Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Analysis of Cannabinoids and Metabolites in Dried Urine Spots (DUS).

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

Determination of Antidepressants and Antipsychotics in Dried Blood Spots (DBSs) Collected from Post-Mortem Samples and Evaluation of the Stability over a Three-Month Period.

An LC-MS/MS method for the identification and quantification of antidepressants and antipsychotics was developed on dried blood spots (DBSs). Moreover, analyte stability on DBSs within a 3-month period was monitored. Aliquots of 85 µL of blood from autopsy cases were pipetted onto DBS cards, which were dried and stored at room temperature. DBSs were analyzed in triplicate immediately, within the following 3 weeks, and after 3 months. For each analysis, a whole blood stain was extracted in phosphate buffer and purified using Solid Phase Extraction (SPE) cartridges in order to avoid matrix effects and injected in the LC-MS/MS system. Thirty-nine molecules were screened. Limits of detection (LODs) ranged between 0.1 and 3.2 ng/mL (g) and 0.1 and 5.2 ng/mL (g) for antidepressants and antipsychotics, respectively. Limits of quantification (LOQs) varied from 5 to 10.0 ng/mL for both. Sixteen cases among the 60 analyzed resulted positive for 17 different analytes; for 14 of these the method was fully validated. A general good agreement between the concentrations on DBSs and those measured in conventional blood samples (collected concurrently and stored at -20 °C) was observed. The degradation/enhancement percentage for most of the substances was lower than 20% within the 3-month period. Our results, obtained from real post-mortem cases, suggest that DBSs can be used for routine sample storage.

Death of a seven-month-old child in a washing machine: a case report.

The authors present a case which brings out a unique modality of child homicide by placing the baby in a washing machine and turning it on. The murder was perpetrated by the baby's mother, who suffered from a serious depressive disorder. A postmortem RX and then a forensic autopsy were performed, followed by histologic examinations and toxicology. On the basis of the results of the autopsy, as well as the histology and the negative toxicological data, the cause of death was identified as acute asphyxia. This diagnosis was rendered in light of the absence of other causes of death, as well as the presence of typical signs of asphyxia, such as epicardial and pleural petechiae and, above all, the microscopic examinations, which pointed out a massive acute pulmonary emphysema. Regarding the cause of the asphyxia, at least two mechanisms can be identified: drowning and smothering. In addition, the histology of the brain revealed some findings that can be regarded as a consequence of the barotrauma due to the centrifugal force applied by the rotating drum of the washing machine. Another remarkable aspect is that we are dealing with a mentally-ill assailant. In fact, the baby's mother, after a psychiatric examination, was confirmed to be suffering from a mental illness-a severe depressive disorder-and so she was adjudicated not-guilty-by-reason-of-insanity. This case warrants attention because of its uniqueness and complexity and, above all, its usefulness in the understanding of the pathophysiology of this particular manner of death.

Ethyl glucuronide in hair: A 5-year retrospective cohort study in subjects sanctioned for driving under the influence of alcohol and psychoactive substances.

The evaluation of drinking behaviors can help in limiting high-risk situation, such as driving under the influence (DUI). We investigate ethyl glucuronide in hair (hEtG) levels to evaluate alcohol consumption behavior in subjects followed up after having been charged for DUI of psychoactive substances and/or alcohol. We performed a retrospective observational cohort study on 4328 subjects over 18 years old who underwent hEtG analysis in the period 2015-2019 in the Italian Province of Pavia. hEtG level was used as a proxy for the alcohol consumption behavior. Effects of age, sex, and district on alcohol drinking behavior were investigated with an ordinal logit model. A state sequence analysis was used to study people's alcohol consumption behavior over time. hEtG was found ≥7.0 pg/mg in 22.2% of the drivers (of which 7% has an hEtG ≥30.0 pg/mg). Among positive cases, a prevalence of males (96.3%) aged 35-44 (32.6%), coming from main city and hinterland (38.2%), was observed. The propensity to drink was higher for males (odds ratio [OR] ≈ 2.28, p < 0.001) and for subject coming from the district devoted to the cultivation of vineyards. Young age classes have a reduced drinking risk if compared to the drivers over 55 years old (p < 0.001). A general decreasing trend over time in hEtG values was observed. Being male, age ≥ 55 years, and coming from rural areas are potential risk factors related to alcohol drinking habits among drivers. Ethyl glucuronide in hair test in the driving license reissuing protocol contributed to decrease alcohol misuse behaviors.

