Long-Term Stability of Benzodiazepines and Z-Hypnotic Drugs in Blood Samples Stored at Varying Temperatures.

Benzodiazepines (BZDs) and Z-drugs are among the most commonly prescribed pharmaceuticals in the world and are considered standard care for various mental illnesses and for the treatment of sleeping and anxiety disorders, alcohol withdrawal, muscle spasms and epilepsy. Some BZDs are not allowed as pharmaceuticals in many countries, and they are used as designer benzodiazepines (DBZDs). All these compounds are typically screened in routine toxicological analyses for forensic purposes. Knowledge of time-dependent decreases in drug concentrations during storage or transport of samples is of considerable significance and allows forensic toxicologists to achieve reliable data, proper interpretation and high-quality results. The aim of this study was to evaluate changes in the amounts of selected BZDs, DBZDs and Z-drugs in blood samples stored at various temperatures. The study involved BZDs (19), DBZDs (3) and Z-drugs (2) spiked into blank blood. Subsequently, the blood samples were stored at various temperatures (room temperature, 4°C, -20°C and -80°C) for up to 6 months. Analyses were performed at 1- to 2-week intervals using liquid chromatography-tandem mass spectrometry. The stability of compounds was evaluated under four temperature conditions over a 6-month period. Some BZDs were stable at all temperatures tested (e.g., diazepam, oxazepam, nordazepam and prazepam) with a degradation rate of only 0-10%. The highest instability was observed for analyte samples kept at room temperature, and the losses in content for some compounds, e.g., lorazepam and chlordiazepoxide, were almost 100%. For other compounds, the stability was clearly different at each tested temperature. To the best of our knowledge, this is one of the first such comprehensive study of the long-term stability of BZDs covering a wide range of different storage temperatures.

Fatal Suicidal Intoxication with Pentoxifylline Complicated by Cardiovascular Disorders.

Pentoxifylline is a xanthine derivative used in vascular disorders that is recognized as a safe drug for patients. The paper describes a rare case of fatal and suicidal pentoxifylline poisoning in an 82-year-old man with multiple preexisting diseases (arterial hypertension, coronary artery disease, type 2 diabetes, and intermittent claudication). The patient was admitted to the clinical toxicology unit approximately 2 h after the overdose and died 36 h after the admission despite intensive care. Multiple arterial blood gas analyses and other laboratory tests were performed during the hospitalization and are reported in the paper. Postmortem examination of the biological material was carried out with the use of histopathological techniques. The toxicological studies using chromatographic techniques coupled with mass spectrometry showed that postmortem blood levels of pentoxifylline have been found in the range which is described in the available literature to be toxic and lethal. The analysis of test results and clinical data showed that the patient died as a result of increasing circulatory and respiratory failure, complicated by disorders of the acid-base and electrolyte balance (respiratory alkalosis, concomitant lactic acidosis, and hypokalemia), hyperglycemia, and coagulation disorders.

Development and validation of a method for the simultaneous analysis of fatty acid ethyl esters, ethyl sulfate and ethyl glucuronide in neonatal meconium: application in two cases of alcohol consumption during pregnancy.

Alcohol consumption during pregnancy constitutes one of the leading preventable causes of birth defects and neurodevelopmental disorders in the exposed children. Fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG) and ethyl sulfate (EtS) have been studied as potential biomarkers of alcohol consumption. However, most analytical approaches proposed for their analysis in meconium samples consist of separated extraction procedures requiring the use of two meconium aliquots, which is costly in terms of both time and materials. Therefore, the aim of this study was to develop and validate a method for the simultaneous extraction of 9 FAEEs, EtG and EtS from one meconium aliquot. The sample was homogenized using methanol, and then FAEEs were extracted with hexane while EtG and EtS were isolated using acetonitrile. Then, extracts were applied to solid-phase extraction columns and analysed by gas chromatography mass spectrometry (FAEEs) and liquid chromatography tandem mass spectrometry (EtG and EtS). Calibration curves were linear with r values greater than 0.99. The LODs ranged from 0.8 to 7.5 ng/g for FAEEs and were 0.2 ng/g and 0.8 ng/g for EtS and EtG, respectively. LOQs ranged from 5 to 25 ng/g for FAEEs and were 1 ng/g and 2.5 ng/g for EtS and EtG, respectively. Accuracies and precisions were between 93.8 and 107% and between 3.5 and 9.7%, respectively. The recovery values ranged from 89.1 to 109%. The method proved to be sensitive, specific, simple and fast and allowed for the reduction of the amount of organic solvent used for extraction compared to other published data while higher recoveries were obtained. The method was used for analysis of meconium samples in two cases of mothers who were consuming alcohol during pregnancy.

