Measurement uncertainty of ethyl alcohol concentration in the exhaled breath of drivers and determination of sobriety at the time of incident. / Niepewnosc pomiaru stezenia alkoholu etylowego w powietrzu wydychanym kierowców oraz ustalenia stanu trzezwosci w chwili zdarzenia.

The aim of the study was to determine the components of measurement uncertainty in the concentration of alcohol in exhaled breath and to determine the state of sobriety at the time of incident. Based on the literature review and the authors' experience in providing opinions for law enforcement and the judiciary, the influence of various factors on the final interpretation of sobriety state is described on the basis of measurement uncertainty of breath analyzers, uncertainty of retrospective and prospective calculations, and uncertainty related to the conversion of alcohol concentrations detected during breath and blood tests. The paper pays particular attention to interpreting the concentrations of ethanol in exhaled breath close to the legal limits of the state of sobriety and the state after alcohol use, or the state after alcohol use and the state of insobriety. Analyzing the results of an exhaled breath test concerning concentrations close to the values of 0.1 mg/dm3 and 0.25 mg/dm3, it is necessary to take into account the factors affecting the measurements obtained, including the measurement uncertainty of the determination of alcohol in exhaled breath, the processes of absorption, distribution and metabolism of ethyl alcohol, and the possibility of the presence of alcohol lingering in the oral cavity. The incorrect execution of measurements of the tested person's alcohol concentration is also a problematic issue. When determining sobriety state by means of retrospective and prospective calculations, it is important to remember that the uncertainty of the result is affected by a number of factors and depends, among other things, on the information provided by the suspect. Hence, the expert should draw conclusions particularly cautiously and any overestimation or underestimation of the components of uncertainty can lead to erroneous conclusions. Awareness of the uncertainties inherent in the results of a sobriety test or alcohol calculation allows for meaningful interpretation of test results and determination of the sobriety state of the person tested.

Analysis of fragmentation pathways of fentanyl derivatives by electrospray ionisation high-resolution mass spectrometry.

RATIONALE: During the last decade there has been a large increase in the availability of new synthetic opioids on the European drug market. Fentanyl analogues accounted for a significant proportion of these compounds. When there is a rapid introduction of new compounds from a given chemical class onto the market, a computer-assisted library search is not recommended and the spectra have to be interpreted individually. Therefore, the knowledge of how different groups of new psychoactive substances are fragmented can be very helpful in identifying new compounds. METHODS: In this study, the fragmentation patterns of 33 fentanyl derivatives were investigated using electrospray ionisation (ESI). The analyses were conducted using liquid chromatography quadrupole time-of-flight mass spectrometry (LC/QTOFMS). Based on measurements carried out under various conditions, the fragmentation pathways of the tested compounds that were divided into groups due to their chemical structure were established. RESULTS: The performed study allowed for the determination of characteristic ions that were formed during the fragmentation of fentanyl derivatives using ESI. Due to the high mass accuracy of the LC/ESI-QTOFMS technique, it was proved that the cleavage of the tested molecules occurred mostly on the bonds adjacent to the nitrogen atoms. Based on the proposed fragmentation scheme, the general structure for fentanyls, and the presence of some characteristic ions, it is possible, after applying simple mathematical operations, to calculate the masses of individual substituents in the formulas of the new fentanyl analogues, which may appear on the drug market. Furthermore, based on the exact masses, it is possible to determine the formulas of these substituents. CONCLUSIONS: Knowledge of the specific fragments generated under ESI conditions can be used in forensic laboratories to determine the structures of novel compounds from the group of fentanyl derivatives.

Analysis of Fragmentation Pathways of New-Type Synthetic Cannabinoids Using Electrospray Ionization.

Recently, dozens of new psychoactive substances have appeared on the European drug market every year. The most abundant group of these compounds is synthetic cannabinoids. In the first few years of the "legal highs" phenomenon, JWH (John W. Huffman) compounds were especially popular among drug users. However, the group of synthetic cannabinoids is constantly expanding, as new compounds are created by replacing known structural elements with different chemical groups. The problem with the identification of novel substances in forensic laboratories results from the structural similarity of the compounds and the rapid introduction of newer designer drugs on the black market. In this study, the fragmentation patterns of 29 new-type synthetic cannabinoids using electrospray ionization were investigated. The analysis was performed using quadrupole time-of-flight mass spectrometry. Based on measurements carried out under various conditions, the way of fragmentation of the tested compounds that were divided into groups due to their chemical structure was established. The study showed that the bond between the carbon atom of the carbonyl group and the ring or NH group attached to the ring was mainly cleaved. This mechanism was adequate for the fragmentation of first-generation synthetic cannabinoids. This paper presents characteristic ions formed by synthetic cannabinoids (i.e., ions originating from an indole/indazole ring and an adamanyl/naphthalene/quinoline ring) using electrospray ionization. Knowledge of these specific fragments can be used in forensic laboratories to determine the structure of novel compounds from the group of synthetic cannabinoids. Graphical Abstract ᅟ.

