The metabolic profile of the synthetic cannabinoid receptor agonist ADB-HEXINACA using human hepatocytes, LC-QTOF-MS and synthesized reference standards.

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl using LC-QTOF-HRMS together with synthesized references.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 µM) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the ß position of the phenylpropanoyl moiety was highly favored; ß-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

The metabolism of the synthetic cannabinoids ADB-BUTINACA and ADB-4en-PINACA and their detection in forensic toxicology casework and infused papers seized in prisons.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50 , 11.5 nM and ADB-4en-PINACA (EC50 , 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50 , 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.

Structure Elucidation of Urinary Metabolites of Fentanyl and Five Fentanyl Analogs using LC-QTOF-MS, Hepatocyte Incubations and Synthesized Reference Standards.

Fentanyl analogs constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism, and studies utilizing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis of hepatocyte incubations and/or authentic urine samples do not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study, seven motifs (2-, 3-, 4- and ß-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogs, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC-QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogs, ß-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. ß-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/ß-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogs where nothing is known about the metabolism.

Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards.

MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for the detection of synthetic cannabinoids in humans, since they are generally extensively metabolized. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesized metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesized metabolites were incubated with human liver microsomes (HLM) and the supernatants were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the fourth most abundant metabolite. The metabolic pathways were investigated using synthesized metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg and t1/2 was 2.1 min, indicating a low metabolic stability. M15, M8, and potentially M5 are suggested as suitable urinary targets.

LC-QTOF-MS Identification of Major Urinary Cyclopropylfentanyl Metabolites Using Synthesized Standards.

Cyclopropylfentanyl is a fentanyl analog implicated in 78 deaths in Europe and over 100 deaths in the United States, but toxicological information including metabolism data about this drug is scarce. The aim of this study was to provide the exact structure of abundant and unique metabolites of cyclopropylfentanyl along with synthesis routes. In this study, metabolites were identified in 13 post-mortem urine samples using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Samples were analyzed with and without enzymatic hydrolysis, and seven potential metabolites were synthesized in-house to provide the identity of major metabolites. Cyclopropylfentanyl was detected in all samples, and the most abundant metabolite was norcyclopropylfentanyl (M1) that was detected in 12 out of 13 samples. Reference materials were synthesized (synthesis routes provided) to identify the exact structure of the major metabolites 4-hydroxyphenethyl cyclopropylfentanyl (M8), 3,4-dihydroxyphenethyl cyclopropylfentanyl (M5) and 4-hydroxy-3-methoxyphenethyl cyclopropylfentanyl (M9). These metabolites are suitable urinary markers of cyclopropylfentanyl intake as they are unique and detected in a majority of hydrolyzed urine samples. Minor metabolites included two quinone metabolites (M6 and M7), not previously reported for fentanyl analogs. Interestingly, with the exception of norcyclopropylfentanyl (M1), the metabolites appeared to be between 40% and 90% conjugated in urine. In total, 11 metabolites of cyclopropylfentanyl were identified, including most metabolites previously reported after hepatocyte incubation.

Attribution of fentanyl analogue synthesis routes by multivariate data analysis of orthogonal mass spectral data.

Chemical attribution signatures (CAS) can be used to obtain useful forensic information and evidence from illicit drug seizures. A CAS is typically generated using hyphenated chemical analysis techniques and consists of a fingerprint of the by-products and additives present in a sample. Among other things, it can provide information on the sample's origin, its method of production, and the sources of its precursors. This work investigates the possibility of using multivariate CAS analysis to identify the synthetic methods used to prepare seized fentanyl analogues, independently of the analogues' acyl derivatization. Three chemists working in two labs synthesized three different fentanyl analogues, preparing each one in duplicate by six different routes. The final collection of analogues (96 samples) and two intermediates (16 + 32 samples) were analysed by GC-MS and UHPLC-HRMS, and the resulting analytical data were used for multivariate modelling. Independently of analogue structure, the tested fentanyls could be classified based on the method used in the first step of their synthesis. The multivariate model's ability to classify unknown samples was then evaluated by applying it to six new fentanyl analogues. Additionally, seized fentanyl samples was analysed and classified by the model.

