Åse Marit Leere Øiestad

Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid-liquid extraction and UPLC-MS/MS

A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 μL whole blood were added 500 μL of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut(®) columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d(5), flunitrazepam-d(7) and nitrazepam-d(5), respectively. The analytes were separated using UPLC with a 2.1×100 mm BEH C(18)-column, 1.7 μm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 μM for flunitrazepam to the highest of 20 μM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R(2) values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 μM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.

Gas phase reactivity of small cationic cobalt clusters towards methanol

Abstract The gas phase reactivity of small cationic cobalt clusters, Co n + (n=1–12) , towards methanol has been investigated using Fourier transform ion cyclotron resonance mass spectrometry. Sequential addition of methanol molecules to the clusters was observed to be the dominating process, with the exception of Co4+ and Co5+, for which dehydrogenation of methanol was the dominating initial reaction step. Isotopic labelling studies with CD3OH showed that the dehydrogenation is a 1,1-elimination reaction involving the methyl group. Loss of a cobalt atom upon methanol addition was also observed to be a facile process, being most pronounced for Co2+, Co3+ and Co6+.

Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography–tandem mass spectrometry ☆

An ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid-liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), a flow rate of 0.5mL/min, and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode, using two transitions for the compounds and one transition for the IS. The run time of the method was 8min including equilibration time. The calibration curves had R(2) values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0nmol/L to 0.10μmol/L (1.3-26ng/mL) and the limits of detections (LODs) from 1.0 to 20nmol/L (0.24-5.3ng/mL). LLOQ corresponds to 0.28-5.5pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC-MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC-MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.

Lethal poisonings with AH-7921 in combination with other substances

AH-7921 is a synthetic μ-opioid agonist, approximately equipotent with morphine. We report the death of two young individuals after ingestion of AH-7921 in combination with other psychoactive drugs. In the first case a young man died shortly after ingesting Internet drugs. Toxicological analysis of post mortem peripheral blood revealed AH-7921 (0.43 mg/L), 2-FMA (0.0069 mg/L) and 3-MMC (0.0021 mg/L) as well as codeine (0.42 mg/L), codeine-6-glucuronide (0.77 mg/L) and acetaminophen (18.7 mg/L). The second case involved a young female found dead at home. The only positive finding at medicolegal autopsy was needle marks. Toxicological analysis revealed AH-7921 (0.33 mg/L), methoxetamine (MXE) (0.064 mg/L), etizolam (0.27 mg/L), phenazepam (1.33 mg/L), 7-aminonitrazepam (0.043 mg/L), diazepam (0.046 mg/L), nordiazepam (0.073 mg/L), and oxazepam (0.018 mg/L) in blood. In both cases intoxication with AH-7921 in combination with other psychoactive drugs was considered to be the cause of death.

Evaluation of 13C- and 2H-labeled internal standards for the determination of amphetamines in biological samples, by reversed-phase ultra-high performance liquid chromatography–tandem mass spectrometry

Stable isotope-labeled internal standards (SIL-ISs) are often used when applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze for legal and illegal drugs. ISs labeled with (13)C, (15)N, and (18)O are expected to behave more closely to their corresponding unlabeled analytes, compared with that of the more classically used (2)H-labeled ISs. This study has investigated the behavior of amphetamine, (2)H3-, (2)H5, (2)H6-, (2)H8-, (2)H11-, and (13)C6-labeled amphetamine, during sample preparation by liquid-liquid extraction and LC-MS/MS analyses. None or only minor differences in liquid-liquid extraction recoveries of amphetamine and the SIL-ISs were observed. The chromatographic resolution between amphetamine and the (2)H-labeled amphetamines increased with the number of (2)H-substitutes. For chromatographic studies we also included seven additional (13)C6-amphetamines and their analytes. All the (13)C6-labeled ISs were co-eluting with their analytes, both when a basic and when an acidic mobile phase were used. MS/MS analyses of amphetamine and its SIL-ISs showed that the ISs with the highest number of (2)H-substitutes required more energy for fragmentation in the collision cell compared with that of the ISs with a lower number. The findings, in this study, support those of previous studies, showing that (13)C-labeled ISs are superior to (2)H-labeled ISs, for analytical purposes.

