Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of 25 psychoactive drugs in cerumen and its application to real postmortem samples.

PURPOSE: In the present study, a method for the detection of 25 psychoactive substances in cerumen was developed and validated. This method targets opiates, cocaine, antidepressants, benzodiazepines, antipsychotics and antiparkinsons. METHODS: Analysis was performed on a SCIEX Triple Quad 6500+ system after liquid-liquid extraction. Methanol with 1% acetic acid was chosen as the extraction solvent. After the addition of the solvent, samples were vortexed, sonicated, centrifuged and directly injected into the liquid chromatography-tandem mass spectrometry system. RESULTS: The method was found to be selective and sensitive (limit of detection: 0.017 ng-0.33 ng/mg), the assay was linear for all analytes with linear regression coefficient ranging 0.9911-1.00. The values for intra-assay precision was between 4.34 and 14.6% and inter-assay precision between 5.81 and 17.7%, with accuracy within the acceptable criteria for all analytes. All analytes in cerumen specimens were stable for 48 h at 4 °C and 72 h at - 20 °C, whilst no significant matrix effect or carryover was observed. Applicability was proven by analyzing cerumen samples from 25 deceased with a history of drug abuse. All analytes were detected in real samples, thus confirming the sensitivity of the developed method. CONCLUSIONS: According to our knowledge, it is the first time that a method for the simultaneous detection of 25 psychoactive drugs in cerumen was developed, fully validated and finally applied to 25 postmortem samples.

Analysis, Stability and Distribution of Pharmaceuticals and Drugs of Abuse over a Period of One Year in Formalin-Fixed Liver and Formalin Solutions.

The present study reports a thorough research on the stability of drugs of abuse and pharmaceuticals over a time period of 12 months. Fixed-liver tissues and formalin solutions where the tissues were preserved were analyzed using an ultra high performance liquid chromatography--tandem mass spectrometry method that has been developed and validated for this purpose. The method monitors 84 drugs in a 13-minute run. The concentrations of the drugs found were compared with their concentrations determined in the fresh liver tissues in a previous study. In the study, 14 cases with forensic interest were included with the main objective of the analysis and the study of the stability and the distribution of drugs of abuse and pharmaceuticals in the human liver and the formalin solution during preservation. The results showed that the number of detected compounds in the first month was significantly lower than the compounds found in fresh tissues. The effect of formalin was catalytic, and few substances could be detected. Specifically, out of the 86 positive detections of the monitored substances in the fresh tissues (in which 25 different substances were found), only 32 (37%) remained detectable 3 months after, 20 (23%) 6 months after and 15 (17%) 12 months after.

Determination of fentanyl and norfentanyl in cerumen in the setting of postmortem investigation.

Cerumen is an emerging alternative biological matrix in the field of forensic toxicology. An ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry [UHPLC-MS/MS] method for the determination of fentanyl and norfentanyl in cerumen was developed and applied in a mixed drug toxicity fatal case. The method was found to be selective and sensitive (LOQ: 0.05 ng/mg for fentanyl and 0.02 ng/mg for norfentanyl), while validation included recovery, carryover, short-term stability, matrix effect, accuracy, and precision (RSD%). Accuracy ranged from 83.1% to 103.5%, while intra- and inter-day precision ranged from 8.6% to 13.1% and from 8.3% to 15.8%, respectively. Matrix effect experiments showed that matrix did not significantly affect signal intensity (82.3%-96.8%). Short-term stability concerning sample extracts was found satisfactory. Fentanyl and norfentanyl were detected in cerumen at a concentration of 1.17 and 0.36 ng/mg respectively. The findings in cerumen corroborate the cause of death and suggest that cerumen is a potential specimen for detecting drugs of abuse in forensic cases.

Detection and determination of C12-, C14-, C16-alkyldimethylamines in human blood using gas chromatography-mass spectrometry.

RATIONALE: N,N-Dimethyldodecylamine is produced from lauryl alcohol and dimethylamine. C12-C16 alkyldimethylamines are used as intermediates for the manufacture of amineoxides and quaternary amino compounds. In the present study a gas chromatography-mass spectrometry (GC/MS) method for the determination of C12-C16 alkyldimethylamines in blood was developed and validated. The reason for this study was the detection of the above compounds in the postmortem blood sample of a fatal suicide case. METHODS: Analysis of amines was performed using a gas chromatograph (Agilent Technologies 7890A) with an MS 5975C inrXL, EI/CI MSD with triple-axis detector in selected ion monitoring mode, after liquid-liquid extraction. Four different organic solvents (butyl acetate, ethyl acetate, n-hexane and n-heptane) were used for the optimization of the extraction procedure, resulting in ethyl acetate being the solvent of choice for the extraction procedure. A QuEChERS step was applied (20 mg of MgSO4 , 5 mg of NaCl) to 1 mL of blood and pH was adjusted at 12 (K2 CO3 buffer solution). After the addition of the extraction solvent, samples were vortexed, centrifuged and directly injected into the GC/MS system. RESULTS: In validation, the method was found to be selective and sensitive (limit of detection from 0.3 to 0.5 ng/mL, limit of quantitation from 10.0 to 20.0 ng/mL), whilst validation included recovery, stability, accuracy and precision (relative standard deviation). Validation results were found satisfactory: intra- and interday precision ranged from 0.4% to 2% and from 0.6% to 1.9% respectively, while intra- and interday accuracy ranged from 87% to 109% and from 86% to112.8%. C12-C16 alkyldimethylamines were detected in blood samples at a concentration of 8.39 µg/mL (C12), 3.01 µg/mL (C14) and 0.42 µg/mL (C16). CONCLUSIONS: A rapid, sensitive and reliable method was developed for the determination of C12-C16 alkyldimethylamines in postmortem blood, after optimization of the sample preparation procedure, and finally successfully applied to a real postmortem blood sample from a fatal case involving these compounds.

