Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high-resolution mass spectrometry detection.

Despite prevention efforts, many cases of mushroom poisoning are reported around the world every year. Among the different toxins implicated in these poisonings, muscarine may induce parasympathetic neurological damage. Muscarine poisonings are poorly reported in the current literature, implying a lack of available data on muscarine concentrations in human matrices. A validated liquid chromatography with high-resolution mass spectrometry detection (Orbitrap technology) method was developed to determine muscarine concentrations in human urine, plasma, and whole blood samples. Muscarine was determined using 100 µL of biological fluids, and precipitation was used for sample preparation. Liquid chromatography-mass spectrometry was performed using an Accucore Phenyl-X analytical column with the electrospray source in positive ion mode. Muscarine was quantitated in parallel reaction monitoring (PRM) mode with D9-muscarine as the internal standard. The method was validated successfully over the concentration range 0.1-100 µg/L for plasma and whole blood and 1-100 µg/L for urine, with acceptable precision and accuracy (<13.5%), including the lower limit of quantification. Ten real cases of suspected muscarine poisoning were successfully confirmed with this validated method. Muscarine concentrations in these cases ranged from 0.12 to 14 µg/L in whole blood,

Assessment of 19 years of a prospective national survey on drug-facilitated crimes in France.

Drug facilitated-crime or chemical submission (DFC/CS) is defined as the concealed or forced administration of psychoactive substances to a victim for criminal purposes. This is a national program set up in the early 2000 s in the form of a prospective multicenter survey, the results of which this manuscript presents. Over this 19-year period, 5487 cases were collected, analyzed and classified into 54 % of suspected cases, 29 % of chemical vulnerability (CV) cases and 17 % of proven DFC/CS cases. In the overall data, the most prevalent victims were female (81 %), with an average age of 27 years. Sexual assault was the most frequent aggression (77 %), followed by theft (14 %). Victims of proven DFC/CS cases were from of all ages including children and elderly. In 934 victims of DFC/CS, 100 various psychoactive substances were detected mostly represented by benzodiazepines and z-drugs (55 %), various sedatives including antihistamines (16 %) and non-therapeutic substances (16 %). Gamma-hydroxybutyric acid (GHB) was found in 4 % cases. In CV cases, alcohol (90 %) and cannabis (32 %) intake were mainly involved. In France, despite prevention messages, DFC/CS has been an epidemic for many years and has been proven by our national study. This national program has the aim to identifying the substances used but unfortunately not the goal to fight against this phenomenon. Since 2009, we observed a new modus operandi of the aggressors who pose as taxi drivers facilitating the reception of the victims leaving nightclubs. We can emphasize that GHB is not the "date rape drug" but rather the benzodiazepine class is.

Determination of Orellanine in Human Biological Matrices Using Liquid Chromatography with High-Resolution Mass Spectrometry Detection: A Validated Method Applied to Suspected Poisoning Cases.

Consumption of mushrooms can become unsafe for the consumer in case of confusion. Some fungi of Cortinarius genus contain the nephrotoxic mycotoxin orellanine responsible for their toxicity. Related case poisoning diagnosis is a challenge for both clinicians and analysts because of a long latency period between intake and toxic syndrome, the lack of available information in literature and the numerous pitfalls of orellanine identification/quantification in biological samples. In this situation, we propose an analytical method designed for the orellanine detection and/or quantification in biological matrices such as plasma, urine and whole blood, in a context of related intoxication suspected case. Using 1 mL biological sample volume, this liquid chromatographic with high-resolution mass spectrometry detection method (i) exhibits a limit of quantification for orellanine of 0.5 µg/L in plasma and urine and (ii) enables orellanine detection in whole blood with a limit of detection of 0.5 µg/L. This validated analytical method was successfully applied to 10 suspected intoxication cases.

Human Poisoning from Poisonous Higher Fungi: Focus on Analytical Toxicology and Case Reports in Forensic Toxicology.

Several families of higher fungi contain mycotoxins that cause serious or even fatal poisoning when consumed by humans. The aim of this review is to inventory, from an analytical point of view, poisoning cases linked with certain significantly toxic mycotoxins: orellanine, α- and ß-amanitin, muscarine, ibotenic acid and muscimol, and gyromitrin. Clinicians are calling for the cases to be documented by toxicological analysis. This document is therefore a review of poisoning cases involving these mycotoxins reported in the literature and carries out an inventory of the analytical techniques available for their identification and quantification. It seems indeed that these poisonings are only rarely documented by toxicological analysis, due mainly to a lack of analytical methods in biological matrices. There are many reasons for this issue: the numerous varieties of mushroom involved, mycotoxins with different chemical structures, a lack of knowledge about distribution and metabolism. To sum up, we are faced with (i) obstacles to the documentation and interpretation of fatal (or non-fatal) poisoning cases and (ii) a real need for analytical methods of identifying and quantifying these mycotoxins (and their metabolites) in biological matrices.

