•Isotonitazene quantification in human post-mortem tissues, blood and hair.•Isotonitazene accumulation in brain and hearth and very low concentration in liver.•Isotonitazene powder determination ...using GC-MS, NMR.•Very low isotonitazene concentration in blood can be fatal.
The paper describes the first three deaths reported in Europe involved in isotonitazene consumption, a potent benzimidazole derivate opioid consumed in the recreational drug scene. Isotonitazene powder and purity determination was performed on the sample collected in the first death scene by NMR, HRMS, GC-FTIR, ATR-FTIR and GC–MS. Isotonitazene purity was determined by GC–MS analysis and proton NMR, and was defined to be above 95 % and 98 %, respectively.
Quantification of isotonitazene in biological samples was performed using a targeted analysis based on SPE extraction and ultra-high performance liquid chromatography tandem mass spectrometry.
The isotonitazene median concentration in femoral whole blood was 1.20ng/mL. Isotonitazene concentration in hair was similar or even lower compared to that seen in fentanyl abusers. Isotonitazene distribution in tissues converges in the brain, lungs and heart, respectively. Surprisingly, isotonitazene concentration in liver is the lowest measured for all tissues and fluids analyzed.
Based on circumstantial evidence, autopsy findings and the results of the toxicological analysis, the medical examiner concluded that the cause of all three deaths was an acute intoxication with isotonitazene.
Since isotonitazene toxic concentration levels are very low, the consumption of this new psychoactive drug is a real hazard for human health.
•Methadone enantiomers separation and quantitation using SFC-MS/MS in DBS and DMS.•Methadone enantiomers separation and quantitation using LC-MS/MS in DBS and DMS.•SFC-MS/MS and LC-MS/MS comparison ...between method validation parameters.•Quantification and ratio of R- and S-methadone in real post mortem samples fluids.
This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of rs = 0.9978 for R-methadone and rs = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results.
Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation.
For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ...ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC–QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., β-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.
The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may ...contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C₁₈ reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.
Recent census data has found that roughly 40% of adults 65 years and older not only consume alcohol but also drink more of it than previous generations. Older drinkers are more vulnerable than ...younger counterparts to the psychoactive effects of alcohol due to natural biological changes that occur with aging. This study was specifically designed to measure the effect of long-term moderate alcohol consumption on cognitive health in older adult drinkers. An extensive battery of validated tests commonly used in aging and substance use literature was used to measure performance in specific cognitive domains, including working memory and attention. An age (young, old) (*) alcohol consumption (light, moderate) factorial study design was used to evaluate the main effects of age and alcohol consumption on cognitive performance. The focus of the study was then limited to light and moderate older drinkers, and whether or not long-term moderate alcohol consumption exacerbated age-related cognitive decline. No evidence was found to support the idea that long-term moderate alcohol consumption in older adults exacerbates age-related cognitive decline. Findings were specific to healthy community dwelling social drinkers in older age and they should not be generalized to individuals with other consumption patterns, like heavy drinkers, binge drinkers or ex-drinkers.
The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may ...contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C
18 reversed-phase column in two gradients of 9
min (including two 3
min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500
ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.
Among the different techniques enlisted for metabolome analysis, ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS) represents a powerful platform for ...rapid and sensitive metabolite fingerprinting which provides very reproducible datasets in both chromatographic and mass dimensions. This technique is well established for bio fluid analysis 1 but has only scarcely be used for plant metabolomics. In our search for new bioactive stress-induced plant constituents, a generic UPLC-TOF-MS approach has been devised 2 and consists of (i) high throughput metabolite fingerprinting involving rapid gradients on numerous control and stressed plants (ii) high resolution metabolite profiling of selected pool samples on high peak capacity UPLC columns after efficient gradient transfer. Multivariate analysis applied to the fingerprinting data enables group discrimination and evidences ions (
m/z
) responsible for the main differences. High resolution profiling with long gradients provides a precise localisation and deconvolution of the putative biomarkers. Applied to the study of the wound response in
Arabidopsis thaliana
(Brassicaceae), this sequential strategy has been used to detect new key minor biomarkers and enabled the discrimination of different stress states in relation with global metabolome variations. Microgram amount of the biomarkers of interest was obtained by LC-MS triggered microfractionation. Their full structure determination was ensured by 1D and 2D CapNMR experiments 3. Original derivatives of the plant hormone jasmonic acid 4 were thus identified and a global picture of their temporal and spatial dynamics was obtained. The defence gene expression potential of these oxylipins was evaluated based on DNA microarray experiments.
Acknowledgements: Swiss National Science Foundation (Grants no. 205320–107735/1 to J-L.W. and S.R.).
References: 1. Wilson, I.D. et al. (2005)J. Prot. Res. 4:5911. 2. Grata, E. et al. (2007)J. Sep. Sci. 30:2268. 3. Glauser, G. et al. (2008)J. Chromatogr. A 1180:90. 4. Farmer E.E. et al. (2003) Curr. Opin. Plant Biol. 6:1.
Detailed metabolite profiling of crude plant extracts, mandatory for both quality control and metabolomics purposes, requires high-resolution separation and sensitive detection with a reasonable ...sample throughput. In this respect, the use of ultra-high pressure liquid chromatography working at high temperature and coupled to time-of-flight mass spectrometry (HT-UHPLC-TOF-MS) was evaluated in terms of achievable peak capacity for a given analysis time.
In a first step, it was shown that the longest column does not compulsory provide the maximal peak capacity for a given analysis time in UHPLC, using representative natural products. From a theoretical point of view, a 150mm column should be preferentially selected for gradient lengths up to 60min at 30°C, while longer columns are attractive only for higher analysis times. Compared to 30°C, peak capacities were increased by about 20–30% for a constant gradient length at 90°C and gradient time decreased by 2-fold for an identical peak capacity 1.
In a second step, profiling of natural crude sample, as example of complex mixtures, was evaluated. Extracts from the model plant
Arabidopsis thaliana
and from a
Ginkgo biloba
phytopreparation were analyzed. For metabolites spread over a large polarity range (e.g., methanolic extract of
Arabidopsis thaliana
) the use of high temperature (HT) was found beneficial with similar improvements as those recorded with the standard mixture. On the other hand, for the analysis of extracts containing more polar analytes (e.g.,
Ginkgo biloba
), HT was found detrimental and causes a decrease in retention and thus resolving power 2.
Stability under HT conditions was evaluated and no apparent degradation was evidenced for both standard mixtures and crude extract analyses 2. HT represents thus an additional parameter that can be considered for improving high-resolution profiling of extracts with metabolites spread over a large polarity range.
References:
1 Guillarme, D. et al. (2009)J. Chromatogr. A 1216:3232–3243.
2 Grata, E. et al. (2009)J. Chromatogr. A, submitted.
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