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Vitreous humour as a complementary sample to blood for the detection/confirmation of diazepam: ante-mortem and post-mortem studies in an animal model

The quantification of medical or toxic substances in vitreous humour (VH) could be very useful in forensic toxicology when blood sample determinations are impossible due to absence or deterioration. However, few studies have been made in this area and even fewer have tried to find a relationship between drug levels in both samples. To determine a correlation ratio between blood and VH diazepam (DZ) levels, we performed an experimental study using rabbits administered with a sub-toxic dose of DZ under known and controlled conditions. Blood and VH samples were collected 0.5, 1, 2, 3 and 6 hours after the drug administration in order to determine DZ and its main active metabolite, desmethyldiazepam (DMD). In addition, we have studied an animal group sacrificed 2 hours after intramuscular (i.m.) drug administration with blood and VH collection 24 hours later, to evaluate the existence of possible post-mortem changes. After DZ administration, a fast absorption phase was observed with a plasma Cmax value 1 hour after, followed by a rapid concentration decrease, with a half-life of 1 hour, indicating that, besides elimination, a fast distribution to other organs and tissues and/or hepatic metabolism occurred. Diazepam Cmax value in VH was achieved between 1 and 2 hours, when plasma concentrations had already decreased to half the value. The plasma/VH DZ ratio calculated at this time was 10. In the post-mortem study, while plasma DZ concentration at 24 hours was smaller, DMD levels were higher than those at the time of death. In the VH, both DZ and DMD concentrations at 24 hours were higher than those obtained at the time of death. That is, in both fluids DZ and DMD concentrations were different from those at the time of death and post-mortem distribution and redistribution phenomena occurred. The combination of antemortem and post-mortem studies has allowed the determination of a correlation ratio for DZ in the rabbit of 6×, comparing the concentrations in VH collected 24 hours after death with the concentrations detected in plasma at the time of death. This study opens new perspectives for the use of VH as a complementary sample to blood for DZ detection and confirmation. The putative relevance of the correlation ratio obtained, for forensic toxicology practice with medical substances, namely benzodiazepines, recommends further studies in humans.

UPLC-MS/MS determination in blood of a mixed-drug fatal intoxication: a case report.

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.

An UPLC–MS/MS method for the determination of valproic acid in blood of a fatal intoxication case

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.

Qualitative and quantitative analysis of a group of volatile organic compounds in biological samples by HS-GC/FID: application in practical cases

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.

A fatal forensic intoxication with fenarimol: analysis by HPLC/DAD/MSD.

Fenarimol (Rubigan) is a pyrimidine ergosterol biosynthesis inhibitor used as a systemic fungicide. The authors present a fatal fenarimol intoxication case analysed in the Forensic Toxicology Service of the National Institute of Legal Medicine. The results were used to compare two different HPLC techniques, regarding selectivity and sensitivity: an HPLC system with a diode array detector (DAD) and an HPLC system with a DAD and a mass spectrometry detector (MSD) with an electrospray interface. All biological samples were submitted to a solid-phase extraction procedure. The detection and quantification limits of fenarimol, linearity, precision and accuracy were evaluated. The fenarimol concentration levels determined were of 89.0 mg/ml in gastric contents, 1.9 mg/g in liver and 0.4 mg/g in kidney. Blood was not available at autopsy. No published data related to fenarimol self-poisoning were found, so it was not possible to interpret the results obtained by comparison with toxic/lethal levels.

Validated UPLC-MS/MS assay for the determination of synthetic phosphodiesterase type-5 inhibitors in postmortem blood samples

The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) 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(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 μm 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.

Interference of anesthetics in blood alcohol analysis by HS-GC-FID: A case report

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.