Fatal N-Ethylhexedrone Intoxication.

N-Ethylhexedrone [2-(ethyloamino)-1-phenylhexan-1-one; α-ethylaminohexanophenone (NEH)] is one of the most recent synthetic cathinones that appeared on the illegal market in late 2015. The majority of information concerning the model of consumption of NEH and its impact on the body originates only from self-reports from gray literature websites and drug forums. There are only limited data associated with the concentrations of NEH in blood samples available in the literature. This article presents a case of fatal NEH intoxication and a method for the determination of this substance in whole blood. A 21-year-old man without any diagnosed diseases was admitted to the hospital due to disorientation, aggression and finally loss of consciousness. Hyperthermia (>41°C), tachycardia (>160 beats per minute), tachypnea (20 breaths per minute), blood pressure (110/60 mmHg) and acute kidney failure were diagnosed. After a few hours of hospitalization, the patient died. A plastic bag with a white powder was found in his underwear. Analysis of the powder by another laboratory revealed cocaine hydrochloride; however, no cocaine or its metabolites were found in the biological material upon testing in our laboratory. Therefore, re-analysis of the powder was performed, and NEH was identified. Liquid-liquid extraction followed by liquid chromatography-triple quadrupole-mass spectrometry (LC-MS/MS) analysis were used for the determination of NEH in blood. The validation parameters were as follows: calibration range 1-250 ng/mL, accuracy 106.5-109.9%, precision 3.5-6.3%, recovery 90.1-96.9%, limit of detection 0.07 ng/mL and limit of quantification 1 ng/mL. NEH was quantified in the blood at a concentration of 145 ng/mL. Additionally, amphetamine at low concentrations and 11-nor-9-karboksy-Δ9-tetrahydrokannabinol (THC-COOH) were detected. Our study provided information on the possible lethal concentration and toxidrome that clinicians can observe for NEH-intoxicated patients and can be helpful during the preparation of toxicology analysis reports for a court of law for proper data interpretation.

Rapid and simple multi-analyte LC-MS/MS method for the determination of benzodiazepines and Z-hypnotic drugs in blood samples: Development, validation and application based on three years of toxicological analyses.

Benzodiazepines (BZDs) and Z-drugs have been particularly important treatments for sleeping and anxiety disorders for many years. However, recently, a number of new benzodiazepines (named designer benzodiazepines, DBZDs) were synthesised, but some of them have never been used in the clinic; they reached the black drug market as new psychoactive substances and are used for recreational purposes. The abuse of these substances has led to many crimes and even deaths. Therefore, it is necessary to develop new methods for their quantification for forensic and clinical toxicology. A liquid chromatography-tandem mass spectrometry-based method was developed for the simultaneous determination of 20 classical BZDS, 4 DBZDs and 3 Z-hypnotic drugs in human whole blood. As a sample preparation step, liquid-liquid extraction requiring the use of only 0.5 mL of blood sample and 1 mL of extraction solvent was applied. The selectivity, linearity, carry-over effects, limits of detection (LOD) and quantification (LOQ), precision, accuracy (both intra- and inter-day assays) and recovery were evaluated for validation. Calibration curves were linear with r values > 0.98. The LODs ranged from 0.01 to 0.33, and the LOQs were assumed to be 1 ng/mL. Inter-day precisions and accuracies were in the ranges of 87.8% - 108.5% and 1.8% - 11.2%, respectively. The recovery values ranged from 81.0% to 106.7%. The developed method proved to be sensitive, specific, simple, and fast and can be quickly modified and expanded for new compounds by the optimization of MRM. The method was applied for analysis of blood samples in 145 toxicological cases over a three-year study (2017 - 2019), which allowed us to obtain information on the prevalence of the use of these substances. The most frequently determined compounds were nordazepam (87 cases; 60%), diazepam (81 cases; 55.9%), temazepam (72 cases; 49.7%), oxazepam (56 cases; 38.7%), and midazolam (36 cases; 24.8%). The ranges of concentrations were wide and are presented as box plots. The results were used for the preparation of medico-legal opinions, which proved the utility of the method for routine toxicology analyses.

