SWGTOX Guidelines Research Articles

Determination of 465 psychoactive substances, drugs and their metabolites in urine by LC-MS/MS.

Due to the emergence of novel psychoactive substances on the drug market, there is a growing demand for analytical methods allowing identification and determination of as many psychoactive substances as possible in the shortest possible time, which can be easily expanded to include additional analytes appearing in street trade. Immunochemical methods are not sufficient to meet constantly growing requirements. Therefore, the aim of the study was to develop an analytical method enabling quick analysis of urine samples for psychoactive substances, drugs and their metabolites. Liquid-liquid extraction (LLE) and liquid chromatography coupled with mass spectrometry (LC-MS/MS) were used for this purpose. Using available analytical standards, the operating parameters of the mass spectrometer were selected for each of the 477 analytes, and MRM (multiple reaction monitoring) acquisition was selected for each of them. The use of analytical standards allowed for the identification of analytes separated on the chromatography column. The exceptions are methylmethcathinone isomers (3-MMC and 4-MMC), which we were unable to separate using the gradient elution method used. Extraction using acetonitrile with the addition of ammonium formate and formic acid allowed us to obtain high recoveries without the use of β-glucuronidase. Recovery values ranged from 18.43 to 119.94%. The matrix effect was eliminated by obtaining a calibration curve in the matrix. The developed analytical method was validated in accordance with SWGTOX guidelines. Only 12 substances did not meet the validation criteria (CV: ±20% and bias: ±20%). Thus the validated method makes it possible to determine 465 psychoactive substances in just 30 minutes. In the validation process, values such as the limit of detection (LOD) and the limit of quantification (LOQ) were also determined. The LODs are in the range of 3-30 ng mL-1, and the LOQs are in the range of 10-100 ng mL-1. The method was successfully applied to toxicological analyses of urine samples, which was an opportunity to develop it further to meet the needs of toxicology.

Dispersive liquid-liquid microextraction (DLLME) for determination of tricyclic antidepressants in whole blood and plasma samples and analysis by liquid chromatography with diode array detector (LC-DAD)

Microextractions have been developed for the tricyclic antidepressants (TCAs) analysis in biological matrices, including dispersive liquid-liquid microextraction (DLLME). The proposed DLLME employed 490 µL of biological sample (whole blood or plasma), which were added 15 mg of NaCl, 10 µL of medazepam as internal standard (10 µg/mL) and 100 µL of 2 M NaOH. This mixture was homogenized by vortex (2800 rpm/10 s) and 400 µL of hexane (extractor solvent) with 600 µL of methanol (dispersing solvent) were added to the sample. After the vortex step (2800 rpm/5 s), an ultrasonic bath for 300 s was employed. Then, this content was centrifuged (10 min/10000 rpm), organic phase was collected and dried under air flow. After, 30 µL of the mobile phase was used for resuspension and 20 µL is injected into LC-DAD. This method was optimized and fully validated according to UNODC and SWGTOX guidelines, reaching limits of detection equivalent to analytical methodologies that employ mass spectrometry (MS). Also, it was applied in real cases involving suspected exposure to TCAs. So, the developed DLLME for the determination of TCAs in whole blood and plasma samples proved to be a simple, reliable, robust and reproducible method that can be used in toxicology and clinical laboratories.

Exploring the potential of fabric phase sorptive extraction in postmortem toxicology: Green analysis of pheniramine in a forensic case of its fatal intoxication

