Scientific Working Group For Forensic Toxicology Research Articles

Development and Validation of a Uplc-Qtof-Ms Method for Blood Analysis of Isomeric Amphetamine-Related Drugs

The identification of isomeric drugs is gaining increasing importance in forensics and doping control. Isomers vary in terms of safety, effectiveness, and regulation, particularly for amphetamine-related drugs (ARDs). This study developed and validated a pseudo-isocratic UPLC-qTOF-MS analytical method for the identification of isomeric Amphetamine-related drugs (ARDs) in blood following mixed-mode solid-phase extraction (MMSPE). The procedure requires 250 μL of blood to achieve a limit of quantification (LOQ) and detection (LOD) of 20 ng/mL for all analytes. In aged animal blood samples, extraction recoveries of 63–90% and matrix effects of 9–21% were observed. Precision and accuracy for all analytes were within 20% and 89–118%, respectively. The analytical method was developed and validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard. It has acceptable accuracy and precision for use in doping control and forensic toxicology.

Determination of Ethyl Glucuronide and Ethyl Sulfate in Dried Blood Spots by UHPLC-MS-MS: Method Validation and Assessment of Ethanol Exposure in Postmortem Samples from Road Traffic Victims.

The determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood has been proposed in clinical and forensic applications to identify recent alcohol consumption. Also, there is a growing interest on the use of dried blood spots (DBS) in toxicological analysis, allowing increased stability of the analytes and simplifying sample transportation and storage. This study presents the development and validation of a method for quantifying EtG and EtS in DBS using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The DBS samples were extracted with a mixture of methanol and acetonitrile (80:20 v/v) and analyzed using UHPLC-MS-MS with electrospray source in negative mode, after separation with a fluoro-phenyl stationary phase. Validation was performed according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, with calibrations ranging from 0.10 to 18 µg/mL for EtG and 0.02 to 6 µg/mL for EtS. The analytes were stable in DBS stored from -20 to 45°C for 21 days. The method was successfully applied to capillary and venous DBS samples from 20 volunteers after ethanol ingestion and to DBS samples from 99 fatal victims of road traffic injuries. Capillary DBS was comparable to venous DBS and fresh whole blood in Passing-Bablok and Bland-Altman analysis, with correlation coefficients >0.91 (P < 0.001) for all comparisons. In postmortem application, the DBS EtG and EtS analysis indicated positive exposure to ethanol in 72.7% of the cases (EtG: 0.10-24.0 µg/mL and EtS: 0.03-4.11 µg/mL). The identification of ethanol consumption from blood alcohol concentrations (BACs) and EtG/EtS in DBS was in agreement in 98.6% of positive and 96.3% of negative cases (kappa 0.877, P < 0.001), indicating a high level of concordance with BAC in assessing alcohol use in postmortem samples.

Validation of gas chromatography mass spectrometry (GC-MS) qualitative method for detection of paraquat in postmortem samples by sodium borohydride-platinum chloride chemical reduction

