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.

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.