Sample preparation is vital in postmortem toxicology to extract and purify analytes from complex matrices. Challenges like interfering substances and limited sample quantity can be overcome with effective preparation, ensuring accurate results. Hence, a simple, sustainable, affordable, and high-throughput method using fabric phase sorptive extraction (FPSE) - gas chromatography - mass spectrometry (GC–MS) was developed for pheniramine (PHN) analysis in postmortem biological samples (blood and liver). FPSE extraction efficiency was optimized by studying factors like membrane type, stirring time and speed, back-extraction solvent, sample pH, and ionic strength. The optimized method was validated following SWGTOX guidelines for linearity, precision, sensitivity, recovery, matrix effect, stability, and other relevant parameters. The method exhibited linearity in the range of 0.1–10 µg mL−1 for blood and 0.1–10 µg g−1 for liver samples under optimal conditions. The limit of quantification (LOQ) was 0.066 µg mL−1 and 0.073 µg g−1 for PHN in blood and liver, respectively. Intra-day and inter-day precision were < 5.8% and < 9.8%, respectively. Recovery and accuracy ranged from 74 to 80% and 95–100%, respectively. Real autopsy samples were analyzed using this method, compared to conventional solid-phase extraction (SPE), demonstrating its effectiveness. Additionally, the environmental impact and sustainability were assessed using ComplexGAPI and Analytical Eco-Scale tools. The FPSE-GC–MS method showed superior performance, marking a notable advancement in postmortem toxicology sample preparation. To the best of our knowledge, this is the first application of FPSE in the analysis of postmortem tissue samples. In conclusion, FPSE-GC–MS method offers a simple, sustainable, and affordable approach to sample preparation for postmortem forensic samples, overcoming challenges such as interfering substances and limited sample quantity. These advantages of FPSE make it a promising alternative to traditional methods, positions it as a significant advancement in the field, poised to shape the future of sample preparation in forensic toxicology.

UPLC-MS/MS Based Identification and Quantification of a Novel Dual Orexin Receptor Antagonist in Plasma Samples by Validated SWGTOX Guidelines.

Lemborexant (LEM) is a novel dual orexin receptor antagonist (DORA), recently approved for the treatment of insomnia. As with other DORAs, LEM has potential of abuse and therefore placed in Schedule IV class by the United States Drug Enforcement Administration (USDEA). In this study, a sensitive and accurate UPLC-MS/MS assay was developed for the quantification of LEM in human plasma sample using losartan as an internal standard (IS). The chromatographic separation was performed by using gradient elution of mobile phase, comprising of 10 mM ammonium acetate and acetonitrile with a flow rate of 0.3 mL/min. An Acquity UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) column was used for separation of LEM and IS by maintaining the oven temperature of 40 °C. The electrospray ionization in positive mode was used for sample ionization. The precursor to product ion transition of 411.12 > 175.09 (qualifier) and 411.1 > 287.14 (quantifier) was used for detection and quantification of LEM, respectively, in multiple reaction monitoring mode. Being a drug of abuse, the assay was validated according to "Scientific Working Group for Toxicology" (SWGTOX) guidelines, including limit of detection (LOD), limit of quantification (LOQ), precision and bias, calibration model, interferences, carry-over effects, matrix effects, and stability parameters. The LOD and LOQ of the assay were 0.35 and 1.0 ng/mL, respectively. The linear range was between 1-300 ng/mL with correlation coefficient of ≥0.995. The method was also cross validated in rat plasma samples with acceptable ranges of precision and accuracy before its application for pharmacokinetic study in rats.

Determination of cannabinoids content in light cannabis inflorescences sold in France.

In the last 2 years, the number of shops selling CBD-rich THC-deprived cannabis flowers (CrTd) has increased considerably in France as in many European countries. The objective of this study was to determine the actual composition of the samples sold in these stores and to discuss regulatory consequences that may affect users. Samples were provided from shops in the region Provence-Alpes Cote d'Azur (PACA), France. Pictures of the samples were taken before they were weighed then crushed. Twenty milligrams were diluted in 10ml heptane ethyl acetate (7:1; v:v) for analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was validated according to SWGTOX guidelines for the quantification of cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC) and cannabinol (CBN). Thirty-nine samples obtained between November 2021 and January 2022 in the PACA region were analyzed in this study. Mean content was 0.32% (0.03%-0.77%; STDV = 0.17%; n = 39) for THC, 2.23% (0.01%-5.97%; STDV = 1.29%; n = 39) for CBD and 0.01% (0.004%-0.025%; STDV = 0.01%; n = 19) for CBN. THC content over the threshold defined by the European legislation (>0.3%) was found in 18 of the 39 samples analyzed together with a CBD content <1% in nine samples (23%). None of the products analyzed had health risk messages on the packaging. The consumption of these products may lead to the presence of THC in biological fluids, which can be detected by screening. Users may then find themselves in breach of the law particularly when driving. Consumers should therefore be informed both about the actual composition of these products and about the legal and health risks they run.

