitrite through alternative means. Additionally, another occurrence contributing to nitrite poisoning is the recreational utilization of nitrites in the form of volatile aliphatic esters of nitrous acid, commonly referred to as "poppers". Based on current available papers and reports on the subject of nitrates, nitrites, and poppers intoxications, an epidemiological analysis and evaluation of analytical methods were performed. A total of 128 papers, documenting a collective count of 492 intoxication cases, were identified. Additionally, in order to complete the epidemiological profile of nitrite poisoning, the authors briefly examined six cases of nitrite intoxication that were under investigation in our laboratory. Furthermore, a review of nitrite poisoning cases over the past 100 years shows that the old poison is still in use and poses a substantial risk to society.

A fatal case involving the highest ever reported 4-CMC concentration.

Synthetic cathinones comprise a large amount of substances present on the dark market, which creates an undeniably worldwide problem and still is posing a threat. A 22-year-old man was brought to the Emergency Room from a party, where he had ingested orally 20 g of mephedrone. The man exhibited a disorder of consciousness with no logical verbal contact and dilated pupils. Moreover, a metabolic acidosis was present. The patient died after an hour from an admission to the ER. Blood and vitreous humor collected during an autopsy were analyzed with the use of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-QqQ-MS/MS) with the use of C18 column in multiple reaction monitoring (MRM) mode. Both biological specimens were prepared using liquid-liquid extraction (LLE) with the use of ethyl acetate and 0.5 M ammonium carbonate water solution (pH 9). The limit of quantification (LOQ) of the method was 0.5 ng/ml in both matrices; precision and accuracy values did not exceed ±15%. Recovery of the method was in the range of 86.1%-102.7%. Determined concentrations of 4-CMC were 8542 and 9874 ng/ml in blood and vitreous humor, respectively. Other substances present in both biological materials were: atropine, diazepam, lidocaine, and its metabolite norlidocaine, as well as methcathinone and ethyl alcohol. The concentration presented in here described case is the highest ever reported 4-CMC concentration. Important aspect is also receiving other NPS by recreational users than intended, which lead to accidental poisoning (in presented case user assumed 4-CMC was 4-MMC).

Methyl (S)-2-(1-7 (5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-MDMB-PICA) intoxication in a child with identification of two new metabolites (ultra-high-performance liquid chromatography-tandem mass spectrometry).

PURPOSE: Methyl (S)-2-(1-7 (5-fluoropentyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (5F-MDMA-PICA) intoxication in 1.5-year-old child was presented, together with diagnostic parameters discussion and 5F-MDMB-PICA determination in biological material. Furthermore, 5F-MDMB-PICA metabolites were identified in a urine sample as markers of exposure in situation when a parent compound is not present in specimens. METHODS: Drugs and metabolites were extracted from serum and urine with ethyl acetate both under alkaline (pH 9) and acidic (pH 3) conditions. Hair, after decontamination and pulverization, were incubated with methanol (16 h, 60 °C). The analysis was carried out using ultra-high-performance liquid chromatography-tandem mass spectrometry. For the identification of 5F-MDMB-PICA metabolites, an urine sample was precipitated with cold acetonitrile. Analysis was performed using ultra-high-performance liquid chromatograph with quadrupole time-of-flight mass spectrometer. RESULTS: 5F-MDMB-PICA was determined only in serum sample at concentration of 298 ng/mL. After 1 year, when analysis was repeated, concentration of synthetic cannabinoid in the same sample was only 17.6 ng/mL which revealed high instability of 5F-MDMB-PICA in serum sample. Eight 5F-MDMB-PICA metabolites were identified in urine sample, including two potentially new ones with m/z 391.18964 and m/z 275.14016. CONCLUSIONS: Toxicological analysis confirmed a 1.5-year-old boy intoxication with 5F-MDMB-PICA. Besides the parent drug, metabolites of 5F-MDMB-PICA were identified, including two potentially new ones, together with possible metabolic reactions which they resulted from. Metabolites determination could serve as a marker of 5F-MDMB-PICA exposure when no parent drug is present in biological material.