Risk factors for fatal drowning in a Greek region: a retrospective case-control study.

BACKGROUND: Fatal drowning is one of the leading causes of unintentional injury mortality worldwide and a persistent public health concern in Greece. While several pathologic and sociodemographic contributing factors have been previously identified, these have not been extensively investigated in conjunction with the effects of psychoactive substances. METHODS: A retrospective case-control study of drowning deaths was conducted in the Greek regions of Northern Greece and Thessaly during a 10-year period. A regression model was constructed examining differences in detected substances, autopsy findings and sociodemographic characteristics between 240 victims of unintentional fatal submersion and 480 victims of other causes of sudden or violent death. RESULTS: The majority of victims were males (69.4%) and foreign nationality was associated with increased odds of drowning. Cardiomegaly and coronary bypass grafts were significantly more likely to have been recorded among drowning victims, while the frequency of other circulatory system disorders was also elevated. Several of these findings were potential arrhythmogenic substrates which could adversely interact with the diving reflex. Selective serotonin reuptake inhibitors (SSRIs) were the most commonly detected pharmacological group (9.0%), and along with tramadol, there was an increased likelihood of exposure to them. These drugs have been previously associated with QT prolongation and other adverse effects which may contribute to fatal outcomes in a seawater environment. In contrast, there was a decreased risk of exposure to dependence-inducing drugs and paracetamol. CONCLUSIONS: Male sex, older age, foreign nationality and cardiovascular disease predisposed individuals to an elevated risk of fatal submersion. SSRI antidepressants and tramadol may contribute to this outcome.

A UHPLC-MS-MS Method for the Determination of 84 Drugs of Abuse and Pharmaceuticals in Blood.

The analysis of blood samples for forensic or clinical intoxication cases is a daily routine in an analytical laboratory. The list of 'suspect' drugs of abuse and pharmaceuticals that should be ideally screened is large, so multi-targeted methods for comprehensive detection and quantification are a useful tool in the hands of a toxicologist. In this study, the development of an ultra-high performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method is described for the detection and quantification of 84 drugs and pharmaceuticals in postmortem blood. The target compounds comprise pharmaceutical drugs (antipsychotics, antidepressants, etc.), some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines, benzodiazepines and new psychoactive substances. Sample pretreatment was studied applying a modified Mini-QuEChERS single step, and the best results were obtained after adding a mixture of 20 mg MgSO4, 5 mg K2CO3 and 5 mg NaCl together with 600 µL of cold acetonitrile in 200 µL of sample. After centrifugation, the supernatant was collected for direct injection. LC-MS analysis took place on a C18 column with a gradient elution over 17 min. The method was found to be selective and sensitive, offering limits of detection ranging from 0.01 to 9.07 ng/mL. Validation included evaluation of limit of quantification, recovery, carryover, matrix effect, accuracy and precision of the method. The method performed satisfactorily in relation to established bioanalytical criteria and was therefore applied to the analysis of blood obtained postmortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

Metabolic Phenotyping Study of Mouse Brains Following Acute or Chronic Exposures to Ethanol.

The chronic and acute effect of ethanol administration on the metabolic phenotype of mouse brain was studied in a C57BL/6 mouse model of ethanol abuse using both untargeted and targeted ultra performance liquid chromatography-tandem mass spectrometry. Two experiments based on either chronic (8 week) exposure to ethanol of both male and female mice or acute exposure of male mice for 11 days, plus 2 oral gavage doses of 25% ethanol, were undertaken. Marked differences were found in amino acids, nucleotides, nucleosides, and related metabolites as well as a number of different lipids. Using untargeted metabolite profiling, acute ethanol exposure found significant decreases in several metabolites including nucleosides, fatty acids, glycerophosphocholine, and a number of phospholipids, while chronic exposure resulted in increases in several amino acids with notable decreases in adenosine, acetylcarnitine, and galactosylceramides. Similarly, targeted metabolite analysis, focusing on the hydrophilic fraction of the brain tissue extract, identified significant decreases in the metabolism of amino acids and derivatives, as well as purine degradation especially after chronic exposure to ethanol.

Development of a UHPLC-MS/MS method for the determination of 84 pharmaceuticals and drugs of abuse in human liver.

