Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry methodology (LC-MS-MS-MS3) was developed for the determination and quantification of increment (9)-tetrahydrocannabinol (THC), 11-hydroxy- increment (9)- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy- increment (9)-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC & REG; HHS T3 (100 mm x 2.1 mm i.d., 1.8 & mu;m) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS/MS-MS3 analysis, a SCIEX QTRAP(& REG;) 6500(+) triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r(2) > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

Alcohol is the most determined substance in postmortem toxicology and its interpretation is especially complicated. Although the determination and quantification of ethanol is preferably carried out on blood samples, the determination of this parameter in other biological matrices can prove to be quite important. This may be true, for example, in cases where the blood sample available is insufficient or is in a state deemed unsuitable (e.g. putrefaction or contamination by other biological fluids such as gastric contents). Among the biological samples which can be considered as an alternative to blood in the determination of ethanol, the vitreous humor (VH) appears as the most interesting. Therefore, it is of high relevance the analysis of other biological samples, as urine and VH, that in normal situations are only positive for alcohol when antemortem ingestion has occurred. This is important mostly when the blood alcohol concentration (BAC) is lower than 0.5 g/L. Another study reported that with a BAC of 0.5 g/L there is an 87% probability of a positive result for alcohol in VH and with the increase of this concentration the probability goes up to 99%. In the present study, the authors made a statistical survey, within the period of three years, of the cases that indicated putrefaction in which the medical expert required the analyses of the BAC. From all the cases evaluated, three forensic cases were selected, which represent situations that might occur when studying the BAC of a putrefied corpse. These cases were chosen to highlight the possible benefits of analyzing multiple biological samples, when present, for confirmation of the BAC. The samples analysis was made using an Agilent 6890N gas chromatograph equipped with a flame ionization detector and coupled to an Agilent G1888 headspace injector of fixed volume (1 mL) (HS-GC/FID). Prior to gas-chromatography analysis, all specimens, including the calibrators, were diluted 1:10. With this purpose, 100 μL vitreous humor, urine and blood were diluted with 1 mL aqueous solution of n-propanol (100 mg/L), used as internal standard. In the period of 3 years (2018–2020), there were 4264 medical-legal autopsies executed in which the medical expert required the determination of the BAC. Of these cases, a total of 130 had information stating the presence of putrefaction. They were related to different causes of death (intoxication, drowning, trauma, natural deaths and undetermined cause) and had a postmortem interval between one and six days. BAC measured determined 64 cases with a negative result and 66 with a positive result. Of these, 66.7% had a BAC between 0.1 g/L and 0.5 g/L, relevant as endogenous production might have happened, making the interpretation of these values challenging. However, this interpretation can benefit from the integration of the available case information, especially the search for alcohol in other biological samples. Using this data, it was determined how many cases presented one, two and three biological samples and three forensic cases were selected, representing each of these categories, exemplifying situations that can easily occur when studying the BAC of putrefied corpses. In this study we discussed how the determination of the alcohol concentration in urine and VH can be a valuable tool, especially in the presence of putrefied corpses and low concentrations of alcohol in the blood, when trying to determine whether the BAC was of exogenous origin, endogenous production or both. This suggests that the analysis of these three types of biological samples (blood, urine, VH) can be of use in reaching a conclusion regarding the origin of the alcohol and making it less susceptible to dispute.

Antipsychotic drugs (AP) are widely prescribed for the treatment of schizophrenia and psychosis. The pharmacological treatment of schizophrenia is often performed with the simultaneous use of two or more antipsychotic agents to achieve the desired control of psychotic symptoms Available AP include both conventional (typical) and new (atypical) antipsychotic medications. Atypical AP, such as quetiapine, now account for the vast majority of AP prescriptions. In forensic toxicology, AP are of considerable interest because of their potential abuse and their involvement in intoxications and suicides. The authors retrospectively examined AP positive cases detected in samples collected during autopsies performed in the Forensic Clinical and Pathology Service of National Institute of Legal Medicine and Forensic Sciences Centre Branch or in other autopsies carried out in the central region of Portugal, between January 2016 and December 2018. A quantitative liquid chromatography-tandem mass spectrometry assay was developed for the simultaneous determination of 16 AP (amisulpride, aripiprazole, chlorpromazine, clozapine, cyamemazine, fluphenazine, haloperidol, levomepromazine, melperone, olanzapine, paliperidone, promethazine, quetiapine, risperidone, sulpiride and ziprasidone) in blood samples of postmortem cases. The Laboratory of Forensic Chemistry and Toxicology received 3,588 requests for toxicological analysis: 1,413 cases were positive for drugs from which 351 (24.8%) cases were positive for AP, 60.1% from male individuals and 39.9% from female. Quetiapine was the most prevalent AP (36.5%) followed by olanzapine (20.8%). During this period, there were 25 postmortem cases with AP blood concentrations above therapeutic range, in which 36% of those are in agreement with the information received (psychological history or acute intoxication suspicion) and the manner of death was suicide. Our results point that antipsychotics are an increasingly prevalent class of drugs. AP must be measured not only in toxic concentrations but also in therapeutic levels in postmortem cases; therefore, it is important to come up with a sensitive method to cover the low therapeutic range in which AP are usually present.