Prediction of designer drugs: Synthesis and spectroscopic analysis of synthetic cathinone analogs that may appear on the Swedish drug market.

The use of hyphenated analytical techniques in forensic drug screening enables simultaneous identification of a wide range of different compounds. However, the appearance of drug seizures containing new substances, mainly new psychoactive substances (NPS), is steadily increasing. These new and other already known substances often possess structural similarities and consequently they exhibit spectral data with slight differences. This situation has made the criteria that ensure indubitable identification of compounds increasingly important. In this work, 6 new synthetic cathinones that have not yet appeared in any Swedish drug seizures were synthesized. Their chemical structures were similar to those of already known cathinone analogs of which 42 were also included in the study. Hence, a total of 48 synthetic cathinones making up sets of homologous and regioisomeric compounds were used to challenge the capabilities of various analytical techniques commonly applied in forensic drug screening, ie, gas chromatography-mass spectrometry (GC-MS), gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR), nuclear magnetic resonance (NMR), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Special attention was paid to the capabilities of GC-MS and GC-FTIR to distinguish between the synthetic cathinones and the results showed that neither GC-MS nor GC-FTIR alone can successfully differentiate between all synthetic cathinones. However, the 2 techniques proved to be complementary and their combined use is therefore beneficial. For example, the structural homologs were better differentiated by GC-MS, while GC-FTIR performed better for the regioisomers. Further, new spectroscopic data of the synthesized cathinone analogs is hereby presented for the forensic community. The synthetic work also showed that cathinone reference compounds can be produced in few reaction steps.

Analysis of Explosives by GC-UV.

A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178-330 nm. The gas-phase UV spectra of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.

Prediction of designer drugs: synthesis and spectroscopic analysis of synthetic cannabinoid analogues of 1H-indol-3-yl(2,2,3,3-tetramethylcyclopropyl)methanone and 1H-indol-3-yl(adamantan-1-yl)methanone.

In this work, emergence patterns of synthetic cannabinoids were utilized in an attempt to predict those that may appear on the drug market in the future. Based on this information, two base structures of the synthetic cannabinoid analogues - (1H-indol-3-yl(2,2,3,3-tetramethylcyclopropyl)methanone and 1H-indol-3-yl(adamantan-1-yl)methanone) - together with three substituents - butyl, 4-fluorobutyl and ethyl tetrahydropyran - were selected for synthesis. This resulted in a total of six synthetic cannabinoid analogues that to the authors' knowledge have not yet appeared on the drug market. Spectroscopic data, including nuclear magnetic resonance (NMR), mass spectrometry (MS), and Fourier transform infrared (FTIR) spectroscopy (solid and gas phase), are presented for the synthesized analogues and some additional related cannabinoids. In this context, the suitability of the employed techniques for the identification of unknowns is discussed and the use of GC-FTIR as a secondary complementary technique to GC-MS is addressed. Examples of compounds that are difficult to differentiate by their mass spectra, but can be distinguished based upon their gas phase FTIR spectra are presented. Conversely, structural homologues where mass spectra are more powerful than gas phase FTIR spectra for unambiguous assignments are also exemplified. This work further emphasizes that a combination of several techniques is the key to success in structural elucidations. Copyright © 2015 John Wiley & Sons, Ltd.

Economic Burden of Psoriasis and Potential Cost Offsets with Biologic Treatment: A Swedish Register Analysis.