Determination of Traditional and Designer Benzodiazepines in Urine through LC-MS/MS.

BACKGROUND: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis. OBJECTIVE: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases. METHODS: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode. RESULTS: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine. CONCLUSION: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

A case report on fatal intoxication by tapentadol: Study of distribution and metabolism.

We report a case in which a tapentadol acute intoxication was suspected as the cause of death of a 39-year-old man: approximately two days after death, cardiac and femoral blood, as well as urine, bile, gastric content and chest hair, were collected during the autopsy. Tapentadol was detected before and after hydrolysis in femoral (530 ng/mL unconjugated and 1570 ng/mL conjugated) and cardiac (680 ng/mL unconjugated and 3440 ng/mL conjugated) blood, and additionally in bile (3200 ng/mL), urine (9300 ng/mL), chest hair (2850 pg/mg) and gastric content. LC-QTOF screening analysis confirmed the presence of five different tapentadol metabolites (tapentadol-O-glucuronide, tapentadol-O-sulfate, N-desmethyltapentadol, N-desmethyltapentadol-glucuronide and N-desmethyltapentadol-O-sulfate), in urine, bile, cardiac and femoral blood. Positivity of body hairs allowed us to conclude that the man had used tapentadol in the last weeks/months. Autopsy and toxicological results (also positive for clotiapine, diazepam and chlordesmethyldiazepam) suggested that tapentadol could have caused, even at low concentrations, a severe respiratory depression, which contributed to the death of the subject. This is one of the few cases in literature where tapentadol was detected in blood, together with its metabolites, and the only one in which the parent drug was identified in hairs.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples.

Purpose: Whatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results. Methods: Twenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography-mass spectrometry methods. Results: We were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent. Conclusions: This study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood. Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-020-00567-2.

Distribution of Fluvoxamine and Identification of the Main Metabolite in a Fatal Intoxication.

Fluvoxamine is a selective serotonin reuptake inhibitor, with a half-life of about 30 hours, that is commonly prescribed in the treatment of depression and obsessive and compulsive disorders. Though its more favorable adverse effect profile in comparison to tricyclic antidepressants, overdosages could lead to severe central nervous system depression. We hereby report the case of a 48-year-old woman with psychiatric disorders, who died in the Protected Community where she lived. An autopsy, during which multiorgan congestion and aspiration of gastric content were found, was performed 9 days after the death. Femoral and cardiac blood, urine and bile were collected for toxicological analysis. GC-MS, LC-MS-MS and LC-HRMS screenings were performed on blood samples. The analysis allowed to identify the following drugs: fluvoxamine, clotiapine, 7-aminoclonazepam, propranolol, gabapentin and haloperidol. Quantification of the detected drugs in blood was performed by means of a validated LC-MS-MS analytical procedure, and the following results were achieved: fluvoxamine (2.20 mg/L), gabapentin (41.00 mg/L), 7-aminoclonazepam (0.24 mg/L), clotiapine (0.07 mg/L), haloperidol (<0.01 mg/L) and propranolol (0.24 mg/L). Fluvoxamine concentration in blood exceeded ~10 times the upper limit of therapeutic blood levels (0.23 mg/L). Contributory causes of death, such as due to multiple drug use, however, cannot be excluded. The distribution of fluvoxamine in all biological fluids was evaluated and a postmortem redistribution effect was observed (C/P blood ratio: 1.86). Fluvoxamine acid metabolite was identified in urine, bile and in cardiac blood, through an LC-QTOF analytical procedure.

Importance of segmental hair analysis in a suspected case of attempted homicide by flocoumafen and difenacoum.