Determination of tryptamine analogs in whole blood by 96-well electromembrane extraction and UHPLC-MS/MS

Tryptamines are a class of new psychoactive substances relevant for analysis in forensic laboratories. Tryptamine analogs are diverse in structure and chemical properties, and prone to display instability. This puts high demands on sample preparation, especially from complex matrices such as whole blood. In the present paper, a prototype 96-well electromembrane extraction (EME) device was employed for the simultaneous extraction of 20 tryptamines from whole blood prior to UHPLC-MS/MS analysis. For the EME procedure, spiked whole blood diluted 3-fold with 1% ascorbic acid in 25 mM formic acid was extracted for 60 min across a supported liquid membrane (SLM) containing 5% bis(2-ethylhexyl) ether (DEHP) in 2-nitrophenyloctylether (NPOE) into an acceptor solution containing 1% ascorbic acid in 25 mM formic acid. The method was validated for 16 of the tryptamines in the range 5-500 nM according to SWGTOX guidelines and the analytes displayed limits of detection from >0.15 nM to 0.75 nM. An AGREEprep analysis was performed to evaluate method greenness with a final greenness score of 0.76. 96-well EME offered fast, efficient and green extraction of tryptamines from whole blood, demonstrating good potential for implementation in forensic laboratories when commercial devices will become available.

Comparison of screening with the use of Biochip Array Technology to the direct analysis of 521 psychoactive compounds and their residues with the use of LC-MS/MS method – does it mean the end of an era for immunochemical research?

The classical strategy for toxicology testing of road users involves preliminary immunochemical screening and in case of obtaining the positive result, the instrumental tests are necessary to confirm the result. Immunochemical tests are a cheap, quick and simple tool to confirm the presence of a particular substance in the biological material such as saliva, urine or blood. Their fundamental disadvantages are the limited specificity and the scope of analyses. The field tests allow to determine the presence of a few groups of compounds in one single test. Laboratory tests ELISA usually allow for detecting a few epitopes in one test. In the era of many NPS (novel psychoactive substances) and psychoactive drugs used for recreational purposes, the limited multiplexity factor related to immunochemical arrays is becoming a more serious problem. Therefore, some solution is the application of matrix screening. Biochip Array Technology matrix test allows to analyse 20 various groups of compounds on one chip. Another approach is to completely abandon screening for the sake of definite identification and quantitative determination. In order to satisfy the current situation, the multiplex method with the use of LC-MS/MS method to detect 521 psychoactive compounds and their metabolites was worked out. The experimental material consisted of 300 blood samples taken from road users in Poland, routinely tested in the laboratory of the Institute of Forensic Genetics from 2019 to 2021. The immunochemical tests were carried out with the use of Randox Evidence MultiSTAT analyser. The research with the use of LC-MS/MS method was carried out to detect the presence of 521 psychoactive compounds and their residues in blood taken from the drivers. Blood extraction was carried out using a one-step solid-liquid method. Validation was carried out for quantitative analysis of 521 analytes following the SWGTOX guidelines. Two MRM pairs were selected for each analyte according to the valid mass spectrometry standards and optimal analysis parameters were determined. The analyses were carried out using LC-MS/MS (Kinetex C18 100 × 3 mm, 2.6 μm chromatographic column, mobile phases (in formate buffer) water/methanol, QTRAP 5500 system operated in ESI mode). Comparison of results determined with the use of immunochemical and LC-MS/MS methods indicates that screening immunochemical tests may generate both false positive and false negative results. In spite of the application of the matrix immunochemical test, which allowed the detection of 20 groups of psychoactive substances and metabolites, the LC-MS/MS method made it possible to detect and determine the concentration in a greater number of cases. Taking into account the enormous number of compounds which detection is required while performing screening tests and the need to carry out the confirmation tests, it is justifiable to consider the change of strategy for the sake of the direct instrumental analysis in order to determine a great number of compounds with the use of multiplex analytical methods.