To develop a GC-MS method for the detection of paraquat by sodium borohydride-platinum chloride reduction in postmortem samples. Paraquat [1,1′-dimethyl-4,4′-bipyridilium dichloride,(PQ)] is a bipyridylium quaternary ammonium contact herbicide. At the Institute of Forensic Science and Legal Medicine (IFSLM), an average of 13 cases of PQ poisoning are submitted annually for postmortem toxicological analysis. Previously a colorimetric method for the identification of PQ was used. A more sensitive method was needed to confirm the presence of PQ. There is limited studies on liquid-liquid extraction (LLE) of postmortem fluids and tissues for determination of PQ by GC-MS. Some methods used a chemical reduction involving sodium borohydride (NaBH4) or sodium borohydride with nickel chloride (NaBH4-NiCl2), prior to LLE, to gain more volatile compounds for analysis. In the absence of NiCl2, platinum (IV) chloride (PtCl4) was used as a substitute catalyst in the validation. The aim of this validation is to identify PQ in suspected poisoning cases from postmortem samples using chemical reduction reaction, followed by LLE and then (GC-MS). The qualitative method involved an initial chemical reduction of PQ to the perhydrogenated product and monoene by-product: 500 μL blood, urine or tissue homogenate was placed in a glass tube, followed by 200 μL toluene, 400 μL PtCl4 (1% w/v) and then 500 μL NaBH4 (40% w/v) was added dropwise. The mixture was vortexed then left to stand for 1½ hours at room temperature. 1 mL 5 N sodium hydroxide solution was added to stop the reaction. For LLE, 3 mL hexane: ethyl acetate (4:1) was added to the tube and placed on shaker for 45 minutes at 300 rpm. The sample was centrifuged for 1 hour at 1400 g. The hexane:ethyl acetate layer was then transferred to a beaker and allowed to evaporate to dryness. The residue was reconstituted with 1 mL hexane, transferred to GC vial, and allowed to evaporate to dryness. The residue was then reconstituted with 100 μL hexane, and transferred to a GC vial fitted with glass insert, with 1 μL being injected into the GC-MS system (Agilent 7890A GC coupled with an Agilent 5975C VL MSD with Triple Axis Detector Mass Spectrometer). As recommended by the Scientific Working Group for Forensic Toxicology (SWGTOX), the validation parameters for qualitative confirmation/identification were specificity, recovery, carryover, repeatability and limit of detection. The method was applied to blank blood and urine samples, spiked blood and urine samples, as well as 10 suspected PQ poisoning cases (24 postmortem samples) submitted to the IFSLM. The blank blood and urine samples analysed did not yield any positive results for the analytes of interest. Blood and urine samples spiked with the PQ standard solution resulted in the detection of both the perhydrogenated product and monoene by-product. The limit of detection for both reduction products was 30 ppm. There was no detection of the analytes in the three blanks analysed after an injection of 1000 ppm PQ. The reduction products of PQ were detected in all 10 cases. Four of these cases had previously given negative results using the colorimetric test. The PtCl4 was found to be an adequate substitute catalyst in the reduction procedure. The procedure was proven to be sensitive, repeatable and robust. All sample types assessed by the method gave positive results, however stomach and content produced the best chromatograms with sharp, clearly defined and resolved peaks. In summary, the presented procedure provided excellent performance in the identification of the reduction products of PQ in postmortem samples.

Simultaneous Quantification of 25 Fentanyl Derivatives and Metabolites in Oral Fluid by Means of Microextraction on Packed Sorbent and LC-HRMS/MS Analysis.

Fentanyl and fentalogs’ intake as drugs of abuse is experiencing a great increase in recent years. For this reason, there are more and more cases in which it is important to recognize and quantify these molecules and related metabolites in biological matrices. Oral fluid (OF) is often used to find out if a subject has recently used a psychoactive substance and if, therefore, the person is still under the effect of psychotropics. Given its difficulty in handling, good sample preparation and the development of instrumental methods for analysis are essential. In this work, an analytical method is proposed for the simultaneous determination of 25 analytes, including fentanyl, several derivatives and metabolites. OF was collected by means of passive drool; sample pretreatment was developed in order to be fast, simple and possibly semi-automated by exploiting microextraction on packed sorbent (MEPS). The analysis was performed by means of LC–HRMS/MS obtaining good identification and quantification of all the analytes in less than 10 min. The proposed method was fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) international guidelines. Good results were obtained in terms of recoveries, matrix effect and sensitivity, showing that this method could represent a useful tool in forensic toxicology. The presented method was successfully applied to the analysis of proficiency test samples.

Rapid and quantitative determination of fentanyls and pharmaceuticals from powdered drug samples by paper spray mass spectrometry.

Paper spray mass spectrometry is presented as a direct, quantitative tool for the measurement of pharmaceutical drugs and a variety of fentanyl analogs in solid samples and powder slurries with the ultimate goal of providing meaningful harm-reduction drug checking. Method development and validation was carried out for fentanyl analog slurries as a proxy for street drug samples. Lower limits of quantitation were determined to be 3.6-7.4ng/g for fentanyl analogs in the pharmaceutical slurry matrix. Using 1mg of solid sample, the method can quantify picogram quantities of these drugs, well below required thresholds for even the most potent fentanyl analogs. Quality control samples were prepared and used to assess method validity according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. Performance metrics for both precision and accuracy were found to be within SWGTOX-recommended guidelines. Additionally, pharmaceutical tablets were used to demonstrate the applicability of the developed paper spray methodology for the direct qualitative and quantitative analysis of active ingredients in pharmaceutical powders deposited directly onto the paper spray substrate. A proposed workflow for rapid solid drug sample measurements is presented with potential applications for point-of-use street drug measurements and other solid sample matrices.

