Development of a harmonised method for the profiling of amphetamines: III. Development of the gas chromatographic method

This study focused on gas chromatographic analysis of target compounds found in illicit amphetamine synthesised by the Leuckart reaction, reductive amination of benzyl methyl ketone, and the nitrostyrene route. The analytical method was investigated and optimised with respect to introduction of amphetamine samples into the gas chromatograph and separation and detection of the target substances. Sample introduction using split and splitless injection was tested at different injector temperatures, and their ability to transfer the target compounds to the GC column was evaluated using cold on column injection as a reference. Taking the results from both techniques into consideration a temperature of 250 degrees C was considered to be the best compromise. The most efficient separation was achieved with a DB-35MS capillary column (35% diphenyl 65% dimethyl silicone; 30 m x 0.25 mm, d(f) 0.25 microm) and an oven temperature program that started at 90 degrees C (1 min) and was increased by 8 degrees C/min to 300 degrees C (10 min). Reproducibility, repeatability, linearity, and limits of determination for the flame ionisation detector (FID), nitrogen phosphorous detector (NPD), and mass spectrometry (MS) in scan mode and selected ion monitoring (SIM) mode were evaluated. In addition, selectivity was studied applying FID and MS in both scan and SIM mode. It was found that reproducibility, repeatability, and limits of determination were similar for FID, NPD, and MS in scan mode. Moreover, the linearity was better when applying FID or NPD whereas the selectivity was better when utilising the MS. Finally, the introduction of target compounds to the GC column when applying injection volumes of 0.2 microl, 1 microl, 2 microl, and 4 microl with splitless injection respectively 1 microl with split injection (split ratio, 1:40) were compared. It was demonstrated that splitless injections of 1 microl, 2 microl, and 4 microl could be employed in the developed method, while split injection and splitless injections of 0.2 microl should be avoided.     

Quantification of the amphetamine content in seized street samples by Raman spectroscopy

A Raman spectroscopy method for determining the drug content of street samples of amphetamine was developed by dissolving samples in an acidic solution containing an internal standard (sodium dihydrogen phosphate). The Raman spectra of the samples were measured with a CDD-Raman spectrometer. Two Raman quantification methods were used: (1) relative peak heights of characteristic signals of the amphetamine and the internal standard; and (2) multivariate calibration by partial least squares (PLS) based on second derivative of the spectra. For the determination of the peak height ratio, the spectra were baseline corrected and the peak height ratio (h(amphetamine at 994 cm(-1) )/h(internal standard at 880 cm(-1) )) was calculated. For the PLS analysis, the wave number interval of 1300-630 cm(-1) (348 data points) was chosen. No manual baseline correction was performed, but the spectra were differentiated twice to obtain their second derivatives, which were further analyzed. The Raman results were well in line with validated reference LC results when the Raman samples were analyzed within 2 h after dissolution. The present results clearly show that Raman spectroscopy is a good tool for rapid (acquisition time 1 min) and accurate quantitative analysis of street samples that contain illicit drugs and unknown adulterants and impurities.     

Blood levels of 3-methylfentanyl in 3 fatal poisoning cases

Three fatal poisoning cases due to 3-methylfentanyl are described. In each case, the death was accidental and occurred after injection of the opioid combined with amphetamine, heroin, or other drugs. The victims' ages, ranging from 30 to 41 years, were higher than those typically found in heroin poisonings in Finland. The blood concentrations of cis-3-methylfentanyl, measured here for the first time by a specific tandem mass spectrometric method, ranged from 0.3 to 0.9 microg/L (mean 0.5 microg/L). These values are significantly lower than the levels reported for alpha-methylfentanyl and fentanyl in fatal poisonings. Repeated seizures of fentanyl and its analogs have been reported in Europe close to the Russian border.     