Case report: relevance of metabolite identification to detect new synthetic opioid intoxications illustrated by U-47700.

Today, new psychoactive substances (NPS) producers increasingly appear to be targeting new synthetic opioids (NSOs), and the recent emergence of NSOs is causing considerable concern in North America and in Europe. For toxicologists, NSO detection in a forensic context presents three additional difficulties to the general NPS analytical detection challenge: (i) high frequency of new products, (ii) low concentrations (in µg/L range and under) in biological samples related to their high opioid potency, and (iii) extensive metabolism. In this context, the present work aims to highlight the relevance of NSO metabolite detection in potential intoxication cases. Illustration is given with U-47700, an emerging NSO, (i) that was identified in a powder recently collected in France and in a fatality case, (ii) whose metabolites were in vitro produced using human liver microsomes and their mass spectra (MS) added in our MS/MS and HRMS libraries, and (iii) for which metabolism data were compared to those of the literature: U-47700 was identified in the powder and at 3040 µg/L in peripheral blood in the fatality case. In addition, high amounts of several U-47700 metabolites, especially N-desmethyl-U-47700, were observed in urine. Even if metabolite formation may largely depend on the enzymatic activity as well as on the length of the survival time, confrontation of these results to data found in the literature strongly suggests that this metabolite is regularly a better blood and (mainly) urine biomarker of U-47700 intake than U-47700 itself. Indeed, in this fatality and in other previous reports, N-desmethyl-U-47700 produced the main observed chromatographic signal (i) systematically in vitro and (ii) commonly in vivo, especially in urines. N,N-Didesmethyl-U-47700 is also sometimes a better biomarker of U-47700 intake than U-47700 itself. Accordingly, we suggest adding N-desmethyl-U-47700 (and N,N-didesmethyl-U-47700) in mass spectrum databases used for toxicological screening in order to reduce the risk of false-negative results in intoxication cases involving U-47700.

Suicidal shot in the mouth with rubber bullets.

Rubber bullets weapons can have tragic consequences when used at short range and on vulnerable body areas. A man in his forties was found deceased at his house, covered in blood. A "non-lethal" firearm was found near the bed: the «SAPL GC27¼. This is a single shot handgun with mini Gomm Cogne ammunition: 12 rubber spherical pellets, 7.4 mm in diameter. The findings were consistent with an intra buccal shot, and an ingestion-inhalation of blood and projectiles. Cause of the death was linked to both the hemorrhage due to mouth and aero digestive crossroad trauma, responsible of a mechanical asphyxia, and blood absorption in lungs. This case insists on the deadly potential of this weapon if misused. Fortunately, the term "non-lethal" has been progressively replaced, but sometimes still can be seen, especially in gunsmith and webstores. It should be deleted to avoid confusion among inexperienced people, and at best, this weapon should be restricted to certain professions.

Death From Butane Inhalation Abuse in Teenagers: Two New Case Studies and Review of the Literature.

The ready availability of butane makes butane abuse frequent. Fatalities are rare. This study presents two cases of death by butane overdose. The postmortem analyses were carried out using headspace gas chromatography-mass spectrometry. It revealed femoral blood butane concentrations of 18 and 22 mg/L, respectively, as well as specific combinations of adjuvants in each victim. In one of the victims, brain and fatty tissue also contained butane, pointing to chronic consumption. The originality of this study is to show that the identification of specific combinations of adjuvants can be helpful for identifying the brand of deodorant used. Also, sampling the skin and mucosa can help identify the method of drug delivery. The histological examination documented both the direct toxic effect of the gas on the respiratory mucosa and signs of chronic abuse. Volatile substance intoxications should be systematically considered in case of sudden death in a teenager.

Fatal heat stroke in a child entrapped in a confined space.

We report the case of a child succumbing to heatstroke caused by confinement in an icebox. The post mortem examination found cyanosis and hematomas indicating that the child had tried to get out of the container. The temperature of the body was higher than it should have been considering the rigor and delay before post mortem examination. The autopsy showed no significant injury and toxicological tests were negative. A physiological study etablished that death resulted from heatstroke, not a lack of oxygen or CO2 poisoning. We conclude that heat stroke should be considered as a possible mechanism of death even in the absence of context of environmental hyperthermia. We recommend that in these situations involving confinement, establishing the mechanism of death should be done not only on the basis of a detailed post-mortem examination to rule out other causes of death, but also based on complete physiological investigations.