One of the techniques most widely used in ethanol analysis in forensic laboratories is undoubtedly the headspace gas chromatography with flame-ionization detection (HS-GC-FID) since the determination of this substance is carried out directly, without the need for additional purification procedures, which leads to increased productivity. This is a very important factor due to the high number of alcohol analysis requested to these laboratories. The presence of other volatile substances can cause a problem given the fact that they can be interferents in ethanol analysis by HS-GC-FID, which can have legal consequences related with driving under the influence of alcohol. The authors report a case of a routine analysis by HS-GC-FID for the determination of ethanol of a driver who has suffered an accident in which the use of two chromatographic columns with different polarities was essential to obtain an unequivocally identification of this substance in presence of an interfering volatile anesthetic administered in the hospital. The method was validated according to international recommendations before being introduced into routine laboratory in terms of selectivity, limits of detection (LOD), limits of quantification (LOQ), linearity, repeatability, intermediate precision, accuracy, robustness and carryover.

A simple and sensitive procedure, using n-propanol as internal standard (IS), was developed and validated for the qualitative and quantitative analysis of a group of 11 volatile organic substances with different physicochemical properties (1-butanol, 2-propanol, acetaldehyde, ethyl acetate, acetone, acetonitrile, chloroform, diethyl ether, methanol, toluene and p-xylene) in whole blood, urine and vitreous humor. Samples were prepared by dilution with an aqueous solution of internal standard followed by Headspace Gas Chromatography with a Flame-ionization Detector (HS GC-FID) analysis. Chromatographic separation was performed using two capillary columns with different polarities (DB-ALC2: 30m×0.320mm×1.2μm and DB-ALC1: 30m×0.320mm×1.8μm), thus providing a change in the retention and elution order of volatiles. This dual column confirmation increases the specificity, since the risk of another substance co-eluting at the same time in both columns is very small. The method was linear from 5 to 1000mg/L for toluene and p-xylene, 50–1000mg/L for chloroform, and 50–2000mg/L for the remaining substances, with correlation coefficients of over 0.99 for all compounds. The limits of detection (LOD) ranged 1 to 10mg/L, while the limits of quantification (LOQ) ranged from 2 to 31mg/L. The intra-day precision (CV<6.4%), intermediate precision (CV<7.0%) and accuracy (relative error ±10%) of the method were in conformity with the criteria normally accepted in bioanalytical method validation. The method developed has been applied to forensic cases, with the advantages that it uses a small sample volume and does not require any extraction procedure as it makes use of a headspace injection technique.

Trends in forensic toxicology show the introduction of rapid analytical methods for the simultaneous quantitative analysis of drugs. The authors present a fatal case involving a 32-year-old male, found dead in bed by his mother, with several blue, white and orange pills next to the body. Empty tablets were found in the trash bin and a suicide note was on the desk. He was diagnosed with bipolar disorder and had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed to determine 55 different medicines and 32 benzodiazepines in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in positive and negative ion mode. Chromatographic analysis was preceded by an optimized solid-phase extraction procedure using Oasis® HLB (3 cc, 60 mg) extraction columns. The extracted analytes were separated by UPLC (Waters) with a reversed-phase Acquity UPLC® HSS T3 (2.1 × 100 mm id, 1.8 μm) column with acetonitrile and 0.1% formic acid in water as mobile phase, at 0.5 mL/min flow rate and a chromatographic run-time of 8 min. Analytes detection was achieved with a triple-quadrupole mass spectrometer in positive and negative electrospray ionization mode with multiple reaction monitoring (MRM). Two MRM transitions were monitored for each target-compound and one for each deuterated internal standards. Toxicological results showed high blood concentrations of antipsychotics (haloperidol, olanzapine and quetiapine), antidepressants (fluoxetine and paroxetine) and anxiolytics (bromazepam and lorazepam). Risperidone and other benzodiazepines were also present in therapeutic concentrations. Neither alcohol nor illicit drugs were present in the analyzed samples. The UPLC-MS–MS method showed to be appropriate for screening, identification and quantitation of antipsychotics, antidepressants, anxiolytics and antiepileptic drugs in blood after intake of therapeutic as well as toxic doses. The autopsy and toxicological results led the pathologist to rule that death was due to a mixed-drug intoxication. The manner of death was determined to be suicide.