The 4-hydroxycoumarin derivatives are the most used rodenticides and act as classical anticoagulants, interfering with the production of clotting factors in liver by antagonizing the action of vitamin K reductase, thereby inhibiting recycling of vitamin K1, involved in activation of blood clotting factors, resulting in massive bleeding. In this paper, we present the case of a 72-year old man providing abnormal coagulation parameters (PT-INR between 16.1 and 19.1) after hospitalization. Blood samples tested positive for flocoumafen and difenacoum, two superwarfarin rodenticides. Patient's hair specimens, sampled 19 days after his hospitalization, showed that traces of both difenacoum and flocoumafen were detected in the first 1 cm; in the intermediate segments (1-2 and 2-3 cm), both difenacoum and flocoumafen were absent, while in the distal segment (3-4.5 cm), only difenacoum was found, but in significant amounts (140 pg/mg). Exposure to difenacoum in the past months, at least 4-5 before hospitalization, was confirmed by the presence of the rodenticide in the distal segment. Moreover, among the seized material, two specimens resulted compatible with the two rodenticides.

Determination of fentanyl and 19 derivatives in hair: Application to an Italian population.

Nowadays fentanyl and its analogs represent the most numerous group among synthetic opioid and, due to their higher potency in comparison to traditionl opioids, the main cause of the critical increase of fatal intoxications opioids-intake related in the USA. We developed an LC-MS/MS method for the detection and quantification of fentanyl and its analogs in hair, then applied to 117 real samples, 97 collected from drugs users and 20 from postmortem cases of drugs addicts. The ionization and MRM parameters have been optimized for 27 molecules: 20 reached the acceptance criteria for identification and quantification. LODs and LOQs of 0.2 and 0.5 pg/mg, respectively, were reached for most of the substances, except for five compounds for which were set at 0.5 and 1.0 pg/mg. 2 out of the 97 samples collected from drug users tested positive; one for carfentanil, butyryl fentanyl, THFF and ocfentanil; the other one for 3-methyl norfentanyl. 2 out of the 20 postmortem samples show positive results: one only for fentanyl, the other for furanyl fentanyl, acetyl fentanyl, methoxyacetyl fentanyl, methoxyacetyl norfentanyl, ocfentanil and 4-ANPP. Despite the relatively small number of samples, the results suggest that the method should be included in routine hair analyses for monitoring the new synthetic opioids potential intake by drug users.

Fatal poisoning of four workers in a farm: Distribution of hydrogen sulfide and thiosulfate in 10 different biological matrices.

We evaluate the distribution of sulfide and thiosulfate (TS) in biological samples of four dairy farmers died inside a pit connected to a manure lagoon. Autopsies were performed 4 days later. Toxicological analyses of sulfide and TS were made using an extractive alkylation technique combined with gas chromatography/mass spectrometry (GC/MS). Autopsies revealed: multiorgan congestion; pulmonary edema; manure inside distal airways of three of the four victims. Sulfide concentrations were cardiac blood: 0.5-3.0 µg/mL, femoral blood: 0.5-1.2 µg/mL, bile: <0.1-2.2 µg/mL; liver 2.8-8.3 µg/g, lung: 5.0-9.4 µg/g, brain: 2.7-13.9 µg/g, spleen: 3.3-6.3 µg/g, fat: <0.1-1.5 µg/g, muscle: 2.6-3.5 µg/g. TS concentrations were cardiac blood: 2.1-4.9 µg/mL, femoral blood: 2.1-2.3 µg/mL, bile: 2.5-4.4 µg/mL, urine: <0.5-1.8 µg/mL; liver <0.5-2.6, lung: 2.8-5.4 µg/g, brain: <0.5-1.9 µg/g, spleen: 1.2-2.9 µg/g, muscle: <0.5-5.6 µg/g. The cause of death was assessed to be acute poisoning by hydrogen sulfide (H2S) for all the victims. Manure inhalation contributed to the death of three subjects. The measurement of sulfide and TS concentrations in biological samples contributed to better understand the sequence of the events. Subjects 3 provided the highest concentration of sulfide in brain, thus, supporting the hypothesis of a rapid loss of consciousness and respiratory depression. One by one, the other farmers entered the pit in attempts to rescue the coworkers but collapsed. Despite the rapid death, subject 3 was the only one with TS detectable in urine. This could be due to differences in metabolism of H2S.