Hollow-fibre liquid-phase microextraction and gas chromatography-mass spectrometric determination of amphetamines in whole blood

Here, we present a fully validated method using a hollow-fibre liquid-phase microextraction technique for the determination by gas chromatography-mass spectrometry (GC–MS) of amphetamine (AMP), methamphetamine (MET), fenproporex (FEN), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxyethylamphetamine (MDEA) in whole blood. The validation parameters presented successful values within those recommended by the Scientific Working Group for Forensic Toxicology (SWGTox) in the Standard Practices for Method Validation in Forensic Toxicology. The limits of detection ranged from 1 to 3 ng/mL, and the limits of quantification ranged from 2 to 5 ng/mL. The determination coefficients (r2) ranged from 0.990 to 0.997, and the method presented good intraday and interday accuracy (from 90.4% to 97.2%) and satisfactory recovery (from 68% to 110%). No carryover was observed. The heteroscedasticity was tested, and only AMP presented homoscedasticity. Weighting factors were applied to correct the linearity of MET (1/x2), MDA (1/x), FEN (1/x1/2), MDMA (1/x2) and MDEA (1/y). Dilution integrity was tested at ratios of 1:2, 1:5 and 1:10, and all maintained intraday precision (from 94.9% to 99.3%) and interday precision (from 89.4% to 94.9%). The validated method was applied to six real whole blood samples from individuals suspected of consuming ecstasy, and MDMA, MDA and amphetamine were successfully identified and quantified.

A Mechanism-Based Forensic Investigation into the Postmortem Redistribution of Morphine.

The interpretation of postmortem drug levels is complicated by the change in drug blood levels during the postmortem period, a phenomenon known as postmortem drug redistribution. We investigated the postmortem redistribution (PMR) of morphine, morphine-3-glucuronide and normorphine in the rat. Morphine (10mg/kg) was intravenously injected into rats, followed by euthanasia 1h post-injection. The carcasses were placed in a supine position at room temperature, and tissues including heart blood, femoral blood, liver, lung and brain were collected at different time points: 0, 8, 16 or 24h postmortem. The samples were analyzed with a validated (following modified Scientific Working Group for Forensic Toxicology (SWGTOX) (20) guidelines) liquid chromatography-tandem mass spectrometry method. The use of a mechanism-based approach (involving the used set doses of drug with the study performed in controlled environment) to assess PMR using systematic and statistical analyses provides important information that has not previously been presented in PMR literature. While previous human studies focus on central to peripheral ratios as well as peripheral to tissue ratio, this work focused on the change in morphine and metabolite concentrations over the course of the postmortem interval in relation to each other in addition to the comparison to additional matrices at each postmortem interval. Postmortem redistribution was identified in several tissues across the postmortem interval; however, there was minimal statistical difference observed among each matrix at a given postmortem interval with the exception of normorphine and morphine-3-glucuronide. Combined, our study provides a valuable resource and reference information that can aide toxicologists, medical examiners or coroners when assessing postmortem drug concentrations of morphine and metabolites when they are making determinations of cause of death.

Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250μL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Validation of the Neogen ELISA Benzodiazepine Kit using Clonazepam as the Target Molecule for Blood and Urine.

This study demonstrates the validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using clonazepam as the target molecule for the Neogen® Benzodiazepine kit. Decision points were validated at 10.0 ng/mL for whole blood and 25.0 ng/mL for urine. The validation design was based on the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation and included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness and a case sample evaluation. The experimental limit of detection for clonazepam was determined to be at least 5.0 ng/mL in whole blood and at least 10.0 ng/mL in urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels ±50% and +100% of the decision point. Although the method was optimized and exceptional precision was demonstrated at each level, the current SWGTOX validation requirements for a valid decision point were not fulfilled. However, both the blood and urine matrix did meet the proposed revision of the SWGTOX requirements for determining a valid decision point promulgated by the American Academy of Forensic Sciences Standards Board and the assay was reliably able to detect benzodiazepines without interference from matrix components or other compounds routinely detected in authentic case samples. Case sample results were comparable with those obtained when the samples were initially screened using oxazepam as the target molecule. The Neogen® Benzodiazepine kit using clonazepam as the target molecule exhibited cross-reactivity for 29 different benzodiazepines and demonstrated excellent precision and sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for benzodiazepines, even though the validation did not fulfill current SWGTOX requirements for a valid decision point.