Biomarkers of Alcohol Consumption in Body Fluids - Possibilities and Limitations of Application in Toxicological Analysis.

BACKGROUND: Ethyl alcohol is the most popular legal drug, but its excessive consumption causes social problems. Despite many public campaigns against alcohol use, car accidents, instances of aggressive behaviour, sexual assaults and deterioration in labor productivity caused by inebriated people is still commonplace. Fast and easy diagnosis of alcohol consumption is required in order to introduce proper and effective therapy, and is crucial in forensic toxicology analysis. The easiest method to prove alcohol intake is determination of ethanol in body fluids or in breath. However, since ethanol is rapidly metabolized in the human organism, only recent consumption can be detected using this method. Because of that, the determination of alcohol biomarkers was introduced for monitoring alcohol consumption over a wider range of time. OBJECTIVE: The objective of this study was to review published studies focusing on the sample preparation methods and chromatographic or biochemical techniques for the determination of alcohol biomarkers in whole blood, plasma, serum and urine. METHODS: An electronic literature search was performed to discuss possibilities and limitations of application of alcohol biomarkers in toxicological analysis. RESULTS: Authors described the markers of alcohol consumption such as: ethanol, its nonoxidative metabolites (ethyl glucuronide, ethyl sulfate, phosphatidylethanol, ethyl phosphate, fatty acid ethyl esters) and oxidative metabolites (acetaldehyde and acetaldehyde adducts). We also discussed issues concerning the detection window of these biomarkers, and possibilities and limitations of their use in routine analytical toxicology for monitoring alcohol consumption or sobriety during alcohol therapy.

A rapid and sensitive method for the quantitative analysis of ibuprofen and its metabolites in equine urine samples by gas chromatography with tandem mass spectrometry.

Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 µL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optimized and validated according to the principles for bioanalytical methods. The assay achieved satisfactory validation parameters, namely, recovery (92.2-105%), interday accuracy (92.5-106%), and precision (0.3-4.4%) for all investigated compounds as well as limits of quantification of 50 ng/mL for ibuprofen, 2-hydroxyibuprofen, and carboxyibuprofen, 25 ng/mL for 1-hydroxyibuprofen and 100 ng/mL for 3-hydroxyibuprofen. The applicability of the method was evaluated by the analysis of five real urine samples collected from different horses after drug administration. In view of the low limits of quantification, high selectivity, repeatability, and recovery, the procedure can be utilized for laboratory applications, including the control of ibuprofen abuse in equestrian sports for anti-doping purposes and drug/pharmaceutical mentality investigations.

Blood concentrations of a new psychoactive substance 4-chloromethcathinone (4-CMC) determined in 15 forensic cases.

PURPOSE: The 4-chloromethcathinone (4-CMC) is a synthetic derivative of cathinone and belongs to new psychoactive substances. Neither data on the effects of 4-CMC on the human body, nor on nontoxic, toxic and lethal concentrations in biological materials have been published in the literature. This paper describes the results of an analysis of the blood concentrations of 4-CMC determined in 15 forensic cases related to nonfatal intoxication including driving under the influence, and fatalities including overdoses, suicide and traffic accidents. METHODS: A new method for the quantification of 4-CMC using gas chromatography-mass spectrometry (GC-MS) was developed. The symptoms of 4-CMC use were also studied based on an analysis of the documents prepared during the collection of samples or at autopsies. RESULTS: The limits of detection and quantification of the method for blood samples were 0.3 and 1 ng/mL, respectively. The calibration curve was linear in the studied concentration range (1-500 ng/mL) with the correlation coefficient at 0.9979. The extraction recoveries varied in the range of 94.3-98.8%. The accuracy and precision were acceptable. The determined concentrations in nonfatal cases ranged from 1.3 to 75.3 ng/mL, and in fatalities from 56.2 to 1870 ng/mL. CONCLUSIONS: Our study can assist in the recognition of the possible effects caused by 4-CMC and can be helpful during the preparation of forensic toxicological opinions for courts of law. The validation parameters indicate the sensitivity and accuracy of the method. This is the first work presenting a validated method for the determination of 4-CMC in blood samples by GC-MS.