An ultra-sensitive UHPLC-QqQ-MS/MS method for determination of 54 benzodiazepines (pharmaceutical drugs, NPS and metabolites) and z-drugs in biological samples.

Benzodiazepines exhibit central nervous system depressive activity as well as sedative, hypnotic, and anticonvulsant properties, which enable to use them as medical treatment in anxiety, epilepsy, insomnia and alcohol withdrawal syndrome. However, from 2000s illegal benzodiazepine derivatives have started to emerge on illicit drug market as new psychoactive substances (NPSs) monitored in many countries. Analysis of both pharmaceutical drugs and NPSs from benzodiazepines group could be challenging, as usually very low concentrations need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of 54 benzodiazepines (pharmaceutical drugs, NPS and their metabolites) and 3 z-drugs with one metabolite in biological fluid samples. The lower limit of quantification for most substances was 50 pg/mL, whereas for 17 substances as low as 10 pg/mL was achieved. Together with reduced sample volume to 100 µL it makes the developed method suitable for a sensitive multidrug toxicological analysis. Presented method was applied in routine toxicological practice as well as for the determination of benzodiazepines, z-drugs and their metabolites in 25 authentic biological fluids (blood, urine, vitreous humor and bile), both antemortem and postmortem. 19 different compounds, including benzodiazepines, their metabolites and z-drugs were determined. Antemortem blood concentrations were within 0.2-114.5 ng/mL, whereas concentrations in antemortem urine samples were 0.03-102.6 ng/mL. In postmortem specimens, concentrations ranged within 0.2-473.2 ng/mL, 0.5-94.1 ng/mL, 1.3-208.8 ng/mL and 41.5-42.0 ng/mL in blood, vitreous humor, urine and bile, respectively. The developed method is suitable for a forensic toxicology analysis, as well as clinical toxicology which is evidenced by the positive results of international proficiency tests.

Severe poisoning after smoking a mixture of 4-fluoroisobutyryl fentanyl (4-FiBF) and alpha-pyrolidinoisohexaphenone (α-PiHP).

CONTEXT: Intoxications after ingestion of new psychoactive substances are currently one of the most challenging issues in clinical toxicology. Synthetic cathinones represented the largest group of drugs seized in 2020, but the increasing distribution of fentanyl analogues is resulting in a growing global opioid crisis. In addition, synthetic opioids may be intentionally combined with psychostimulants by drug manufacturers to reduce depressive effects. We report a case of severe poisoning after smoking a mixture of 4-fluoroisobutyryl fentanyl (4-FiBF) and alpha-pyrrolidinoisohexaphenone (α-PiHP). CASE DETAILS: A 29-year-old male was found out of conscious in his apartment and taken to the Intensive Care Unit. Examinations revealed pinpoint pupils, slight respiratory acidosis, leukocytosis as well as body temperature of 39.4 °C and increased creatinine with decreased eGFR level. Toxicological analysis of biological samples revealed presence of 4-FiBF and α-PiHP in concentrations: 87.7 ng/mL and 5.0 ng/mL (blood) and 2291.0 ng/mL and 722.2 ng/mL (urine), respectively. After 4 days, the patient was discharged home. DISCUSSION: Unique combination of clinical symptoms was a result of a simultaneous 4-FiBF and α-PiHP intoxication. To our knowledge, this is the first case of ingestion such unusual mixture of new psychoactive substances with a full description of medical treatment.

A Case of Amphetamine and Methamphetamine Intoxication in Cat.