Reversed-Phase Liquid Chromatographic Internal Standard Method Using Losartan Potassium for Quantitative Estimation of Ivabradine Hydrochloride in Pharmaceutical Tablet Dosage Form and Plasma

A reversed-phase liquid chromatographic method for ivabradine hydrochloride using Diode Array Detector (DAD) and internal standard technique was developed and validated according to ICH and SWGTOX guidelines. The prime objective of this study was to develop a precise and accurate method that can be equally applicable to biological (plasma) as well as non-biological (active pharmaceutical ingredient and pharmaceutical tablets) matrices. Losartan potassium was used as an internal standard due to its easy availability. After liquid-liquid extraction using acetonitrile, the ivabradine hydrochloride and internal standard were chromatographed on Agilent 1200 series HPLC system equipped with DAD detector, auto-sampler and chemstation software. Analytical separation was achieved on Agilent C-18 (5µm, 25cm x 4.6mm) reversed-phase column at 30°C column oven temperature, 10μL injection volume and 286nm wavelength. Isocratic mobile phase system comprised of 60:40 v/v ratio of HPLC grade methanol and water adjusted to pH 6.8 using orthophosphoric acid was employed with 1mL/min flow rate. The method linear range was 0.025-3µg/mL (25-3000ng/mL) for pharmaceutical tablets and plasma with the coefficient of linearity ranged 0.997-0.999. Results for precision, accuracy, recovery, stability and matrix effect studies were within acceptable limits for both plasma and tablets. Method was successfully applied to the commercial tablet products and patient plasma samples to estimate the amount of ivabradine hydrochloride.

Accelerated Extraction and Analysis of Ethyl Glucuronide in Hair by Means of Pressurized Liquid Extraction Followed by Liquid Chromatography–Tandem Mass Spectrometry Determination

The measurement of ethyl glucuronide (EtG) in hair is an established practice to evaluate alcohol consumption habits of the donors; nevertheless, analytical variability has shown to be an important factor to be considered: measured EtG values can vary significantly as a consequence of analyte washout during decontamination, pulverization of samples, extraction solvent and incubation temperature. In the present study, we described a new method for automated hair decontamination and EtG extraction from the inner core of the hair by using pressurized liquid extraction (PLE), followed by solid-phase extraction (SPE) cleanup; validation was performed according to SWGTOX guidelines. The extraction efficiency of the new method was evaluated by comparing the results with those obtained by a validated and ISO/IEC 17025:2005 accredited method; an average positive difference of + 32% was observed when the extraction was performed by PLE. The effect of hair pulverization was also studied, and a good correlation between cut and milled hair was observed, implying that PLE allowed a highly efficient extraction of EtG from the inner keratin core of the hair, no matter if it has been cut or pulverized. Finally, to verify the results, paired aliquots of 27 real hair samples were analyzed with both PLE and a protocol optimized by design-of-experiment strategies planned to maximize the extraction yield; in this case, a comparable efficiency was observed, suggesting that exhaustive EtG extraction was obtained with both approaches. This finding opens new perspectives in the eligible protocols devoted to hair EtG analysis, in terms of speed, automation and reproducibility.

Distribution of synthetic opioids in postmortem blood, vitreous humor and brain

In the US, the use of synthetic opioids (e.g. fentanyl and derivatives) has become an increasing health issue with thousands of overdose deaths being observed since 2013. With the high mortality rate associated with these substances, postmortem analyses and interpretation of synthetic opioids has become extremely important. However, due to the novelty of these compounds, the available data are limited and provides challenges to toxicologists. The objectives of this study were (1) to develop and validate analytical methods for the determination of synthetic opioids in vitreous humor and brain, and (2) to investigate the postmortem distribution of new synthetic opioids in blood, vitreous humor, and brain tissue. Vitreous humor (0.5mL) and brain tissue (5g) homogenized in water (diluted 1:3, w/w) were extracted by mixed mode cation exchange-reversed phase solid phase extraction. Extracts were analyzed by liquid chromatography tandem mass spectrometry (LC–MSMS). The chromatographic separation was performed by reversed-phase with 0.1% formic acid in water and in acetonitrile as mobile phases in gradient mode, with a total run time of 21min. Data were acquired with ESI+ in dynamic multiple reaction mode (dMRM), monitoring 2 transitions per compound. The methods were succesfully validated following SWGTOX guidelines, with limits of quantification of 0.1ng/mL in vitreous humor and 0.1ng/g in brain. Fifty-eight authentic case samples from the New York City Office of the Chief Medical Examiner (NYC-OCME) were analyzed to assess the distribution and detectability of synthetic opioids in these postmortem samples. Of the fifteen synthetic opioids included in the method, six synthetic opioids and metabolites (4-ANPP, acetylfentanyl, fentanyl, furanylfentanyl, norfentanyl, U-47700) were detected in the authentic cases. Concentrations for most analytes were within the 0.1 to 100ng/mL or ng/g calibration range across all three matrices, with only concentrations from acetylfentanyl and U-47700 exceeding 100ng/mL or ng/g. The highest concentrations were observed in brain (except norfentanyl), followed by blood and vitreous humor. Most analytes were detected in all three matrices in a given case. This was followed by detection of an analyte in combinations of brain and another matrix or brain only. Through the case analyses, vitreous humor and brain demonstrated to be viable alternatives to blood when performing postmortem analyses of synthetic opioids. Brain exhibited a higher detectability for most analytes when compared to blood and vitreous humor.