Loss of H2 from CH2NH2+ and NHNH2+. Reaction mechanisms and dynamics from observation of metastable ion fragmentations and ab initio calculations

Abstract On the basis of the measurement of the translational energy release distribution, previous isotope labelling experiments, extensive ab initio calculations of relevant parts of the potential energy surface and an ab initio direct dynamics calculation, it is concluded that the loss of H 2 from CH 2 NH 2 + ( 1 ) occurs in two steps. The first step, which is irreversible, is a hydrogen rearrangement giving the high energy isomer CH 3 N + ( 2 ). The existence of this isomer is transient and it decomposes “on the fly” to give the products CHNH + ( 3 ) and H 2 ( 4 ) via the transition structure TS 2 3 + 4 . Passage of this transition structure brings the reacting system to a part of the potential energy surface which provides a strong repulsive force between the two fragments. This gives rise to the observed large translational energy release. The mechanism also explains why the reaction is a specific 1,2-H 2 elimination. NHNH 2 + ( 5 ) decomposes in one step via TS 5 6 + 4 to give NNH + ( 6 ) and H 2 ( 4 ), also with a large translational energy release. There is an alternative higher energy route which occurs in two steps via the isomer NNH 3 + ( 7 ).

Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC-MS/MS

Collection of oral fluid (OF) is easy and non-invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high-throughput ultra-high-performance liquid chromatography-tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96-well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry-over. Inter-assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7-amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium-labelled and 13 C-labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12-month period. Copyright

Validated methods for determination of neurotransmitters and metabolites in rodent brain tissue and extracellular fluid by reversed phase UHPLC–MS/MS

Fast and sensitive methods for simultaneous determination of dopamine (DA), the two DA-metabolites homovanillic acid (HVA) and 3-methoxytyramine (3-MT), serotonin (5-HT) and the 5-HT-metabolite 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), acetylcholine (ACh), glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rodent brain tissue (1.0-4000nM) and extracellular fluid (ECF) (0.5-2000nM) based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) have been developed. Of the three different sample preparation methods for brain tissue samples tested, a simple and rapid protein precipitation procedure with formic acid was found to give the best results. The neurotransmitters (NTs) and NT metabolites were separated using UHPLC with an Acquity UPLC HSS T3 C18 column (2.1×100mm, 1.8μm particle size) with acidic mobile phase. Gradient elution with methanol was used and quantification was performed using multiple reaction monitoring (MRM). The total run time was 5.2min including equilibration time. The methods were validated by determining calibration model, intra- and inter-day precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), matrix effects (ME), carry-over and stability. Surrogate analytes were used to enable determination of the recovery and ME of the endogenous analytes in brain tissue. The methods were applied for determination of NTs at basal levels in rodent brain ECF and brain tissue homogenate. The developed methods are valuable tools in the studies of mechanisms of drugs of abuse, and neurologic and psychiatric disease.

Oral fluid drug analysis in the age of new psychoactive substances

Oral fluid has become an important matrix for drugs of abuse analysis. These days the applicability is challenged by the fact that an increasing number of new psychoactive drugs are coming on the market. Synthetic cannabinoids and synthetic cathinones have been the main drug classes, but the diversity is increasing and other drugs like piperazines, phenethylamines, tryptamines, designer opioids and designer benzodiazepines are becoming more prevalent. Many of the substances are very potent, and low doses ingested will lead to low concentrations in biological media, including oral fluid. This review will highlight the phenomenon of new psychoactive substances and review methods for oral fluid drug testing analysis using on-site tests, immunoassays and chromatographic methods.

Loss of H 2 from CH 3 NH 3 + , CH 3 OH 2 + and CH 3 FH + . Reaction mechanisms and dynamics from observation of metastable ion fragmentations and ab initio calculations

The distributions of the translational energy (T) released during loss of H2 from metastable CH3NH3+ and CH3OH2+ ions have been measured. For both reactions the most probable T value accounts for approximately 3/4 of the reactions reverse critical energy. Subject to the same experimental conditions CH3FH+ ions do not give rise to any measurable signal for H2 loss. The relevant parts of the potential energy surfaces of all three reactions were investigated using various ab initio quantum chemical computational schemes. Ab initio direct dynamics calculations were performed to obtain representative reaction trajectories. Translational energy releases computed at the end of these trajectories (where the fragments have separated) agree with the corresponding experimental figures. The three reactions follow a common polar mechanism which involves an initial transfer of a proton from the most basic centre (N, O or F) towards one of the hydrogen atoms of the methyl group. During this stage the proton polarizes the electrons around the methyl hydrogen to give it some hydride character, and in the transition state this has resulted in an embryonic H–H bond. Further electron reorganization during the concerted bond breaking and bond making process leads to a strong repulsive force along the reaction coordinate as the two fragments depart. This accounts for the highly non-statistical partitioning of the available potential energy into relative translation between the two fragments formed.