Evaluation of flavour profiles in e-cigarette refill solutions using gas chromatography-tandem mass spectrometry.

Many flavour compounds that are present in e-liquids for e-cigarettes are responsible for specific tastes and smoking sensations for users. Data concerning content and specific types of flavours is often limited and unknown to users. The aim of the research was to define and compare flavour profiles of e-liquids with the same group taste from different manufacturers. Gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was used to separate and identify 90 popular compounds (98, including isomers) of interest. The developed method was validated in terms of accuracy (88-113%) for three spiking levels and the intra-day (0.2-13%) and inter-day precision (1-10%). Limits of quantitation were in the range of 10-816 ng/mL, while the matrix effects for 80% of the compounds were at negligible levels. The proposed method is rapid, simple and reliable and uses a green and modern GC-MS/MS technique. Twenty-five samples of five different flavours (tobacco, strawberry, cherry, menthol and apple) from five different producers were analysed, and the determined compounds were categorized and differentiated. The approach proposed in this study allowed for the evaluation of which compounds/group of compounds are responsible for taste and to distinguish common flavour compounds among the investigated brands for each flavour. Furthermore, the presented research can be considered in future toxicological studies.

Application of gas chromatography-tandem mass spectrometry for the determination of amphetamine-type stimulants in blood and urine.

Amphetamine, methamphetamine, phentermine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) are the most popular amphetamine-type stimulants. The use of these substances is a serious societal problem worldwide. In this study, a method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) with simple and rapid liquid-liquid extraction (LLE) and derivatization was developed and validated for the simultaneous determination of the six aforementioned amphetamine derivatives in blood and urine. The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. The most important advantage of the method is the minimal sample volume (as low as 200µL) required for the extraction procedure. The validation parameters, i.e., the recovery (90.5-104%), inter-day accuracy (94.2-109.1%) and precision (0.5-5.8%), showed the repeatability and sensitivity of the method for both matrices and indicated that the proposed procedure fulfils internationally established acceptance criteria for bioanalytical methods The procedure was successfully applied to the analysis of real blood and urine samples examined in 22 forensic toxicological cases. To the best of our knowledge, this is the first work presenting the use of GC-MS/MS for the determination of amphetamine-type stimulants in blood and urine. In view of the low limits of detection (0.09-0.81ng/mL), limits of quantification (0.26-2.4ng/mL), and high selectivity, the procedure can be applied for drug monitoring in both fatal and non-fatal intoxication cases in routine toxicology analysis.

Comprehensive determination of flavouring additives and nicotine in e-cigarette refill solutions. Part II: Gas-chromatography-mass spectrometry analysis.

Flavouring compounds are an essential part of e-liquid products for cigarettes. In general, they are regarded as safe for ingestion, but they may have unrecognized risks when they are inhaled. In some cases, manufactures do not currently abide by the Tobacco Products Directive (2014/40/EU) and do not declare the detailed contents of e-liquids on their labels. To help evaluate the health impact of flavouring substances, there is a need for comprehensive approaches to determine their concentrations in e-liquids. For this purpose, a GC-EI-MS method was developed and validated for the simultaneous determination of 46 commonly used flavour additives in e-liquids. The proposed method performed well in terms of the key validation parameters: accuracy (84-113%), inter-/intra-day precision: 0.1-10% and 1-11%, respectively, and sensitivity (limit of detection: 3-87ng/mL). The sample preparation step was based on a simple "dilute & shoot" approach. This study is a complementary method to the LC-MS/MS procedure described in Part I. Both approaches are suitable for the comprehensive determination of 88 flavouring compounds and nicotine and can be used as tools for the rapid evaluation of the quality and safety of e-cigarette products.