Validation and preliminary application of a GC–MS method for the determination of putrescine and cadaverine in the human brain: a promising technique for PMI estimation

IntroductionAmong the several techniques proposed for the estimation of the Post Mortem Interval (PMI), the analysis of odorous amines has been applied in the past, with conflicting results. The aims of this study are: (i) to develop and validate a GC–MS method for the determination of putrescine (PUT) and cadaverine (CAD) in the human brain (validation study) and (ii) to study the relation of PUT and CAD concentration in the human brain and the PMI (decomposition study). Materials and methodsValidation study. Analysis has been performed through GC–MS after a liquid-liquid extraction and a single step-derivatization for the identification and quantification of odorous amines in brain cortex samples. The standard protocol used in forensic toxicology, slightly modified for endogenous compounds according to recent guidelines, was used for validation. Decomposition study. Three uninjured human brains were sampled during the autopsy of three fatal traumatic cases. Along a 120-hour period of decomposition under experimental conditions, each brain was sampled along predetermined time intervals. ResultsValidation study. Both PUT and CAD validation parameters were within the acceptable values defined by the Scientific Working Group for Forensic Toxicology (SWGTOX), with better selectivity, linearity, accuracy and precision values for PUT. Decomposition study. A significant relationship between PUT and CAD levels and PMI has been demonstrated through statistical analysis with a correlation coefficient of 0.98 for PUT and 0.93 for CAD (p < 0.0001). ConclusionAlthough further experimental studies on a wider number of samples are necessary, the results of this study suggest a possible role of polyamine levels in brain cortex for the estimation of PMI.

Quantification of seven novel synthetic opioids in blood using LC–MS/MS

The objective of this study was to develop, optimize, and validate a method for the simultaneous quantification of U-47700, AH-7921, U-49900, U-50488, MT-45, W-18, and W-15 in whole blood using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Blood samples (500 µL) were fortified with mixed calibrator or quality control (25 µL) and internal standard (10 µL) solutions. Analytes were isolated via a solid-phase extraction procedure. Analysis was performed using an Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. The method was validated in accordance with Scientific Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology and applied to the analysis of postmortem blood specimens. Lower limits of quantification were 0.25–1 ng/mL and the upper limits of quantification were 100 ng/mL. The coefficients of determination (R2) for the calibration curves were > 0.99. Analytical bias, within-run imprecision, and between-run imprecision were within ± 15%, ≤ 16%, and ≤ 17%, respectively. All analytes were found to be stable at room temperature for 24 h, refrigerated (4 °C) for 72 h, and in the autosampler (4 °C) for 72 h. Authentic blood samples (n = 30) were analyzed using the validated method. Mean (range) U-47700 concentrations were 214 (3.2–1448) ng/mL in 15 cases. A quantification method for seven synthetic opioids (U-47700, AH-7921, U-49900, U-50488, MT-45, W-18, and W-15) in whole blood was developed, optimized, and validated in accordance with SWGTOX standard practices for method validation in forensic toxicology. This sensitive method was successfully applied to postmortem casework.

New analytical approach to determine organophosphorus insecticides in blood by dried matrix spots sampling and GC-MS/MS

This work describes the optimization of a new method for the determination of five organophosphorus insecticides in whole blood. The analytes were extracted from the matrix (50μL) using the dried blood spot (DBS) approach and were analyzed by gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS). The studied compounds (diazinon, chlorpyrifos, parathion-ethyl, chlorfenvinphos and quinalphos) were chosen based on the statistics of intoxications in Portugal, and ethion was used as internal standard. The method was fully validated, taking into account international guidelines for bioanalytical method validation, such as those of the Food and Drug Administration (FDA), International Conference on Harmonization (ICH) and Scientific Working Group for Forensic Toxicology (SWGTOX). A linear range of 0.1-25μg/mL was obtained for all compounds, except for diazinon and quinalphos (0.05-25μg/mL and 0.25-25μg/mL, respectively), presenting determination coefficients above 0.99. Concerning precision, the coefficients of variation (CVs) were lower than 14% for all compounds. Those compounds were found to be stable in the samples. Although the values obtained for recovery were low (between 1 and 12%), the method proved to be sensitive, since detection limits between 0.05 and 0.1μg/mL were obtained. The novelty is the use of the DBS approach in the extraction of these compounds, and this is the first paper reporting it: DBS is a recent technique of bioanalysis in the field of toxicology, and in addition to its simplicity and sensitivity, it is applicable routinely in both clinical and forensic toxicology situations. Graphical abstract ᅟ.