Quantification of hypoglycin A in serum using aTRAQ(®) assay.

BACKGROUND: Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. METHODS: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. RESULTS: Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50µmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. CONCLUSIONS: The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.

Screening approach by ultra-high performance liquid chromatography-tandem mass spectrometry for the blood quantification of thirty-four toxic principles of plant origin. Application to forensic toxicology.

Plant poisonings have left their mark on history and still cause many deaths, whether intentional or accidental. The means to show toxicological evidence of such poisonings should be implemented with great care. This article presents a technique for measuring thirty-nine toxic principles of plant origin in the blood, covering a large amount of toxins from local or exotic plants: α-lobeline, α-solanine, aconitine, ajmaline, atropine, brucine, cephalomannine, colchicine, convallatoxin, cymarine, cytisine, digitoxin, digoxin, emetine, gelsemine, ibogaine, jervine, kavain, lanatoside C, lupanine, mitragynine, neriifolin, oleandrin, ouabain, paclitaxel, physostigmine, pilocarpine, podophyllotoxin, proscillaridin A, reserpine, retrorsine, ricinine, scopolamine, senecionine, sparteine, strophanthidin, strychnine, veratridine and yohimbine. Analysis was carried out using an original ultra-high performance liquid chromatography separation coupled with tandem mass spectrometry detection. Extraction was a standard solid phase extraction performed on Oasis(®) HLB cartridge. Thirty-four of the thirty-nine compounds were put through a validation procedure. The assay was linear in the calibration curve range from 0.5 or 5 µg/L to 1000 µg/L according to the compounds. The method is sensitive (LOD from 0.1 to 1.6 µg/L). The within-day precision of the assay was less than 22.5% at the LLOQ, and the between-day precision was less than 21.5% for 10 µg/L for all the compounds included. The assay accuracy was in the range of 87.4 to 119.8% for the LLOQ. The extraction recovery and matrix effect ranged from 30 to 106% and from -30 to 14%, respectively. It has proven useful and effective in several difficult forensic cases.

A validated method for quantifying hypoglycin A in whole blood by UHPLC-HRMS/MS.

Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a nutritious and readily available fruit which is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of several species of maple (Acer spp.) which are suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy in Europe, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning. Analysis was carried out using solid-phase extraction (HILIC cartridges), dansyl derivatization and UHPLC-HRMS/MS detection. The method was validated in whole blood with a detection limit of 0.35 µg/L (range: 0.8-500 µg/L). This is the first method applicable in forensic toxicology for quantifying HGA in whole blood. HGA was quantified in two serum samples from horses suffering from atypical myopathy. The concentrations were 446.9 and 87.8 µg/L. HGA was also quantified in dried arils of unripe ackee fruit (Suriname) and seeds of sycamore maple (Acer pseudoplatanus L.) (France). The concentrations were 7.2 and 0.74 mg/g respectively.

Modern toxic antipersonnel projectiles.

In the spring of 1944, Kurt von Gottberg, the SS police chief in Minsk, was shot and injured by 2 Soviet agents. Although he was only slightly injured, he died 6 hours later. The bullets were hollow and contained a crystalline white powder. They were 4-g bullets, semi-jacketed in cupronickel, containing 28 mg of aconitine. They were later known as akonitinnitratgeschosse. The Sipo (the Nazi security police) then ordered a trial with a 9-mm Parabellum cartridge containing Ditran, an anticholinergic drug with hallucinogenic properties causing intense mental confusion. In later years, QNB was used and given the NATO code BZ (3-quinuclidinyl-benzylate). It was proven that Saddam Hussein had this weapon (agent 15) manufactured and used it against the Kurds. Serbian forces used the same type of weapon in the Bosnian conflict, particularly in Srebrenica.The authors go on to list the Cold War toxic weapons developed by the KGB and the Warsaw pact countries for the discreet elimination of dissidents and proindependence leaders who had taken refuge in the West. These weapons include PSZh-13 launchers, the Troika electronic sequential pistol, and the ingenious 4-S110T captive piston system designed by the engineer Stechkin. Disguised as a cigarette case, it could fire a silent charge of potassium cyanide. This rogues gallery also includes the umbrella rigged to inject a pellet of ricin (or another phytalbumin of similar toxicity, such as abrin or crotin) that was used to assassinate the Bulgarian writer and journalist Georgi Markov on September 7, 1978, in London.During the autopsy, the discovery of a bullet burst into 4 or 5 parts has to make at once suspecting the use of a toxic substance. Toxicological analysis has to look for first and foremost aconitine, cyanide, suxamethonium, Ditran, BZ, or one of the toxic phytalbumins. The use of such complex weapons has to make suspect a powerful organization: army, secret service, terrorism. The existence of the Russian UDAR spray gun in the present day, however, shows that these weapons are still present. The possibility that one might be used to spray a charge of cyanide is still very real, especially as it would not be very difficult for an informed amateur to produce homemade toxic ammunition by adapting existing civil or military cartridges.