Development of a harmonized method for the profiling of amphetamines. I. Synthesis of standards and compilation of analytical data

Reference material was synthesised for 21 substances that are frequently present as synthetic impurities, i.e. by-products, in illicitly produced amphetamine. Each of these substances is a typical by-product for at least one of the three approaches most often used to synthesise amphetamine, namely, the Leuckart, the reductive amination of benzyl methyl ketone, and the nitrostyrene routes. A large body of data on the substances was recorded, including the following: mass spectra, ultraviolet spectra, Fourier transform infrared spectra, infrared spectra in gas phase, and 1H NMR and 13C NMR spectra.     

Development of a harmonized method for the profiling of amphetamines. II. Stability of impurities in organic solvents

The present study focused on the stability of 22 amphetamine impurities dissolved in six organic solvents: isooctane, toluene, ethanol, dichloromethane, ethyl acetate, and diethyl ether. The aim was to find the most inert, and thereby most suitable, solvent for amphetamine profiling. Mixtures of the impurities were prepared in the different solvents, and changes in the concentrations of the individual compounds over-time were monitored by gas chromatographic analysis after 0, 4, 12, 24, 48, and 96 h. Isooctane and toluene provided the most inert conditions, although, a few of the impurities were insufficiently stable in these two solvents. The present experiments were performed as a part of the development of a harmonized method for profiling of amphetamine. The results can be used to support the choice of organic solvents for sample preparation. They also provide information about the stability of the impurities that are found in profiles of illicit amphetamine. This is essential due to the fact, that unstable compounds can have a negative influence on the comparison of profiles.     

Ballistic skin simulant

Hydrogels prepared from water solutions containing 10-20 mass% gelatine are generally accepted muscle tissue simulants in terminal ballistic research. They, however, do not have a surface layer which simulates the effect of human skin. The purpose of this research was to find a suitable skin simulant for enhancing the testing fidelity and the credibility of the results with gelatine-based materials when assessing the injury potential of not only high energy bullets, but also especially that of non-penetrating "less lethal" kinetic impact ammunition and relatively low energy ricochet fragments. A skin simulant also permits the simulation and assessment of exit wounds. The mechanical and ballistic properties of human skin and target simulant were established on the basis of results found in the literature. Some errors in these were found. The corrected values are included in this paper for comparison. The target values of the mechanical properties of the skin simulant were the following: threshold velocity v(th)=94+/-4 m/s, tensile strength 18+/-2 N/mm2 and elongation at break 65+/-5%. A selection of synthetic and natural materials was evaluated as skin simulants by analysing their mechanical and ballistic properties. The results were compared to literature values obtained with human cadavers. The tests showed that the best skin simulant of the ones evaluated was semi-finished chrome tanned upholstery "crust" cowhide of 0.9-1.1 mm nominal thickness. Its threshold velocity was 90.7 m/s, tensile strength 20.89+/-4.11 MPa and elongation at break 61+/-9%. These values are the same as the average values of human skin. Of the synthetic materials evaluated, 1mm thick natural rubber can be used on impact side as a threshold velocity filter with some reservations although its theoretical threshold velocity is only 82.9 m/s.     

Post-mortem SNP analysis of CYP2D6 gene reveals correlation between genotype and opioid drug (tramadol) metabolite ratios in blood

Tramadol is an opioid drug metabolised in phase I by cytochrome P450 (CYP) enzymes, of which CYP2D6 is mainly responsible for the O-demethylation of tramadol, but is not involved in N-demethylation. Defects in the genes encoding drug metabolising enzymes (DMEs) may lead to adverse drug effects, even to death. To aid interpretation of the forensic toxicology results, we studied how the genetic variation of the CYP2D6 gene is reflected in tramadol metabolite ratios found in post-mortem samples. In 33 Finnish autopsy cases where tramadol was found, we analysed both the CYP2D6 genotype and the concentrations of tramadol and its metabolites O- and N-demethyltramadol. As expected, we found a correlation between the number of functional CYP2D6 alleles and the ratio of tramadol to O-demethyltramadol. We also found a correlation between the number of functional alleles and the ratio of tramadol to N-demethyltramadol. This can be explained by the complementary nature of the two main tramadol demethylation pathways. No known CYP2D6 inhibitors were associated with exceptional metabolic ratios. Furthermore, no accidental tramadol poisonings were associated with a defective CYP2D6 gene. Our results on the tramadol are among the first to demonstrate that genetic variation in drug metabolising enzymes can be analysed in post-mortem blood, and that it correlates well with the parent drug to metabolite ratios. The results also suggest that genetic factors play, in general, a dominant role over other factors in the metabolism of individual drugs.     