Mast cells influence neoangiogenesis in prostatic cancer independently of ERG status.

A significant proportion of prostatic adenocarcinomas show recurrent translocation leading to ERG expression. Previously we found that ERG+ cases have higher microvessel density than negative ones. One factor influencing angiogenesis in cancer is mast cells. The aim of the present study was to evaluate the relationship between microvessels, mast cells and ERG status. Tissue microarrays prepared from 113 radical prostatectomy specimens were analyzed with immunohistochemistry for CD31, tryptase and chymase. Vascular profiles and tryptase-positive and chymase-positive cells were counted. The average number of tryptase-positive cells was 28.93/mm2 and chymase-positive cells 9.91/mm2. The average number of CD31+ vascular profiles was 352.66/mm2. The average number of tryptase-positive cells was 26.35/mm2 for ERG- cases and 32.12/mm2 for ERG+ cases. The average number of chymase-positive cells was 8.14/mm2 for ERG- cases and 12.06/mm2 for ERG+ cases. The average number of CD31+ vascular profiles was 321.34/mm2 for ERG- cases and 390.74/mm2 for ERG+ cases. The number of CD31+ vascular profiles was positively correlated with the number of tryptase-positive and chymase-positive cells (R = 0.26 and R = 0.20). In summary, we demonstrated an interrelationship between mast cells, microvascular density and ERG status in prostatic carcinoma.

Prostate cancer with different ERG status may show different FOXP3-positive cell numbers.

Prostatic carcinoma is the most frequent cancer in males in the Western world. A significant proportion of these cancers have a recurrent translocation involving ETS family genes, which leads to the overexpression of ERG transcription factor. Prostate cancers, which bear this mutation, differ in a number of features, including tumor microenvironment. One of the components of the tumor microenvironment is FOXP3 positive lymphocytes, which may participate in breaking immunosurveillance and promoting tumor growth. The aim of the study was to analyze the relationships between ERG expression, number of FOXP3 positive cells and other features of the tumor. The study group consisted of 65 cases. Tissue microarrays composed of 2 mm tissue cores were used for immunohistological evaluation. Immunohistochemistry for ERG and FOXP3 was performed according to the routinely applied protocol. The FOXP3 positive cells were counted and the results were expressed as the number of cells per mm2. The average number of FOXP3 positive cells was 33.30/mm2 for all cases, 21.43/mm2 for the ERG negative and 42.28/mm2 for the ERG positive group (p < 0.02). There were no significant relationships between FOXP3 positive cell count and any other parameters studied. Our results suggest that the immune response may differ between ERG negative and ERG positive prostatic carcinomas.

Mast cells in systemic and cutaneous lupus erythematosus.

Mast cells (MCs) are known to be regulators of inflammation and immunity, due to the released mediators and expressed cell surface molecules. Lupus erythematosus (LE) is a group of diseases which can be systemic or limited to the skin. Due to the fact that cytokines and chemokines produced by inflammatory cells contribute to the pathogenesis of LE, we quantified the number of mast cells present in the skin. The aim of the study was to compare the chymase-positive and tryptase-positive mast cell counts within skin biopsies from patients with systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE) and subacute cutaneous lupus erythematosus (SCLE). The material consisted of 45 skin biopsies: 6 with SLE, 34 with DLE and 5 with SCLE. Chymase- and tryptase-positive cells were stained by immunohistochemistry and counted. The mean count of chymase-positive mast cells was 85.14 hpf for the whole group, 35.83 for SLE, 88.48 for DLE and 121.6 for SCLE. The mean count of tryptase-positive cells was 120.05 hpf for the entire group, 59.17 for SLE, 126.42 for DLE and 149.8 for SCLE. The differences between groups were significant for chymase- and tryptase-positive cells.

Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine.

UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which has been detected in many herbal blends, resinous samples and powders seized from the Polish drug market since the beginning of 2012. This paper presents the case of intoxication by this substance. A complete picture of the symptoms observed by a witness, paramedics and medical doctors are given. In the analysis of powder residues from the plastic bag seized from the intoxicated person by gas chromatography-mass spectrometry (GC-MS), UR-144 and its major pyrolysis product [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2-yl)but-3-en-1-one] were detected. Both substances were also identified in a blood sample collected on admission of the patient to hospital using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS). Blood concentration of UR-144 was 6.1 ng/mL. A urine sample collected at the same time was analyzed by liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LC-QTOF-MS). The parent substance and its pyrolysis products were not detected in urine, while their five metabolites were found. The experiments allowed the location of derivative groups to be established, and thus elucidate rough structures of the metabolites; a dihydroxylated metabolite of UR-144 and mono-, dihydroxylated and carboxylated metabolites of its pyrolysis product were identified.

Structural elucidation and identification of a new derivative of phenethylamine using quadrupole time-of-flight mass spectrometry.

RATIONALE: In recent years, the phenomenon of uncontrolled distribution of new psychoactive substances that were marketed without prior toxicological studies has been observed. Because many designer drugs are related in chemical structure, the potential for misidentifying them is an important problem. It is therefore essential to develop an analytical procedure for unequivocal elucidation of the structures of these compounds. The issue has been discussed in the context of 25I-NBMD [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2,3-methylenedioxyphenyl)methyl]ethanamine], a psychoactive substance first discovered on the drug market in 2012. METHODS: The substance was extracted from blotter papers with methanol. Separation was achieved via liquid chromatography. Analysis was conducted by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOFMS). Identification of the psychoactive component was supported by electron impact gas chromatography/mass spectrometry (GC/EI-MS). RESULTS: The high accuracy of the LC/ESI-QTOFMS method allowed the molecular mass of the investigated substance (M(exp) = 441.0438 Da; mass error, ∆m = 0.2 ppm) and the formulae of ions formed during fragmentation to be determined. The main ions were recorded at m/z = 135.0440, 290.9876 and 305.9981. Structures of the obtained ions were elucidated in the tandem mass spectrometry (MS/MS) experiments by comparing them to mass spectra of previously detected derivatives of phenethylamine. CONCLUSIONS: The performed study indicated the potential for using LC/QTOFMS method to identify new designer drugs. This technique can be used supplementary to standard GC/MS. Prior knowledge of the fragmentation mechanisms of phenethylamines allowed to predict the mass spectra of the novel substance--25I-NBMD.

25C-NBOMe--new potent hallucinogenic substance identified on the drug market.

This publication reports analytical properties of a new hallucinogenic substance identified in blotter papers seized from the drug market, namely 25C-NBOMe [2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine]. The identification was based on results of comprehensive study including several analytical methods, i.e., GC-EI-MS (without derivatization and after derivatization with TFAA), LC-ESI-QTOF-MS, FTIR and NMR. The GC-MS spectrum of 25C-NBOMe was similar to those obtained for other representatives of the 25-NBOMe series, with dominant ions observed at m/z=150, 121 and 91. Fragment ions analogic to those in 2C-C (4-chloro-2,5-dimethoxy-ß-phenylethanamine) were also observed, but their intensities were low. Derivatization allowed the determination of molecular mass of the investigated substance. The exact molecular mass and chemical formula were confirmed by LC-QTOF-MS experiments and fragmentation pattern under electrospray ionization was determined. The MS/MS experiments confirmed that the investigated substance was N-(2-methoxy)benzyl derivative of 2C-C. The substance was also characterized by FTIR spectroscopy to corroborate its identity. Final elucidation of the structure was performed by NMR spectroscopy.

Identification and characterization of 2,5-dimethoxy-3,4-dimethyl-ß-phenethylamine (2C-G)--a new designer drug.

This study presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5-dimethoxy-3,4-dimethyl-ß-phenethylamine (2C-G), a new designer drug. A powder sample containing 2C-G was seized in Poland in 2011. The paper focuses on a comparison of the analytical features of 2C-G and other members of the 2C-series, in order to assess the possibility of unequivocal identification. The occurrence of intense peak at m/z = 178 and different intensities of the ions at m/z = 165 and 180 in the gas chromatography-electron impact-mass spectrometry (GC-EI/MS) spectrum of 2C-G made it possible to distinguish it from 2C-E. Differences in relative intensities of the ions at m/z = 192, 179 and 177 were observed for GC-EI/MS spectra of TFAA derivatives of 2C-G and 2C-E. An identical set of ions was recorded for these substances using the liquid chromatography-electrospray ionization/quadrupole time of flight mass spectrometry (LC-ESI/QTOFMS) method in both MS and tandem mass spectrometry (MS/MS) mode, but the distinction was possible based on differences in the ion intensities at m/z = 193.1223 and 178.0988. The Fourier transform infrared (FTIR) spectrum of 2C-G was significantly different from other members of the 2C-series, with a characteristic doublets at 993-1014 cm(-1) and 1099-1124 cm(-1) , and the ratio of bands at higher wavenumbers. Final elucidation of the structure of 2C-G was carried out by (1) H and (13) C NMR spectroscopy. The study indicated that the marketing of analogues of controlled substances poses a real analytical challenge for forensic laboratories, and the application of sophisticated methods is often required for unequivocal identification of a new substance.