The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra (R)), tadalafil (Cialis (R)) and vardenafil hydrochloride (Levitra (R)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health.The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired.Samples were prepared using Oasis (R) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC (R) HSS T3 (100 x 2.1 mm i.d., 1.8 mu m particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate.Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard.Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were <= 3 ng/mL and <= 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples. (c) 2013 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil(®) next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide. A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode. The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC(®) BEH (2.1 × 50 mm id, 1.7 μm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1-143.1 for valproic acid and m/z 296.1-205.0 for hydrochlorothiazide used as an internal standard (IS). The limit of quantification (LOQ) was 0.5 μg/mL and the method was linear in the concentration range of 0.5-100 μg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 μg/mL, 10 μg/mL and 50 μg/mL). Toxicological results showed high concentration of VPA (556 μg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L. A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.

Paraquat (PQ) is a toxic quaternary ammonium compound widely used in agriculture. Numerous cases of paraquat intoxication have been reported either accidentally or intentionally as suicidal attempts.A method for the determination of the herbicide PQ in blood and urine samples was developed using liquid chromatography-(electrospray ionization) mass spectrometry (LC-ESI-MS), following extraction with Oasis WCX solid-phase cartridges. Chromatographic separation was achieved using an Atlantis (R) HILIC silica column, eluted isocratically with acetonitrile and ammonium formate (200mM) buffer, pH 3.8, at a 300 mu L/min flow rate. Quantitation was achieved by the addition of ethyl paraquat as internal standard (IS). The compounds were detected monitoring two ions for PQ (m/z 185 and m/z 171) and m/z 213 for the IS. The method was applied to determine PQ in two cases: a non fatal case, a 42-year-old female with 0.13 mu g/mL PQ concentration in blood and 6.29 mu g/mL in urine and a lethal case, a 51-year-old male with 0.27 pg/mL PQ concentration in blood.The authors developed a specific, sensitive and rapid assay for the identification and quantification of PQ, very important for monitoring suspected paraquat intoxications in hospitals and subsequently help in the treatment of these patients.

Introduction and aims. Adult orthotopic liver transplantation (OLT) is associated with considerable blood product requirements. The aim of this study was to assess the ability of preoperative information to predict intraoperative red blood cell (RBC) transfusion requirements among adult liver recipients.Methods. Preoperative variables with previously demonstrated relationships to intraoperative RBC transfusion were identified from the literature: sex, age, pathology, prothrombin time (PT), factor V, hemoglobin (Hb), and platelet count (plt). These variables were then retrospectively collected from 758 consecutive adult patients undergoing OLT from 1997 to 2007. Relationships between these variables and intraoperative blood transfusion requirements were examined by both univariate analysis and multiple linear regression analysis.Results. Univariate analysis confirmed significant associations between RBC transfusion and PT, factor V, Hb, Pit, pathology, and age (P values all < .001). However, stepwise backward multivariate analysis excluded variables Plt and factor V from the multiple regression linear model. The variables included in the final predictive model were PT, Hb, age, and pathology. Patients suffering from liver carcinoma required more blood products than those suffering from other pathologies. Yet, the overall predictive power of the final model was limited (R(2) = .308; adjusted R(2) = .30).Conclusion. Preoperative variables have limited predictive power for intraoperative RBC transfusion requirements even when significant statistical associations exist, identifying only a small portion of the observed total transfusion variability. Preoperative PT, Hb, age, and liver pathology seem to be the most significant predictive factors but other factors like severity of liver disease, surgical technique, medical experience in liver transplantation, and other noncontrollable human variables may play important roles to determine the final transfusion requirements.