An amazing case of fatal self-immolation.

We present a surprising case of suicide by self-immolation. A surveillance camera filmed the victim's agony. We were able to ascertain that he survived 13 min after ignition. This case was the starting point for a review of the literature of the forensic elements required to correctly analyze cases of suicide by self-immolation and to establish the causal link between the burn lesions and death. The authors will focus on the forensic and medical aspects in favor of suicide by self-immolation and on the forensic aspects required to understand the ignition process, the circumstances surrounding the fire with a particular emphasis on determining whether an accelerant was doused on the victim before ignition.

A validated method for quantifying atractyloside and carboxyatractyloside in blood by HPLC-HRMS/MS, a non-fatal case of intoxication with Atractylis gummifera L.

Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).

The principal toxic glycosidic steroids in Cerbera manghas L. seeds: identification of cerberin, neriifolin, tanghinin and deacetyltanghinin by UHPLC-HRMS/MS, quantification by UHPLC-PDA-MS.

The toxicity of the sea mango (Cerbera manghas L.) is well known. The plant is ranked as one of the deadliest of the southern Asian coastline. Cardenolidic heterosides are responsible for the cardiotoxicity of trees of the Cerbera genus. We have identified and determined the concentration of the principal glycosidic steroids present in the seeds of sea mangos (Thailand). Drug screening of an extract of the seeds was carried out using ultra-high performance liquid chromatography coupled to photodiode array detection and mass spectrometry (UHPLC-PDA-MS) with quantification at 219nm. Identification was confirmed by UHPLC-HRMS. Deacetyltanghinin (m/z 549.3055±2ppm), neriifolin (m/z 535.3259±2ppm), tanghinin (m/z 591.3169±2ppm) and cerberin (577.3375±2ppm) were the most abundant glycosidic steroids present in the sea mango seeds. A seed of the dried ripe fruit had concentrations of 1209.1, 804.2, 621.4 and 285.9µg/g, respectively. A seed of the fresh unripe fruit had concentrations of 49.4, 47.0, 3.5 and 2.3µg/g.

Quantification of pregabalin using hydrophilic interaction HPLC-high-resolution MS in postmortem human samples: eighteen case reports.

Pregabalin is a drug for treating epilepsy, anxiety disorders and neuropathic pain. Cases of poisoning are rare, though some have been fatal. Concentrations of pregabalin in postmortem human samples and its distribution have very rarely been documented. As the literature is so scarce, we propose to report the concentrations in autopsy samples of 18 people who had been taking Lyrica(®), including one case of a mixed overdose involving pregabalin. Analysis was carried out using an original Hydrophilic Interaction LIquid Chromatography (HILIC) technique coupled with a high-resolution mass spectrometer (m/z 160.1334 ± 5 ppm). The sensitivity of the technique enables a quick and simple treatment of the samples by protein precipitation. The method was validated in the whole blood with detection and quantification limits of 0.025 and 0.060 µg/mL, respectively. Pregabalin was a likely factor in the cause of death in 3 of the 18 cases. In the other individuals, the concentrations ranged from 0.4 to 17.0 in the peripheral blood, 1.5 to 11.1 in the central blood, 126.6 to 2004.6 in the urine and 10.5 to 58.3 µg/mL in the bile, with median values of 5.6, 4.6, 534.6 and 17.7, respectively.

Atropine eye drops: an unusual homicidal poisoning.

In March 2009, the body of a 51-year-old man was found in the boot of his car. The body had been frozen before being dismembered at the abdomen. The autopsy failed to determine the cause of death. Systematic toxicological analyses of the victim's peripheral blood and urine showed the presence of atropine, a powerful anticholinergic. Atropine was therefore specifically detected and quantified throughout the victim's biologic samples by HPLC-MS² in the biologic fluids and UHPLC-MS² in the hair. The atropine concentrations were 887 ng/mL in the cardiac blood, 489 ng/mL in the peripheral blood, 6693 ng/mL in the gastric contents (1.1 µg), 6753 ng/mL in the urine, and 2290 pg/mg in the hair. The blood concentrations measured in the decedent were consistent with an overdose of atropine, which was determined as the cause of death. The manner of death was a homicide with criminal intent.