Comparison of six derivatizing agents for the determination of nine synthetic cathinones using gas chromatography-mass spectrometry

Six acylation reagents have been compared for their derivatisation potential towards nine synthetic cathinones by gas chromatography-mass spectrometry (GC-MS). The evaluated reagents were pentafluoropropionic anhydride (PFPA), trifluoroacetic anhydride (TFA), chlorodifluoroacetic anhydride (CLF2AA), heptafluorobutyric anhydride (HFBA), acetic anhydride (AA) and propionic anhydride (PA). The synthetic cathinones included flephedrone (4-fluoromethcathinone or 4-FMC), mephedrone (4-methylmethcathinone or 4-MMC), pentedrone (also known as α-methylamino-valerophenone), methedrone (4-methoxy-N-methcathinone, p-methoxymethcathinone), methylone (3,4-methylenedioxy-N-methylcathinone or bk-MDMA), butylone (β-keto-N-methylbenzodioxolylbutanamine or bk-MBDB), ethylone (3,4-methylenedioxy-N-ethylcathinone MDEC or bk-MDEA), pyrovalerone (4-methyl-β-keto-prolintane) and 3,4-methylenedioxypyrovalerone (MDPV). The derivatizing agents were optimised for incubation time and temperature with some important validation parameters studied to evaluate derivatisation reactions. The anhydrides studied proved to be suitable for synthetic cathinones – all of them showing RSD and accuracy below 20%. PFPA and HFBA followed by TFA are the best choice of derivatising agents based on validation parameters. Five internal standards were evaluated with good results. Three way ANOVA, interference, fragmentation patterns and high peak area values at a concentration of 0.50 μg ml−1 were evaluated and discussed. AA and PA derivatives give high relative abundance for most drugs examined. HFBA gives more ions and multi-fragmentation patterns

The analysis and long-term stability of amphetamine-type stimulants and synthetic cathinones in urine using novel extraction methods and GC−MS

Over the last few years stimulant substances, such as amphetamine-type stimulants (ATS) and synthetic cathinones (SC), have dramatically increased the frequency of lethal intoxications. The spread of these stimulant drugs has caused a complex challenge to the forensic toxicology community. Most analytical methods focus on detection and quantification of a specific class of drugs instead of an extensive variety of compounds. A method for the detection and quantification of 29 ATS and SC drugs in a single procedure was carried out using solid-phase extraction (SPE), pentafluoropropionic anhydride (PFPA) agent and gas chromatography—mass spectrometry (GC−MS). The method was validated in accordance with SWGTOX guidelines using human urine samples. The limits of detection (LOD) and lower limits of quantification (LLOQ) were between (0.5 and 10) ng mL−1, and (5 and 50) ng mL−1, respectively. The linearity range was between 50 and 2000 ng mL−1 with a R2 >0.990 for 20 compounds. The bias and RSD were ≤20%, and no interferences or carryover were observed. The recovery was 80 to 120% for the majority of analytes. Prior to testing the substances, the GC−MS was initially optimised in terms of the oven and injector port temperatures. The sensitivity and selectivity of the GC−MS were then improved using acidified methanol and derivatisation agents. Six acylation reagents were compared and investigated using PFPA, trifluoroacetic anhydride (TFA), chlorodifluoroacetic anhydride (CLF2AA), heptafluorobutyric anhydride (HFBA), acetic anhydride (AA) and propionic anhydride (PA). The derivatisation method was optimised by modifying incubation time and temperature during the reaction and evaporation stages. Several parameters were used to evaluate the performance of the reagents, including the number of ions, relative ion ratio, peak area values, number of unique ions with some validation parameters. The reagents were further inspected using recovery through SPE in whole blood. The results of the comparison study showed that PFPA was the favoured reagent. All the derivatisation reagents were suitable for use on cathinones. Long term stability was investigated for the 29 stimulant compounds in human urine specimens over a period of 381 days at room temperature (RT), refrigerator (4°C) and freezer (−20°C) conditions. ATS were stable under all conditions, and all tested substances were stable at freezer conditions. Most SC at RT had lost more than 20% of the compound after two days, and had completely disappeared after a month. Most SC’s at refrigerator temperatures were unstable after day 21, and gradually decreased until undetected between days 77 and 349. The substances were stable on the autosampler for three days. No concentration-dependent variations were observed. Half-lives of selected drugs were briefly discussed. A sample preparation method that meets green analytic chemistry (GAC) requirements is desirable. Therefore, a method using solid phase microextraction (SPME) tips were initially developed via 13 processing steps and then validated using GC−MS in urine for eight ATS and SC substances. LOD and LLOQ were (5−25) ng mL-1, and (25−100) ng mL-1, respectively. The bias and RSD were <15% error with R2 ≥0.992 for all analytes. Applying green analytical chemistry (GAC) parameters, the procedure had minor effects on health, waste and safety proportionate to LLE and SPE by adding the only microscale amounts of methanol and salt. Attention to the prevalence of new psychoactive substances (NPS) such as SC, is significant to the justice system and the forensic toxicology community. The prevalence of SC was studied using 273 urine specimens collected from Riyadh City in Saudi Arabia. The cathinone compound estimation prevalence rate was 1.01%. No other cathinones were identified. Further prevalence studies should be conducted in the future using a larger sample size and incorporating more drug substances and metabolites