Distribution of quetiapine and metabolites in biological fluids and tissues.

Quetiapine is an atypical antipsychotic drug, frequently found in post-mortem samples. The quantitative determination of active metabolites may help in the interpretation of the potential toxic effects of the parent drug and its role in death. A fully validated LC-MS/MS method was developed for the identification and quantification of quetiapine and two main metabolites (N-desalkylquetiapine and 7-hydroxyquetiapine) in blood, biological fluids and tissues. Then, the distribution of analytes in different matrices was evaluated. LODs of 0.9, 0.3 and 0.3ng/mL were calculated for quetiapine, N-desalkylquetiapine and 7-hydroxyquetiapine respectively; while a LOQ at the concentration of 10.0ng/mL was defined for the three analytes. 13 post-mortem positive real cases have been included in the experiment. The results revealed that quetiapine and N-desalkylquetiapine might undergo a significant post-mortem redistribution, while 7-hydroxyquetiapine is less affected by this factor. N-desalkylquetiapine could be found in blood in relatively high concentrations in comparison to those of quetiapine; therefore, it should be always advisable to measure both the analytes. The analysis of tissues could provide additional data on potential intoxication with quetiapine.

Distribution of the Synthetic Cathinone α-Pyrrolidinohexiophenone in Biological Specimens.

We report the analysis of the synthetic cathinones α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopentiophenone (α-PVP), both pyrovalerone derivatives, in blood, urine, gastric contents, main tissues and hair of a deceased person. Qualitative and quantitative analyses were performed by LC-MS-MS. All the biological samples were collected during autopsy and extracted/purified onto a solid phase extraction cartridge before instrumental analysis. The method was validated for blood and urine and proved to be highly sensitive and specific for both the synthetic cathinones (limit of detection: 0.2 ng/mL and limit of quantification: 0.5 ng/mL). Analyses provided negative results for α-PVP in all biological samples except for the 2-cm proximal hair segment, confirming that the young man had consumed in the last 2 months this compound; instead hair analysis proved that the man was a heavy α-PHP user. α-PHP was identified and quantified in biological fluids and tissues. Interestingly, bile and urine concentrations (1.2 and 5.6 ng/mL, respectively) were fairly lower than blood collected into the thoracic cavity (15.3 ng/mL). The highest concentrations were measured for lung (71.1 ng/mL) and spleen (83.8 ng/mL). Concentrations of 3.5, 7.9, 4.7 and 23.6 ng/mL were measured in liver, kidney, brain and heart, respectively. Even if it is not possible to evaluate the presence of this drug in biological samples as a cause of death, to our knowledge, this is the first case of α-PHP finding in postmortem samples, and its potential toxic effects should be elucidated in the future.

A multi-analyte LC-MS/MS method for screening and quantification of 16 synthetic cathinones in hair: Application to postmortem cases.

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as "bath salts") in human hair has been developed and fully validated using liquid chromatography-tandem mass spectrometry system. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 µL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4-DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone.

A case report on potential postmortem redistribution of furanyl fentanyl and 4-ANPP.

Fatal intoxications due to accidental or voluntary intake of synthetic opioids represent an actual emerging issue. We report a case where we have analyzed furanyl fentanyl and its metabolite 4-anilino-N-phenetyl-piperidine (4-ANPP) in blood, urine, gastric content, bile and cerebrospinal fluid. In this case, a 53-year-old man was found dead at home with a needle still inserted in a vein; a plastic bag containing a white powder (later identified as a furanyl fentanyl-based product) was discovered in the room. Biological samples were collected during autopsy and extracted/purified onto a SPE cartridge before instrumental analysis. Qualitative and quantitative analyses were performed by LC-MS/MS on peripheral and cardiac blood, urine, cerebrospinal fluid (CSF), bile and gastric content. Furanyl fentanyl was identified and quantified in all the biological fluids collected. Interestingly, gastric content revealed an unexpected high amount of furanyl fentanyl; yet, cardiac blood and femoral blood provided significantly different concentrations (11.8 and 2.7 ng/g respectively). The concentration of furanyl fentanyl in CSF was similar to that measured in femoral blood (2.6 ng/mL), thus confirming that CSF could be a good alternative biological fluid whenever a postmortem redistribution is suspected. Concentrations of 93.5, 50.4, 171.7, 41.9, 10.2 ng/mL(g) were measured for 4-ANPP in cardiac blood, femoral blood, urine, bile and cerebrospinal fluid, respectively. The outcomes from the presented case report suggest that the two substances have been not only injected intravenously, but probably also ingested by the man. Fentanyl derivative and its precursor seemed to undergo an extensive postmortem redistribution.