An Ultra-High-Pressure Liquid Chromatographic Tandem Mass Spectrometry Method for the Analysis of Benzoyl Ester Derivatized Glycols and Glycerol.

Presented is an ultra-high-pressure liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method developed for the detection of propylene glycol, glycerol, ethylene glycol and diethylene glycol using isotopically labeled standards in urine as part of ongoing studies to evaluate whether urinary propylene glycol and/or vegetable glycerin concentration are indicators of recent use. Propylene glycol and vegetable glycerol are found in many products that are consumed and used including electronic cigarettes (e-cigarettes). E-cigarettes are battery-powered devices used as an alternative to traditional cigarettes. The liquid formulations aerosolized in these devices largely consist of propylene glycol and/or vegetable glycerol. Published reports regarding the ratio of propylene glycol to glycerol content in these formulations ranged from 50:50 to 100 percent of either. For the analysis of urine specimens from both users and non-users of e-cigarettes, calibrators, controls and specimens were derivatized using benzoyl chloride prior to analysis. They were analyzed using a Waters AcQuity Xevo TQ-S Micro UPLC-MS/MS. Chromatographic separation was performed on an AcQuity UPLC BEH C18 1.7um, 2.1×50mm, column using a 20mM ammonium formate in water and 20mM ammonium formate in methanol as the mobile phase. The method was validated using SWGTOX guidelines for linearity, precision and accuracy, stability, carryover and limit of detection. The linear range was determined using a seven-point calibration curve ranging between 0.5 and 100mcg/mL. The bias for all validation controls was determined to be ±20% of the expected concentrations with CVs of <15%. A total of 124 urine specimens analyzed collected with 50 specimens collected from self-reported non-smokers (cigarettes/e-cigarettes) confirmed cotinine free using the DRI® Cotinine Assay (Thermo Scientific, Waltham, MA) and 74 specimens collected before and after 12hours self-reported e-cigarettes abstinence e-cigarette users. Propylene glycol and glycerol were determined to have concentration ranges of "none detected" to 1470 and "none detected" to 2950mcg/mL, respectively.

Combination of pressurized liquid extraction with dispersive liquid liquid micro extraction for the determination of sixty drugs of abuse in hair

The increasing phenomenon of drug addiction and the introduction of New Psychoactive Substances (NPS) have led to a progressive growth of research in the field of forensic analytical toxicology, with the need to develop modern and faster analytical procedures. Hair testing has gained increasing attention and recognition as a complement to blood and urine analysis, since it is a unique material for the retrospective detection of drugs, due to its large detection window.In this paper, a multiclass method for the simultaneous extraction, identification and quantification of sixty drugs of abuse belonging to different chemical classes in hair is proposed. This method can provide a valid, fast, simple and low-cost alternative to common tests; at the same time, it provides quantitative results, to concurrently confirm the assumption of one or more illicit substances. Both the decontamination step and the extraction of the analytes from the inner core of the hair were carried out by means of pressurized liquid extraction (PLE) while the clean-up was performed by dispersive liquid/liquid microextraction (dLLME), giving the great advantage of a high enrichment factor. The selected chromatographic conditions allowed a satisfying separation of the 60 analytes in 14 min, while the detection was conducted with a high-resolution mass spectrometer with Orbitrap technology. This multiclass method was suitable for analytes with different chemical characteristics allowing to reduce time and cost of analysis, organic solvent volume and the amount of sample required for analysis. The whole method was fully validated as confirmatory method following SWGTOX guidelines.