Origami Chips: Development and validation of a paper-based Lab-on-a-Chip device for the rapid and cost-effective detection of 4-methylmethcathinone (mephedrone) and its metabolite, 4-methylephedrine in urine

4-methylmethcathinone (mephedrone) has emerged in drug seizures as a new psychoactive substance (NPS) causing a public health risk of global concern. Currently, there are no commercial microfluidic devices for the selective detection of mephedrone and so this study presents a simple, low cost and portable paper-based Lab-on-a-Chip (LOC) device with colorimetric detection to fill this gap. Limits of detection for mephedrone in spiked urine and dissolved powder (aqueous) samples are clinically relevant at 4.34 ng mL-1 and 2.51 ng mL-1 respectively. No cross-reactivity for commonly encountered cutting agents, interferents and adulterants were detected. Mephedrone and its main metabolite were detectable in aqueous samples within 3 minutes. Stability and reproducibility measurements showed no significant difference in signal intensity over eight weeks and no significant difference within or between devices. The proposed device has the potential to provide cost-effective, rapid, on-site testing within forensic or clinical settings and therefore has wide global applicability

Determination and long-term stability of twenty-nine cathinones and amphetamine-type stimulants (ATS) in urine using gas chromatography-mass spectrometry

A method was developed for the screening and quantification of seven amphetamine-type stimulants (ATS) and 22 cathinones, including three metabolites, in urine with Gas Chromatography–Mass Spectrometry. This method allowed the detection and quantification of ATS and cathinones group molecules using one procedure. A study of the stability of the drug mixtures for a period of 201 days in human urine samples under three different conditions has been carried. The ATS and cathinones include amphetamine, methamphetamine, MDA, MDEA, MDMA, PMA, PMMA, cathinone, methcathinone, 3′-position-substituted, ring-substituted, methylenedioxy-substituted, N-alkyl-substituted and pyrrolidinyl-substituted. Twenty drugs out of twenty-nine were validated with a quantitative method. This method can be applied to the nine remaining drugs as a screening method. The linearity of the assay was from 50 to 2000 ng/ml, with limits of detection of 0.5 to 10 ng/ml. In terms of accuracy, between-run and within-run precision were ≤20% for 20 compounds with good selectivity. No carryover was seen, and the recovery was between 80 and 120% for most drugs tested. ATS and pyrrolidinyl-substituted groups were conducted to be stable compounds under all conditions. All compounds tested were stable at −20 °C. Some cathinones were primarily degraded after 21 days at 4 °C. They were detectable but unstable after 201 days at 4 °C. Most cathinones were unstable after a day and completely lost after 28 days at RT

Determination of amphetamine-type stimulants (ATSs) and synthetic cathinones in urine using solid phase micro-extraction fibre tips and gas chromatography-mass spectrometry

In recent years, an increasing number of stimulant drugs and new psychoactive substances (NPSs) have caused concern in scientific communities and therefore innovative methods to extract compounds from complex biological samples are required. This work is aimed at developing and validating a clean, convenient and straightforward extraction procedure with microliter amounts of organic solvent using Solid Phase Micro-Extraction tips (SPME tips) and analysis using Gas Chromatography-Mass Spectrometry (GC-MS) in human urine samples. Another aim is to evaluate three different types of SPME fibre tips C18, C18-SCX (mixed mode) and PDMS-DVB. The quantification method examined the different classes of stimulant compounds included Amphetamine-Type Stimulants (ATSs) (amphetamine, methamphetamine, para-methoxyamphetamine (PMA), and (±)-3,4-methylenedioxymethamphetamine (MDMA)) and synthetic cathinones (mephedrone, buphedrine (buphedrone ephedrine metabolite), 4-methylephedrine (mephedrone metabolite), and pentylone). The method was developed with respect to several areas of the experimental design including pH, ionic strength, addition of salts, vial dimensions, analytes and derivatisation, type of solvents, solvent volume, extraction and desorption time, agitation speeds in the extraction and desorption steps and matrix volume. The optimised method was validated for eight compounds using the SPME PDMS/DVB fibre tips with satisfactory linearity and selectivity ranging between 50 and 2000 ng mL−1, and limits of detection (LODs) and low limits of quantification (LLOQs) ranging between (5–25) and (25–100) ng mL−1 respectively. Within-run and between-run accuracy and precision were &lt;15%. The method was applied to real human urine samples indicating its suitability for common stimulant drugs and provided clean chromatograms with no interfering peaks. The assessment of green analytical chemistry for the method used was discussed and compared with Solid Phase Extraction (SPE). According to the results obtained we recommend the method for use in routine laboratories carrying out drug/forensic analysis for confirmation tests of the studied compounds