Estimates of direct and indirect costs of psoriasis are limited. The aim of this study was to estimate: (i) costs in patients with psoriasis compared with controls; and (ii) impact on costs from initiating biologics. The study extracted data from Swedish administrative registers and compared 31,043 patients with 111,645 sex-, age- and residency-matched referents. Mean direct and indirect costs were estimated as US dollars (USD) 1,365 (62%) and USD 3,319 (50%) higher in patients compared with referents, respectively. The study included 352 patients treated with biologics who had at least 1-year follow-up before and after initiation of biologics. Among the 193 patients persistent with biologics for one year, 1-year costs of biologics were estimated at USD 23,293 (95% confidence interval (95% CI) 22,372-24,199). This cost was partially offset, with savings in direct cost estimated to range from USD -1135 (95% CI -2,050 to -328) to USD -4,422 (95% CI -6,552 to -2,771), depending on assumptions. The corresponding estimates for indirect costs savings were from USD -774 (95% CI -2,019-535) to USD -1,875 (95% CI -3,650 to -188). The study suggests that psoriasis is associated with substantial costs, which may be modifiable with treatment.

Spontaneous formation of γ-hydroxybutyric acid from γ-butyrolactone in tap water solutions.

The spontaneous conversion of γ-butyrolactone (GBL) to γ-hydroxybutyric acid (GHB) in seven different Swedish tap waters was investigated. The waters used in the study were selected to represent the diversity among Swedish tap waters as well as possible, which was enabled by principal component analysis (PCA) of a number of water quality parameters. GBL solutions (5, 25 and 50% v/v) were prepared in each of the tap waters and in deionized water and the formation of GHB was followed over time. The GHB quantifications were made using a CZE method, employing a carrier electrolyte consisting of 25mM benzoic acid, 54mM tris(hydroxymethyl)aminomethane (Tris) and 1.7mM tetradecyltrimethylammonium bromide (TTAB), which was developed as a part of the current study. Data evaluation showed that the formation of GHB was largely dependent on the type of tap water. For example, there was a negative correlation between the kinetics of the GHB formation and the alkalinity of the tap waters (r(2)=0.990). This could be explained by a faster decrease in pH in the waters with low buffering capacity (i.e. low alkalinity), which catalysed the hydrolysis of GBL. Equilibrium was reached after 40-250 days depending on the initial GBL concentration and the type of tap water. The level of the equilibrium appeared to be dependent on the initial GBL concentration and ranged from 26 to 37%. Gained knowledge on the levels of the GHB/GBL equilibrium and the kinetics of the formation of GHB in tap water solutions of GBL, including the influence of the tap water quality, may be useful information for casework with the GHB/GBL problem in focus.

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.

Development of a harmonised method for the profiling of amphetamines V: Determination of the variability of the optimised method.

This paper is the fifth in a series of six in relation to the development of a harmonised method for the profiling of amphetamine [L. Aalberg, K. Andersson, C. Bertler, H. Borén, M.D. Cole, J. Dahlén, Y. Finnon, H. Huizer, K. Jalava, E. Kaa, E. Lock, A. Lopes, A. Poortman-van der Meer, E. Sippola, Development of a harmonised method for the profiling of amphetamines I. Synthesis of standards and compilation of analytical data, Forensic Sci. Int. 149 (2005) 219-229; L. Aalberg, K. Andersson, C. Bertler, M.D. Cole, Y. Finnon, H. Huizer, K. Jalava, E. Kaa, E. Lock, A. Lopes, A. Poortman-van der Meer, E. Sippola, J. Dahlén, Development of a harmonised method for the profiling of amphetamines II. Stability of impurities in organic solvents, Forensic Sci. Int. 149 (2005) 231-241]. The third paper [K. Andersson, K. Jalava, E. Lock, L. Aalberg, Y. Finnon, H. Huizer, E. Kaa, A. Lopes, A. Poortman-van der Meer, M.D. Cole, J. Dahlén, E. Sippola, Development of a harmonised method for the profiling of amphetamines III. Development of the gas chromatographic method, Forensic Sci. Int., in press] dealt with the optimisation of the gas chromatographic and detection methods whereas the fourth paper [K. Andersson, K. Jalava, E. Lock, Y. Finnon, S. Stevenson, L. Aalberg, H. Huizer, E. Kaa, A. Lopes, A. Poortman-van der Meer, M.D. Cole, J. Dahlén, E. Sippola, Development of a harmonised method for the profiling of amphetamines IV. Optimisation of sample preparation, Forensic Sci. Int., in press] concerned the optimisation of the extraction method prior to GC analysis. This paper is a study of the optimised method in order to determine its stability. Investigations of within and between day variations were carried out in four laboratories. Moreover, variations between laboratories were also determined. Both flame ionisation detector (FID) and MS detection were used. One laboratory studied nitrogen-phosphorous detector (NPD) detection as well. For this task, 12 batches of amphetamine were prepared. Six of them were synthesised via the Leuckart route, three via the nitrostyrene route and three via the reductive amination route [A.M.A. Verweij, Impurities in illicit drug preparations: amphetamine and methamphetamine, Forensic Sci. Rev. 1 (1989) 2-11]. Taking into account all studied target compounds and the average results from four laboratories, the within day variation was around 6% for FID and 5% for MS, the between days variation was around 10% for FID and 8% for MS. For NPD detection, within day variation was 5% and between days variation 9% (only one laboratory). Finally, the inter-laboratory variation was about 12% for FID (four laboratories) and 10% for MS (three laboratories).