Evidence for nonlinear accumulation of the ultrapotent fentanyl analog, carfentanil, after systemic administration to male rats

The current opioid overdose crisis is being exacerbated by illicitly manufactured fentanyl and its analogs. Carfentanil is a fentanyl analog that is 10,000-times more potent than morphine, but limited information is available about its pharmacology. The present study had two aims: 1) to validate a method for quantifying carfentanil and its metabolite norcarfentanil in small-volume samples, and 2) to use the method for examining pharmacodynamic-pharmacokinetic relationships in rats. The analytical method involved liquid-liquid extraction of plasma samples followed by quantitation of carfentanil and norcarfentanil using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was validated following SWGTOX guidelines, and both analytes displayed limits of detection and quantification at 7.5 and 15 pg/mL, respectively. Male Sprague-Dawley rats fitted with jugular catheters and temperature transponders received subcutaneous carfentanil (1, 3 and 10 μg/kg) or saline. Repeated blood specimens were obtained over 8 h, along with pharmacodynamic measures including core temperature and catalepsy scores. Carfentanil produced dose-related hypothermia and catalepsy that lasted up to 8 h. Carfentanil Cmax occurred at 15 min whereas metabolite Cmax was at 1–2 h. Concentrations of both analytes increased in a dose-related fashion, but area-under-the-curve values were much greater than predicted after 10 μg/kg. Plasma half-life for carfentanil increased at higher doses. Our findings reveal that carfentanil produces marked hypothermia and catalepsy, which is accompanied by nonlinear accumulation of the drug at high doses. We hypothesize that impaired clearance of carfentanil in humans could contribute to life-threatening effects of this ultrapotent opioid agonist.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Quantification of U-47700 and its metabolites in plasma by LC-MS/MS

Novel Synthetic Opioids (NSO) have caused a recent epidemic both nationally and globally. NSO have gained popularity in the illicit drug market and have brought about an increase in fentanyl and its derivatives, as well as other chemically unrelated opioid agonists. U-47700, a non-fentanyl analog analgesic opioid, was first developed by The Upjohn Company and has a reported potency of 7.5 times that of morphine. Like many NSO, U-47700 is usually sold as a research chemical that can be purchased online but can also be found in “Gray Death” which is a mixture of fentanyl(s), heroin, and U-47700. With the emergence of these NSO, there is a need for laboratories to be able to detect these drugs in various matrices. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was optimized and validated to detect and quantify U-47700 and its metabolites, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700, in 100 μL human plasma using an optimized solid phase extraction procedure. A small sample size (100 μL) was utilized for a future pharmacokinetic study in rats. The method was validated according to SWGTOX guidelines, including: precision and bias, linearity, carryover, interferences, matrix effects, limit of detection (LOD), limit of quantification (LOQ), dilution integrity, and stability. The LOD were 0.05 ng/mL for U-47700 and N-desmethyl-U-47700 and 0.1 ng/mL for N,N-didesmethyl-U-47700. Linear ranges for U-47700 and N-desmethyl-U-47700 were 0.1–100 ng/mL and 0.5–100 ng/mL for N,N-didesmethyl-U-47700. Matrix effects were analyzed following the post-extraction addition approach and were <5%, indicating little ion suppression or enhancement. Extraction recovery was >79%. Analytes were stable in all conditions and no endogenous or exogenous interferences were detected. This method was cross-validated in rat plasma with acceptable bias (2.1–6.2%) and precision (−14.7–15.7%) within acceptable limits. Matrix effects and extraction efficiency was comparable to human plasma validation. Postmortem whole blood samples (n = 15) were analyzed with the validated method. U-47700, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 concentration ranges were 1.1–1367 ng/mL, 4.0–1400 ng/mL and 0.7–658 ng/mL, respectively.

Gas Chromatography-Mass Spectrometry Method for the Quantitative Identification of 23 New Psychoactive Substances in Blood and Urine.