Driving under the influence of drugs is an issue of growing concern in the industrialized countries as a risk and a cause for road and labour accidents. The aim of this study was to assess the presence of drugs of abuse among drivers and workers involved in fatal accidents between January 1990 and December 2007, by presenting the requests and toxicological results of corresponding autopsies, from the Pathology Service of the Centre Branch of the National Institute of Legal Medicine and from a Legal Medicine Office. In the Forensic Pathology Service, between 1990 and 2007, a total of 3095 autopsies on road traffic accidents victims and 261 on labour accidents victims were performed and 336 28, respectively, in the office. In both cases, few requests and analyses of drugs of abuse were performed, emphasising the fact that the major percentage of the accidents involved individuals aged between 21 and 30 years-old, ages where the consumption is more often. Nevertheless, from the 261 victims of labour accidents autopsied, only 7 were subject of drug analysis, all with negative results.

The authors report a case of a floating body recovered from a well. A 62-year-old woman committed suicide by drowning herself in a well. According to information given by her relatives she had a 13-year history of depression and she wanted to commit suicide. Liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) was used to determine bromazepam in blood. Samples were extracted using Oasis (R) HLB solid-phase cartridges, and separation and quantitation was done using positive-mode electrospray ionization in the single ion monitoring (SIR) mode. Chromatographic separation was achieved using an Atlantis (R) T3 column (2.1x150 mm, 5 mu m), eluted in a gradient system with acetonitrile and formic acid 0.1%, at a flow rate of 300 mu L/min. Quantitation was achieved by the addition of diazepam deuterated (DZP-d5) as internal standard. The compounds were detected monitoring two ions for bromazepam (m/z 318, and m/z 288) and m/z 290 for the deuterated, DZP-d5.Toxicological results revealed in blood a toxic concentration of bromazepam of 418 ng/mL. This finding might be important for the interpretation, not of the cause of death (since the drowning was confirmed by autopsy), but for the state of mind of the victim that had, somehow, helped her to committ suicide (concomitant existence of a psychotropic substance).

no much information is available on workplace drug testing, including ethanol, being this substance often implicated in labour accidental deaths.the aim of this study was to assess the presence of alcohol among in labour fatal accidents between January 1990 and December 2007, by presenting the requests and toxicological results of corresponding autopsies, from the Pathology service of the centre Branch of the national Institute of Legal medicine and from a Legal medicine office.In the Forensic Pathology service, between 1990 and 2007, a total of 261 autopsies were performed on labour accidents victims.ethanol analysis was requested in only 50% of the cases, with 25% positive results.From the positive studied cases, 24% presented ethanol concentrations above 1.2g/L and 55% under 0.5g/L, with all the cases related to men aged between 41 and 50 years-old (24%).In the Legal medicine office, from April 2001 to 2007, 1219 autopsies were performed, 28 to labour victims, with only one female victim.ethanol analysis was requested in 75% of the cases, with 48% positive results, 70% under 0.5g/L, with all the cases related to men aged between 51 and 60 years-old (24%).

ethanol is the most frequently detected drug in deaths from all causes, particularly traffic and labour accidents.the aim of this study was to assess the presence of alcohol among drivers involved in fatal road traffic accidents between January 1990 and December 2007, by presenting the requests and ethanol results of the road traffic accidents autopsies, from the Pathology service of the centre Branch of the national Institute of Legal medicine and from a Legal medicine office.In the Forensic Pathology service and in the office a total of 9409 and 1219 autopsies were performed, respectively, 3095 and 336 on road traffic accidents victims.ethanol analysis was requested only in 54.5% and 75% of the road traffic accident cases, with 31% and 47% positive results, respectively.the major percentage involved concentrations over the maximum limit legally allowed (1.2g/L), being this undoubtedly present in males with ages between 21 and 30 years-old.Research has identified alcohol as a significant factor related to traffic accidents and this has led to specific legislation and methods of enforcement to prohibit this form of impaired driving.

A fully validated, sensitive and specific method for the extraction and quantification of Δ9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ9-THC (THC-COOH) and for the detection of 11-hydroxy-Δ9-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d3 and THC-COOH-d3, respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r2 > 0.999) within the range investigated. The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC–MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.