Identification of five Mitragyna alkaloids in urine using liquid chromatography-quadrupole/time of flight mass spectrometry

Mitragyna speciosa (Kratom) is a psychoactive plant that has recently emerged as a recreational drug. Mitragyna alkaloids are not within the scope of traditional forensic toxicology screening methods, which may contribute to under-reporting. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS) were used to identify five alkaloids in urine. Target analytes included the two known psychoactive compounds, mitragynine and 7-hydroxymitragynine, in addition to speciociliatine, speciogynine, and paynantheine. Two deuterated internal standards (mitragynine-D3 and 7-hydroxymitragynine-D3) were employed. Using traditional reversed phase chromatography all compounds and isomers were separated in 10 min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 63–96% and limits of quantitation were 0.5–1 ng/mL. Precision, bias and matrix effects were all within acceptable thresholds, with the exception of 7-hydroxymitragynine, which is notably unstable and unsuitable for quantitative analysis. In this paper we present a simultaneous quantitative analytical method for mitragynine, speciociliatine, speciogynine and paynantheine, and a qualitative assay for 7-hydroxymitragynine in urine using high resolution mass spectrometry (HRMS).

Simple and Highly Sensitive UPLC-ESI-MS/MS Assay for Rapid Determination of Suvorexant in Plasma.

Suvorexant is a dual orexin receptor antagonist, recently approved by USFDA for the treatment of insomnia. It is drug-of-abuse and listed in Schedule IV drug of the Controlled Substances Act. In this study, a simple and highly sensitive UPLC-MS/MS assay was developed and fully validated for the determination of suvorexant in rat plasma. Both suvorexant and internal standard (rivaroxaban; IS) were separated on Aquity BEHTM C18 column after the extraction form plasma using diethyl ether as extracting agent. An isocratic mobile phase, consisting of acetonitrile and 10 mM ammonium acetate in composition ratio of 85:15 was eluted at flow rate of 0.3 mL/min. Both suvorexant and IS were eluted within one min with total run time of 1.5 min only. The ionization was performed on electrospray ionization interface in positive mode by multiple reaction monitoring. Precursor to product ion transition of 451.12 > 104.01 for qualifier and 451.12 > 186.04 for quantifier were used for suvorexant whereas 436.10 > 144.93 for IS, respectively. The calibration curves in plasma were linear in the concentration range of 0.33-200 ng/mL (r2 ≥ 0.995) having limit of detection and limit of quantification of 0.10 and 0.33 ng/mL, respectively. All the validation parameters results were found to be within the acceptable limits according to the "Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines" and can be implemented for forensic analysis.

Identification and quantification of synthetic cathinones in blood and urine using liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry.

Synthetic cathinones continue to present a formidable challenge to forensic toxicology laboratories despite the fact that they are often encountered in impaired driving and death investigations. Due to limitations in immunoassay-based screening technologies, many forensic toxicology laboratories must rely on more labor intensive chromatographic-based screening approaches in order to detect these drugs in biological evidence. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry were used to identify twenty-two synthetic cathinones in urine and blood. Target drugs included methcathinone, ethcathinone, pentedrone, buphedrone, 3-fluoromethcathinone (3-FMC), 4-fluoromethcathinone (4-FMC), 4-methylethcathinone (4-MEC), 4-ethylmethcathinone (4-EMC), mephedrone, methedrone, 3,4-dimethylmethcathinone (3,4-DMMC), ethylone, butylone, pentylone, eutylone, methylone, methylenedioxypyrovalerone (MDPV), 4-methylpyrrolidinobutiophenone (MPBP), 3,4-methylenedioxypyrrolidinobutiophenone (MDPBP), α-pyrrolidinopentiphenone (α-PVP), pyrovalerone, and naphyrone. A total of nine deuterated internal standards were employed. Using traditional reversed phase chromatography all positional isomers, including 3-FMC and 4-FMC, were separated in 12min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 84-104% and 81-93% in urine and blood, respectively. Limits of quantitation in both matrices were 0.25-5ng/mL. Precision, bias and matrix effect were all within acceptable thresholds and the assay was free from more than fifty interferences. The validated method was used to identify cathinones in authentic urine case samples (n=20) and these results highlight important considerations for cathinone stability and the subsequent interpretation of results.