Determination of benzodiazepines in blood and in dried blood spots collected from post-mortem samples and evaluation of the stability over a three-month period.

We successfully developed and validated an LC-MS/MS method for the identification of 27 and quantification of 9 benzodiazepines and metabolites in whole blood and DBSs. The results provided a good qualitative and quantitative correlation between DBSs stored at room temperature and whole blood stored at -20°C. A good stability for a three-month period was observed for most of the compounds detected in real post-mortem samples.

Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water.

The study aims the development and validation of a LC-MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1-2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC-MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Two Fatal Cases Involving Cardiovascular Drugs Diltiazem and Amlodipine.

A liquid chromatographic tandem mass spectrometric method for the identification and quantification of 18 cardiovascular drugs was developed in order to evaluate two cases of fatal intoxication involving diltiazem and amlodipine respectively. Samples were simply diluted and centrifuged using a three-steps procedure with methanol, acetonitrile and mobile phase. The method proved to be selective and all the validation parameters fulfilled the acceptance criteria. In particular, linearity was studied in the range limits of quantitation (LOQ)-1,000 ng/mL (LOQ ranging from 0.8 to 33.3 ng/mL for urine and from 0.7 to 41.3 ng/mL for whole blood). The method was successfully applied to two real cases involving diltiazem and amlodipine fatal intoxications, respectively. Though the subject intoxicated by diltiazem did survive several hours after drug intake, central and peripheral blood levels at autopsy were extremely high (23.4 and 13.4 mg/L, respectively); the cause could be due to the formation of a pharmacobezoar that was found in the duodenum and that could have delayed the drug absorption. Moreover, diltiazem showed postmortem redistribution. On the contrary, the amlodipine peripheral blood level in the second case was relatively low (0.17 mg/L), thus confirming that even the uncontrolled intake of a less toxic calcium channel blocker can lead to death. Furthermore, blood samples were analyzed after 2 years of storage at -20°C: both diltiazem and amlodipine showed a significant degradation (70 and 99%, respectively).

Delta-9-tetrahydrocannabinolic acid A (THC-A) in urine of a 15-month-old child: A case report.

INTRODUCTION: The acidic forms of cannabinoids, THC-A and CBD-A are naturally present in cannabis plants and preparations and are generally decarboxylated to the active compounds before the use (e.g. thermally decarboxylated through smoking). Hence, the identification of the acidic compounds in urine could be an evidence of cannabis ingestion rather than a passive exposure to smoke. This case report described a 15-month-old child that suffered an acute intoxication by accidental cannabis ingestion. It is important to assess the ingestion and to discriminate it from a passive exposure to better interpret the clinical findings and to establish the correct therapeutic procedure. METHODS: Urine samples were simply diluted in deionized water and directly injected in the LC-MS/MS system. D3-THCCOOH was used as internal standard. Chromatographic separation of THCCOOH, THC-A and CBD-A was carried out in reversed phase on a c18 column. A triple quad in MRM negative mode was used to monitor the three analytes. RESULTS AND DISCUSSION: The developed LC-MS/MS method was simple and fast. A LOD of 3.0ng/mL and a LOQ of 10.0ng/mL were measured for the three compounds. The analytical procedure was validated accordingly to international guidelines. The two urine samples collected from the 15-month-old child at the hospitalization and after three days provided positive results for THCCOOH (130.0 and 10.0ng/mL respectively). THC-A was found only in the urine sample collected at the hospitalization (concentration: 70.0ng/mL). CONCLUSION: THC-A was detected and quantitated in a urine sample of a 15-month-old child.