Analysis of post-mortem liver for toxicological reasons is a considerable option when blood is unavailable. The development of analytical methods for tissue specimens is not as straightforward as for biological fluids as tissue presents challenges to the analytical chemist. The present study reports the development of a UHPLC-MS/MS method for the detection and quantification of 84 drugs and pharmaceuticals in human liver. The selected target drugs include pharmaceutical drugs and drugs of abuse. Sample preparation was studied using QuEChERS and different ratios of solvent volume and sample mass. Best results were attained by homogenizing 1 g of liver with acetonitrile K2CO3 buffer (pH = 10), QuEChERS salts MgSO4/ NaCl (1st purification step) and PSA/ 150 mg MgSO4 (2nd purification step). The extracted sample was analysed on UHPLC-MS/MS in multiple reaction monitoring mode (MRM) on a reversed-phase (Acquity BEH C18) column. Elution was accomplished by gradient program of mobile phase A: water, 0.1% formic acid and B: methanol, 0.1% formic acid that lasted 17 min. The method was specific, without interferences from the complex matrix. Sensitivity was satisfactory with limit of detection (LOD) ranging from 0.01 ng/g to 4.94 ng/g. Validation study was based on the guidelines of international bodies and included evaluation of recovery, carry-over, matrix effect, accuracy, stability, and precision of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of liver tissue obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

Α Simple Method for the Determination of Lacosamide in Blood by GC-MS.

Lacosamide is a functionalized amino acid with antiepileptic function. Therapeutic drug monitoring (TDM) in patients for lacosamide is critical as it allows clinicians to control epileptic seizures. A single liquid-liquid extraction step was applied for the extraction of lacosamide from whole blood samples which were thereafter analyzed by GC-MS. Optimum extraction conditions were selected on the basis of experiments with various solvents at different pHs, indicating ethyl acetate at pH 12 as the most efficient parameters for the extraction of lacosamide. Method exhibited linearity from 2 to 100 µg/mL with R2  = 0.998. Accuracy and precision were evaluated at three concentrations and found to be within acceptable limits. LOD and LOQ were determined at 0.1 and 0.5 µg/mL, respectively. Lacosamide was found to be stable at storage conditions. The developed method was applied successfully in clinical samples and postmortem blood sample from an overdose case.

Study of Fecal and Urinary Metabolite Perturbations Induced by Chronic Ethanol Treatment in Mice by UHPLC-MS/MS Targeted Profiling.

Alcoholic liver disease (ALD) as a consequence of ethanol chronic consumption could lead to hepatic cirrhosis that is linked to high morbidity and mortality. Disease diagnosis is still very challenging and usually clear findings are obtained in the later stage of ALD. The profound effect of ethanol on metabolism can be depicted using metabolomics; thus, the discovery of novel biomarkers could shed light on the initiation and the progression of the ALD, serving diagnostic purposes. In the present study, Hydrophilic Interaction Liquid Chromatography tandem Mass Spectrometry HILIC-MS/MS based metabolomics analyisis of urine and fecal samples of C57BL/6 mice of both sexes at two sampling time points was performed, monitoring the effect of eight-week ethanol consumption. The altered hepatic metabolism caused by ethanol consumption induces extensive biochemical perturbations and changes in gut microbiota population on a great scale. Fecal samples were proven to be a suitable specimen for studying ALD since it was more vulnerable to the metabolic changes in comparison to urine samples. The metabolome of male mice was affected on a greater scale than the female metabolome due to ethanol exposure. Precursor small molecules of essential pathways of energy production responded to ethanol exposure. A meaningful correlation between the two studied specimens demonstrated the impact of ethanol in endogenous and symbiome metabolism.

Alprazolam and Zolpidem in Skeletal Tissue of Decomposed Body Confirms Exposure.

In several medico-legal cases, bone samples analysis may provide the only source of toxicological information. This case study reports the analysis of a human bone specimen, belonging to a 46-year-old man, found 3 months after his death due to cervical-thoracic injuries in a motorcycle accident. Bone specimen was the only available material for toxicological analysis, among few skull hair and rotten skin. Analysis was performed by a newly developed and validated ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS) method, following simple and efficient sample pretreatment. The results were in accordance with the man's medical record: Alprazolam and zolpidem were found at 2.2 and 5.4 ng/g of bone, respectively. Both these drugs were prescribed to the deceased.

Determination of drugs of abuse and pharmaceuticals in skeletal tissue by UHPLC-MS/MS.

In several medico legal cases bone analysis may provide the only source of toxicological information. The present study reports the development of an UHPLC-MS/MS method for the detection and quantification of 27 drugs and pharmaceuticals in human bones. The target compounds comprise pharmaceuticals (antipsychotics and antidepressants) and some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines and benzodiazepines. Sample pretreatment was studied and the best results were obtained after extraction with methanol, stirring and ultra-sonication. The extract, after filtration, evaporation and reconstitution was analysed on a reversed-phase column (C18) in gradient elution over 17min. The method was found to be selective, and sensitive offering limits of detection (LOD) from 0.03 to 1.35ng/g of bone. Validation included evaluation of limit of quantification (LOQ), recovery, carry-over, matrix effect, accuracy and precision (RSD%) of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of bone and bone marrow obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in bones from legal cases where the analysis of blood was not feasible.