Analytical characterization of three hallucinogenic N-(2-methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs.

This publication reports analytical properties of three new hallucinogenic substances identified in blotter papers seized from the drug market, namely 25D-NBOMe [2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine], 25E-NBOMe [2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine] and 25G-NBOMe [2-(2,5-dimethoxy-3,4-dimethylphenyl)-N-(2-methoxybenzyl)ethanamine]. These substances are N-(2-methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs. The applied procedure covered a variety of analytical methods, including gas chromatography with electron impact mass spectrometry (GC-EI-MS; without derivatization and after derivatization with trifluoroacetic anhydride (TFAA)), liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), which made it possible to identify the active components unequivocally. The GC-MS spectra of analyzed compounds were very similar, with dominant ions observed at m/z = 150, 121, and 91. The remaining ions were analogous to those observed for parent substances, namely 2C-D, 2C-E, 2C-G, but their intensities were low. Derivatization allowed determination of molecular masses of the investigated substances. Their exact masses and chemical formulas were confirmed by LC-QTOF-MS experiments and the fragmentation patterns of these compounds following ESI were determined. The tandem mass spectrometry (MS/MS) experiments confirmed that the studied substances were N-(2-methoxy)benzyl derivatives of the 2C-series compounds. Final elucidation of the structures was performed by NMR spectroscopy. The substances were also characterized by FTIR spectroscopy to corroborate the identity of the compounds.

Identification and characterization of 2,5-dimethoxy-4-nitro-ß-phenethylamine (2C-N)--a new member of 2C-series of designer drug.

The online sale of psychoactive substances, including hallucinogens, is becoming a serious problem in many countries. This paper presents and discusses the mass spectrometric, infrared spectroscopic and nuclear magnetic resonance spectroscopic data of 2,5-dimethoxy-4-nitro-ß-phenethylamine (2C-N), which was identified in a powder sample seized by the authorities in 2011 in Poland. The molecular mass of 2C-N (226.0954 amu) was confirmed in the LC/ESI-QTOFMS experiment. The molecular ion was also observed in the GC-EI/MS spectrum. A characteristic set of ions for the parent substance was found using both chromatographic methods, and when derivatization with trifuluoroacetic anhydride (TFAA) was applied. Two broad dominant bands at 1520 cm(-1) and 1342/1322 cm(-1), observed in the FTIR spectrum of 2C-N, originated from the nitro group. NMR spectroscopy helped unequivocal elucidation of the structure. The applied identification procedure proved to be a powerful tool to determine the structure of a new designer drug.

Identification of naphthoylindoles acting on cannabinoid receptors based on their fragmentation patterns under ESI-QTOFMS.

'Herbal highs' have been advertised as legal and natural substitutes to cannabis, but a detailed examination of these products has revealed that the herbal matrix is laced with synthetic substances that mimic the effects of marijuana. Producers select the ingredients based on the results of scientific studies on the affinities of different chemicals to cannabinoid receptors. Naphthoylindoles have turned out to be the most popular class of substances identified in the products. Legal actions taken in order to tackle the problem of uncontrolled access to one substance have usually resulted in the marketing of derivatives or analogues. In the study, the mass spectral behavior of twelve synthetic cannabinoids from the naphthoylindole family under electrospray ionization (ESI) was investigated. LC-QTOFMS experiments were performed in three modes (low fragmentor voltage, high fragmentor voltage with/without collision energy), and they enabled the identification of protonated molecules and main ions. A general fragmentation pattern under this ionization method was proposed, and mechanisms of ion formation were discussed. The developed procedure allowed the determination of substituent groups of the core naphthoylindole structure and distinction between positional isomers. The obtained results were used for the prediction of the ESI-MS spectra for many naphthoylindoles with a high affinity to cannabinoid receptors. Similarities and differences between ESI-MS and electron impact-MS spectra of naphthoylindoles were discussed. The developed identification process was presented on an example of an analysis of an unknown herbal material, in which JWH-007 was finally identified. Knowledge of the fragmentation mechanisms of naphthoylindoles could also be used by other researchers for identification of unknown substances in this chemical family.