Development of a harmonised method for the profiling of amphetamines VI: Evaluation of methods for comparison of amphetamine

Amphetamine samples were analysed by gas chromatography-mass spectrometry (GC-MS), and the peak areas of 33 target compounds were transformed by applying various pretreatment techniques. The objective was to optimise the ability of a number of distance metrics to establish links between samples of amphetamine originating from the same batch (henceforth referred to as linked distances). Furthermore, partial least squares discriminant analysis (PLS-DA) was used to evaluate the effects of various pretreatment methods on separation of amphetamine batches synthesised by the Leuckart reaction, reductive amination of benzyl methyl ketone, and the nitrostyrene route. The most efficient way to pretreat GC-MS data varied for the different distance metrics, although best results were obtained when data were normalised to the sum of peak areas, and either the fourth root or a logarithm was applied to the normalised data. When pretreating normalised data by fourth root transformation, Pearson correlation was the distance metric that was most successful at finding linked samples. Normalisation and the use of fourth root also represented the best method of pretreating data when employing PLS-DA to separate samples synthesised by different routes. To achieve a faster and more user-friendly procedure for evaluating chromatograms, experiments were performed in which the number of target compounds used to compare samples was reduced. The effect of each compound that was removed was studied by applying PLS-DA and by using Pearson correlation to calculate linked distances as well as unlinked distances (between samples from different batches of amphetamine). Considering both links between samples from the same batch and separation of samples synthesised by different routes, the best results were obtained with the data set comprising 26 compounds. Finally, it was found that the profiling method developed in this work was superior to an existing technique with respect to separating linked and unlinked distances.     

Assessing viability of Finnish reorganization and bankruptcy firms

The objective of the Finnish Bankruptcy Act (FBA) is to give the assets of a non-viable firm to the bankrupted estate and divide them by the creditors of the firm (liquidation). However, the objective of the Finnish Company Reorganization Act (FCRA) is to recover a temporally financially distressed firm that is viable. In these acts, the viability concept is used to refer to a firm that is able to pay its financial obligations in the future. FBA is not efficient if a viable firm is filed for bankruptcy and liquidated. In the same way, FCRA is not efficient if a non-viable firm is filed for reorganization. The purpose of this study is to assess viability of Finnish firms filed for reorganization and bankruptcy. Logistic regression analysis is used to extract two measures for viability. The first measure is based on financial statement information only while the second one takes account of both financial and non-financial information. The estimation sample comprises data from 43636 viable and 98 non-viable firms. The resulted measures are used to assess viability of a sample of firms filed for reorganization and bankruptcy in 2004. In general, the results show that the greater part of firms filed for reorganization is non-viable whereas many of bankruptcy firms are viable. This reflects an occurrence of filtering failure.     

Identification of volatile organic compounds in blood by purge and trap PLOT-capillary gas chromatography coupled with Fourier transform infrared spectroscopy

A purge and trap concentrator with a Tenax trap was coupled to gas chromatography-Fourier transform infrared spectrometry for the identification of volatile organic compounds in blood samples. A styrene-divinyl benzene porous layer capillary column allowed the separation of compounds such as household and medical gases, solvents and alcohol congeners. The identification limits in blood, measured by comparison to an in-house vapour phase spectrum library, generally ranged from 0.05 to 10 mg/1, depending on the analyte structure. Low molecular weight alcohols had identification limits up to 100 mg/1. Six actual casework examples were collected during a 1-year period of routine use to demonstrate the feasibility of the method.