Stimulants belonging to the amphetamine group nowadays pose an undeniable worldwide threat to the life and health of users. Intoxications of domestic animals also occur, which can either be accidental or related to intentional human action. This study presents the first ever reported case of a simultaneous amphetamine and methamphetamine intoxication of a cat, along with the results of toxicological studies. Blood, urine, vitreous humor and liver were collected during the cat's autopsy and analyzed by UHPLC─QqQ─MS/MS. The sample preparation technique was based on one-step precipitation of proteins with cold acetonitrile. The determined amphetamine concentrations in the collected biological materials were 93.4 ng/mL in blood, 496.6 ng/mL in urine, 589.2 ng/mL in the vitreous humor and 291.2 ng/g in liver, respectively. Methamphetamine concentrations were 45.5 ng/mL in blood, 263.1 ng/mL in urine, 351.2 ng/mL in vitreous humor, and 97.7 ng/g in liver. Other substances were also found in the biological material, i.e., diazepam, oxazepam and nordiazepam. Cases of intentional or accidental poisoning of pets with psychoactive substances are a serious problem, carrying the risk to the health and life of the animal. Therefore, it is important to increase awareness of the high risk of poisoning of domestic animals, as well as to learn about the incompletely understood mechanisms of pharmacokinetics of various drugs in animals, including cats.

The dark side of social media: Two deaths related with chloroform intoxication.

A suicide pact is an agreement between people to commit suicide together, which usually takes place at the same time, in the same place, by using the same method. Social media serve as a way of communication between people. Thus, they use such platforms to find potential suicide pact partners. Chloroform, although being regarded to as a slightly forgotten poison, is still linked to homicide and suicide cases. Death due to an acute chloroform ingestion may be a result of central nervous system depression. In this paper, we present application of headspace gas chromatographic method using a dual column/dual flame ionization detector (HS-GC-FID/FID) for the determination of chloroform in two fatal intoxication cases, as well as chloroform stability study. Analysis of biological samples revealed chloroform concentrations of 135.8, 16.1, 8.1, and 37.1 µg/ml in blood, urine, vitreous humor, and bile, respectively. Kidney, liver, and muscle specimens contained 119.5, 99.6, and 28.4 µg/g of chloroform, respectively. The results of stability studies indicate the highest decrease of chloroform in room temperature, so it is advised to store samples in a freezer. The addition of sodium fluoride is recommended as in blood samples collected to the test tubes without any preservative agent, the detection of chloroform after 91 days is almost impossible. It is important to emphasize that even old poisons can cause a lot of concerns today, as here described cases are linked to chloroform intoxication, as well as with possible danger which social media bring about nowadays.

Simultaneous poisoning of 48 birds of prey - bendiocarb determination with the use of UHPLC-ESI-MS/MS method in fatal case from Eastern Europe.

Aim: Bendiocarb is used against a wide range of insects but has already been withdrawn from the market in some countries. It poses a high risk to birds as they can accidentally ingest it while searching for food, followed by toxic effects. This paper presents the results of toxicological and histopathological studies of 48 cases of intentional birds of prey poisoning with bendiocarb in Eastern Europe, specifically Poland. Material and methods: A novel ultra-high-performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for bendiocarb determination in animal liver samples was developed and fully validated. The sample preparation technique was based on one-step precipitation of proteins with cold acetonitrile. The internal standard used was carbaryl-d7. Full time of analysis was less than 10 minutes. The application of the UHPLC-ESI-MS/MS method allowed us to achieve the lowest LOQ (1 ng/g) of bendiocarb in biological samples to date. Results: Necropsies and histopathological examinations of common ravens (Corvus corax), western marsh harriers (Circus aeruginosus), red kites (Milvus milvus), and a white-tailed eagle (Haliaeetus albicilla) revealed multi-organ toxicity manifested as congestion, oedema, or stagnation of blood. An analytical investigation confirmed the presence of bendiocarb in liver in the 1808-7721 ng/g range. Furthermore, the presence of this compound was qualitatively confirmed in the stomach and beak contents and also in the bait located near the deceased animals. Conclusions: A comprehensive forensic examination is crucial to monitor wildlife fatalities, especially applying a combined analytical and histopathological approach to identify and eliminate highly toxic substances which pose a threat to the ecosystem.