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.

Development of a harmonised method for the profiling of amphetamines: IV. Optimisation of sample preparation.

The suitability of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for the preparation of impurity extracts intended for gas chromatographic profiling analyses of amphetamine were evaluated. Both techniques were optimised with respect to the extraction of selected target compounds by use of full factorial designs in which the variables affecting the performance were evaluated. Test samples consisted of amphetamine synthesised by the Leuckart reaction, by reductive amination of benzyl methyl ketone and by the nitrostyrene route. The performance of LLE and SPE were comparable in terms of repeatability and recovery of the target compounds. LLE was considered the better choice for the present harmonised amphetamine profiling method due to the lack of information on the long-term stability of SPE columns.

Development of a capillary zone electrophoresis method including a factorial design and simplex optimisation for analysis of amphetamine, amphetamine analogues, cocaine, and heroin.

A capillary zone electrophoresis (CZE) method was developed for the analysis of amphetamine and 13 amphetamine analogues. A full factorial design was used to screen for important design variables (i.e. carrier electrolyte concentration, pH, and separation temperature), and a modified simplex was employed in a final optimisation step. The resolution values of the target compounds were used as responses in the screening and optimisation phases. This approach made it possible to control the effects of the design variables on the separation of the target compounds. The best results were obtained using a 100mM Tris/phosphate buffer (pH 3.1) at a separation temperature of 10 degrees C, and the analysis time was 23 min under these conditions. After slight modification, the method also enabled baseline resolution of the most commonly encountered amphetamine derivatives, as well as cocaine and heroin, within 7 min. There was a linear relationship between peak area and concentration for all substances, with correlation coefficients in the range of 0.9975-0.9999. Moreover, the technique was repeatable and exhibited relative standard deviation (R.S.D.) values in the ranges of 0.01-0.11% and 0.54-1.60% for relative migration time and corrected peak area, respectively. Lastly, the method was successfully applied to analyse street samples.

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.

Simultaneous analysis of gamma-hydroxybutyric acid, gamma-butyrolactone, and 1,4-butanediol by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was optimised for simultaneous analysis of gamma-hydroxybutyric acid (GHB), gamma-butyrolactone (GBL), and 1,4-butanediol (BD). Best conditions for separation and baseline stability were achieved using a carrier electrolyte comprising 30.0mM sodium barbital and 150.0mM sodium dodecyl sulphate (SDS) at pH 10.2. Calibration functions were linear, giving correlation coefficients (r(2)) >0.998 for the three target compounds. Limits of detection (LOD) defined as three times the noise, were 5.1mg/l, 0.34 and 0.25g/l for GHB, GBL and BD, respectively. The repeatability of migration times and peak areas, expressed as the R.S.D. (n = 9) was better than 0.41 and 3.05%, respectively. Some casework samples were analysed using the optimised conditions.