Tianeptine (Stablon), although structurally similar to tricyclic antidepressants, acts by enhancing the reuptake of serotonin. A fatal case is presented involving a 26-year-old man, found lying in bed with a "mushroom of foam" around his mouth. Empty blister packs of Stablon and a suicide note were found next to the body. A liquid-liquid extraction procedure with n-hexane: ethyl acetate and n-hexane: 2-propanol, followed by LC-DAD-MS analysis, using positive mode electrospray ionization was performed. The detection limit was 0.001 microg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 microg/mL; urine 2.0 microg/mL; liver 23 microg/g; stomach contents 22 mg. Femoral blood analyses also revealed an ethanol concentration of 0.53 g/L. The present method was also developed and validated for the other post-mortem specimens, since no previous published data had confirmed the post-mortem distribution of tianeptine. The absence of other suitable direct causes of death (macroscopic or histological) and the positive results achieved with the toxicological analysis led the pathologist to rule that death was due to an intoxication caused by the suicidal ingestion of tianeptine in combination with alcohol.

Imidacloprid [1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new and potent nitromethylene insecticide with high insecticidal activity at very low application rates. It is the first highly effective insecticide that, like nicotine, acts on the nervous system, causing blockage of postsynaptic nicotinergic acetylcholine receptors. Two fatal cases with this insecticide in two male individuals, of 33 and 66 years old, are presented. An LC/MS with electrospray method for measuring imidacloprid and its metabolites in post-mortem samples is described. In the chromatographic separation, a reverse-phase column XTerra™ MS C18 (2.1 mm i.d. × 150 mm, 5 μm) was used and the mobile phase composed with acetonitrile and 0.1% formic acid (15:85), at a 0.25 mL/min flow rate. Samples were prepared with a liquid–liquid extraction procedure with dichloromethane. Calibration curves for imidacloprid in blood and urine samples were linear from 0.2 to 15 μg/mL. The mean recovery was 86% with a coefficient of variation of ±5.9%. The detection limit was 0.002 μg/mL. Quantitative results were obtained for all post-mortem matrices available of the two fatal cases: blood, urine, stomach contents, lung, liver and kidney. The imidacloprid blood concentrations found in two-cases were 12.5 and 2.05 μg/mL. The authors validated a method to detect and quantify imidacloprid in post-mortem samples, and to our knowledge for the first time a post-mortem tissue distribution was performed on various samples for this insecticide.

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ9-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10cm3, 300mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC+H+], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d3-THC+H+]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100ng/mL showed a linear relationship (r2=0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937ng/mL. However, using Salivette® collection, 26 of the 40 cases had an undetectable THC.

Pesticides are used in most countries around the world to protect agricultural and horticultural crops against damage. Poisoning by these toxicant agents occurs as a result of misuse or accidental exposure, and also by oral ingestion (voluntary or not). In Portugal, pesticide intoxications are still a cause of death, found in a considerable number of cases. The authors retrospectively examined the cases of pesticide poisoning in the Centre of Portugal, from autopsies performed in the Forensic Pathology Service of Coimbra's Delegation of the National Institute of Legal Medicine (NILM) and from other autopsies carried out in the Centre of Portugal, as well as some samples taken in hospitals in cases of suspected intoxication. In this study, the positive cases have been especially studied, in order to identify the pesticide used, as well as the etiology. The frequency of intoxications and its distribution by sex and age were also analyzed. Between January 2000 and December 2002, the Forensic Toxicology Laboratory received 639 pesticide analysis requests. In 2000, in a total of 149 analysis requests, 30 cases were positive, 63.3% from male individuals and 36.7% from female. In 2001, the analysis requests increased to 240 as well as the positive cases (43), 74.4% from male individuals and 25.6% from female and in 2002, the total cases analyzed also increased to 250, with 38 positive (73.6% from male individuals and 26.4% from female). Among the pesticides, organophosphorus insecticides still constitute the most important class detected in forensic intoxications, representing 63% of the total positive cases, followed by herbicides, with 33% of the positive results. Quinalphos is the most important organophosphorus insecticide, present in 32 of the 111 positive cases, followed by the herbicide paraquat, detected in 31 cases. The study emphasizes the increasing number of pesticide analyses, particularly relevant for the organophosphorus compounds and herbicides. Intoxication suspicion, accidental or voluntary, seems to be the most common cause of the incidents, for which analyses are requested, but it is also evident that the putative cause is unknown in a large number of cases. Therefore, more stringent legislation and enforcement regarding the sale and distribution of these toxic substances are needed.