New psychoactive substances (NPSs) have become an integral part of the recreational drug market with "new" compounds being reported by the European Monitoring Centre for Drugs and Drug Addiction weekly. Due to the changing nature of NPSs, it is impractical to carry out single analyte or even simple class quantitation. Although several gas chromatography-mass spectrometry (GC-MS) methods have been developed these are typically class specific. We present a validated GC-MS method for the quantitation of 2-DPMP, 3-MeO-PCE, 3-MeO-PCP, 5-APB, 6-APB, benzedrone, butylone, ethylone, flephedrone, methiopropamine, MDPV, mephedrone, methoxetamine, methylone, naphyrone, 25B-NBOME, 25C-NBOME, 25D-NBOMe, 25E-NBOME, 25H-NBOME, 25I-NBOME, Mescaline-NBOME and 25P-NBOME in blood and urine samples. Sample preparation was carried out using solid-phase extraction followed by derivatisation and analysis by GC-MS. Parameters investigated for validation included bias, precision, linear calibration model, carryover, interferences, limit of detection, limit of quantification, and autosampler and freeze/thaw stability. All drugs yielded successful results for each of these parameters as per SWGTOX guidelines. The GC-MS method was used for the reanalysis of 12 blood samples (eight cases) where 25I-NBOMe, 25C-NBOMe, methoxetamine and methylone had previously been detected by NMS laboratories. This GC-MS method was able to quantitatively detect these drugs in 75% of the blood samples, 42% of which contained either 25C-NBOMe or 25I-NBOMe. This method accurately allows for the simultaneous quantification of a wide variety of compounds via GC-MS, in particular NBOMe compounds which are typically analysed by liquid chromatography-tandem mass spectrometry which is not available in all laboratories.

Addressing the Fentanyl Analogue Epidemic by Multiplex UHPLC-MS/MS Analysis of Whole Blood.

Fentanyl and fentanyl analogues (fentanyls) are very potent opioids posing a serious threat to the public health. Thousands of overdose deaths across the world are caused by fentanyls, and the numbers are increasing. Rapid mapping of current trends in opioid abuse is necessary to accelerate preventive measures. To ensure this, there is a need for sensitive targeted multiplex MS/MS methods to pinpoint drugs of abuse. We present a fully validated UHPLC-MS/MS method for the determination of 26 fentanyls, including several structural isomers, and the opioid antagonist naloxone in human whole blood. Blood samples were prepared by liquid-liquid extraction with ethyl acetate and heptane. The fentanyls were separated with UHPLC, using a Kinetex biphenyl column (2.1 × 100 mm, 1.7 µm; Phenomenex, Verløse, Denmark) with an acidic mobile phase. Quantification was performed by MS/MS. The method was validated according to SWGTOX guidelines. The developed method could successfully separate all 27 analytes, including 7 isomers, and was validated according to SWGTOX guidelines with very low limits of quantification (4-20 pg/mL). The applicability of the method was demonstrated by determination of fentanyls in postmortem blood samples from 2 cases. A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.

Simultaneous Detection and Quantification of Three Novel Prescription Drugs of Abuse (Suvorexant, Lorcaserin and Brivaracetam) in Human Plasma by UPLC-MS-MS.

Suvorexant (SVR), lorcaserin (LCR) and brivaracetam (BVR) have been recently approved for the treatment of insomnia, obesity and epilepsy, respectively. Despite their clinical uses, these drugs have some abuse potential and have been enlisted under the schedule IV (SVR, LVR) and schedule V (BVR) categories of the Controlled Substances Act. A sensitive UPLC-MS-MS assay was developed for simultaneously determining SVR, LCR and BVR in human plasma. The liquid-liquid extraction method, using tert-butyl methyl ether as an extracting solvent, was used for sample preparation. Chromatographic separation was performed by using the Acquity BEH C18 column, using 10 mM ammonium acetate/acetonitrile/formic acid (15/85/0.1%; v/v/v) as the mobile phase. For sample ionization, electrospray ionization was used in the positive-ion mode. The multiple-reaction monitoring mode was used for detecting and quantifying analytes by using separate precursor-to-product ion transitions. The assay was validated following the SWGTOX guidelines, and all validation results were within the acceptable limits. The calibration curves of the analytes in the plasma were found to be linear, and the coefficient of determination (R2) was ≥ 0.992 for all the three analytes. The limit of detection values for SVR, LCR and BVR were 0.08, 0.11 and 0.26 ng/mL, respectively, whereas the limit of quantification values were 0.16, 0.27 and 0.65 ng/mL, respectively. The assay developed in this study is suitable for the identification and quantification of SVR, LCR and BVR in the forensic laboratory.