Multi-class analysis of new psychoactive substances and metabolites in hair by pressurized liquid extraction coupled to HPLC-HRMS.

In this paper, an analytical method has been developed and validated for the analysis of new psychoactive substances (NPS) and metabolites in hair samples. The method was based on pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) clean-up and high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) analysis. To evaluate extraction efficiency and the applicability of the method, hair samples were fortified by soaking in order to obtain a good surrogate for drug users' hair; the amount of incorporated drugs related to their lipophilicity, similarly to in vivo drug incorporation. To the best of our knowledge, this is the first method that allowed for the analysis of both cathinones (5) and synthetic cannabinoids (7) in hair with a single extraction procedure and chromatographic run. A phenethylamine (2C-T-4), 4- fluorophenylpiperazine and methoxetamine were also included showing that PLE coupled to SPE clean-up was suitable for a multi-class analysis of NPS in hair. In addition, the use of PLE significantly reduced hair analysis time: decontamination, incubation, clean-up, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out in approximately 45 min. The method was fully validated according to Scientific Working Group for Forensic Toxicology (SWGTOX) and Society of Hair Testing (SoHT) guidelines. Limit of quantification (LOQ) values ranged from 8 to 50 pg mg-1 for cathinones, phenetylamines and piperazines, and from 9 to 40 pg mg-1 for synthetic cannabinoids (10 pg mg-1 for methoxetamine). Matrix effects were below 15% for all the analytes, demonstrating the effectiveness of the clean-up step. Inaccuracy was lower than 9% in terms of bias. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of illicit drugs and metabolites in oral fluid by microextraction on packed sorbent coupled with LC-MS/MS.

Oral fluid (OF) has become a valuable biologic specimen for toxicological analysis, especially in driving under the influence of drugs (DUID) investigations, because of easy and non-invasive collection procedures. In OF testing, being the sample volume is limited, multi-analyte procedures are particularly advantageous since they save time and resources. In this work, a procedure for the simultaneous analysis of 20 illicit drugs, belonging to the classes of cocaine, amphetamines, natural and synthetic opioids and hallucinogens, is presented. The sample preparation is based on microextraction by packed sorbent (MEPS), a novel technique which is based on the miniaturization of solid-phase extraction (SPE). The presented method, which includes all the most diffused illicit drugs and their metabolites, has been fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. LLOQs ranged from 0.5 to 30ngmL(-1) (diacetylmorphine); the presented method allows the detection of all the selected drugs quite below the cutoff values recommended by Substance Abuse and Mental Health Services Administration (SAMHSA) for abuse identification.

High-Performance Liquid Chromatography with Tandem Mass Spectrometry for the Determination of Nine Hallucinogenic 25-NBOMe Designer Drugs in Urine Specimens

We present a high-performance liquid chromatography triple quadrupole mass spectrometry (HPLC-MS-MS) method for the identification and quantification of nine serotonin 5-HT2A receptor agonist hallucinogenic substances from a new class of N-methoxybenzyl derivatives of methoxyphenylethylamine (NBOMe) designer drugs in human urine: 25H-NBOMe, 2CC-NBOMe, 25I-NBF, 25D-NBOMe, 25B-NBOMe, 2CT-NBOMe, 25I-NBMD, 25G-NBOMe and 25I-NBOMe. This assay was developed for the Virginia Commonwealth University Clinical and Forensic Toxicology laboratory to screen emergency department specimens in response to an outbreak of N-benzyl-phenethylamine derivative abuse and overdose cases in Virginia. The NBOMe derivatives were rapidly extracted from the urine specimens by use of FASt™ solid-phase extraction columns. Assay performance was determined as recommended for validation by the Scientific Working Group for Forensic Toxicology (SWGTOX) for linearity, lower limit of quantification, lower limit of detection, accuracy/bias, precision, dilution integrity, carryover, selectivity, absolute recovery, ion suppression and stability. Linearity was verified to be from 1 to 100 ng/mL for each of the nine analytes. The bias determined for the NBOMe derivatives was 86-116% with a <14% coefficient of variation over the linear range of the assay. Four different NBOMe derivatives were detected using the presented method in patient urine specimens.

Scientific Working Group for Forensic Toxicology (SWGTOX) - Recommendations of the Research, Development, Testing, and Evaluation Committee

Scientific Working Group for Forensic Toxicology (SWGTOX) - Recommendations of the Research, Development, Testing, and Evaluation Committee