UPLC-MS/MS determination of suvorexant in urine by a simplified dispersive liquid-liquid micro-extraction followed by ultrasound assisted back extraction from solidified floating organic droplets

Suvorexant is a novel sedative/hypnotic drug approved for treatment of insomnia. It has significant forensic importance due to its hypnotic and depressant effects on central nervous system. In this study, a highly sensitive UPLC-MS/MS assay was developed and validated for the determination of suvorexant in urine sample. A simplified dispersive liquid-liquid microextraction followed by ultrasound assisted back extraction from solidified floating organic droplets was employed for sample preparation. The 20 μL of 1-undecanol and 200 μL of acetonitrile were used as extraction solvent and dispersive solvent, respectively. An ultrasound assisted back extraction step was employed to enable the cleanup procedure compatible with mass spectrometric detection. Acquity CSH™C18 column with mobile phase composition of 15 mM ammonium acetate: acetonitrile: formic acid (15:85:0.1%; v/v/v) were used for chromatographic separation. The multiple reaction monitoring transition of 451.12 →104.01 and 451.12→186.04 were used for identification and quantification of suvorexant, respectively, whereas 237.06→194.1 was used for IS in positive mode. The assay demonstrated good linearity in the range of 0.27–1000 ng mL−1 with limit of detection (LOD) and quantification (LOQ) of 0.10 and 0.27 ng mL-1, respectively. Assay validation was performed by following SWGTOX guidelines and all validation results were found to be within acceptable limits. This is the first report of dispersive liquid-liquid microextraction based on solidification of floating organic droplets employed to UPLC-MS/MS for application in biological fluids.

Long-term stability of synthetic cathinones in dried blood spots and whole blood samples: a comparative study

The long-term stability of four synthetic cathinones in dried blood spots (DBS) were tested and compared with those of whole blood samples. A method to determine four cathinones (pentylone, butylone, mephedrone and benzedrone) in DBS was developed and validated. For comparison, the traditional liquid-liquid extraction for the same analytes in whole blood was also performed. The calibration curve was found to be linear over 25–1000 ng/mL for DBS samples, with a limit of quantification (LOQ) at 25 ng/mL. The interday imprecision and bias results [up to 7.1% relative standard deviation (RSD) and 5.8%, respectively] were much lower than the maximum data recommended by SWGTOX guidelines. The validation results were similar to those of whole blood samples, which showed interday imprecision and bias results of up to 8.1%RSD and 12.3%, respectively, with a linear calibration curve between 10 and 1000 ng/mL and LOQ of 10 ng/mL. A stability study to compare the degradation rate between both matrices at three storage temperatures [room temperature (25 °C), 4 and − 20 °C] and during 90 days proved the poor stability of cathinones in whole blood, where the methylenedioxy cathinones displayed better stability than those with ring substituents. DBS allowed detection of synthetic cathinones after much longer periods than in whole blood samples. DBS proved, therefore, to be an useful alternative technique to store blood samples. Extraction of DBS is easy and analytes remain stable for much longer periods.

Analysis of new psychoactive substances in oral fluids by means of microextraction by packed sorbent followed by ultra‐high‐performance liquid chromatography–tandem mass spectrometry

In recent years, new drugs, commonly known as new psychoactive substances (NPS), appeared on the market, which include, among others, synthetic cannabinoids, cathinones, and tryptamine analogs of psilocin. The aim of this work was to develop and validate a new method for simultaneous screening and quantification of 31 NPS in oral fluid by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The chosen target analytes represented different chemical and toxicological NPS classes, such as synthetic cathinones, piperazines, phenethylamines, synthetic cannabinoids, and their metabolites. The procedure involved a rapid sample preparation based on protein precipitation followed by clean-up utilizing microextraction by packed sorbent (MEPS); the quantitative analysis was performed by UHPLC-MS/MS. The MEPS clean-up, regardless of non-quantitative recoveries for some analytes, provided an effective removal of interfering compounds, as demonstrated by reduced matrix effects found at different concentrations for all the analytes. The validation protocol, based on SWGTOX guidelines, demonstrated the suitability of the proposed method for quantitative analysis: linearity range ranged over 3 or 4 orders of magnitude; precision and accuracy tests gave RSD% values below 25%, and accuracy ranged from 85.9% to 107%, accomplishing SWGTOX requirements. Limits of detection (LODs) ranged between 0.005 ng/mL and 0.850 ng/mL and limits of quantification (LOQs) from 0.015 to 2.600 ng/mL.