Identifying the challenges of emerging novel psychoactive substances

Novel psychoactive substances (NPS) are compounds similar to common drugs of abuse with slight modifications to their chemical structure. These modifications can be dangerous and sometimes fatal due the lack of knowledge and/or studies of their adverse effects. NPS are sometimes marketed as “legal highs” or “research chemicals” in order to mimic effects of illicit drugs while simultaneously bypassing legislation. Although there are many drugs that are classified as NPS, novel synthetic opioids and synthetic cannabinoids are the focus of this research. Since little is known about these novel psychoactive substances difficulties may arise when analyzing toxicological samples suspected to contain NPS. To address the emergence of novel psychoactive substances, it is necessary to properly identify issues that could hinder analyses by assessing prevalence, examining instability, determining pharmacology, and identifying potential metabolites. The goals of this study were to 1) develop and validate a method to quantify novel synthetic opioids (NSO), buprenorphine, and heroin markers in oral fluid and apply the method to the analysis of oral fluid collected from detainees; 2) using the acquisition method from the oral fluid method, validate a blood method for the NSO and perform a long term stability study; 3) develop and validate a method to detect a NSO (U-47700) and its metabolites in plasma using a small sample volume (100 µL) and cross validate in rat plasma for a pharmacokinetic study in rats; and 4) identify metabolites for two prominent synthetic cannabinoids in vitro verify metabolism with analysis of authentic urine samples. A method was validated for the quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18, and W-15 in oral fluid and was deemed acceptable according to Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. This method was applied to analysis of oral fluid collected from detainees (n=20) in Texas detention centers participating in a drug recognition evaluatoion (DRE) certification program. Although NSO were not detected, valuable data were collected that reinforced oral fluid as viable matrix when compared to presumptive urine results and impairment observations. A blood method was validated for seven NSO and then applied to assess the stability of these analytes over a 36-week study at four temperature conditions (-20°C,4°C, 25°C , and 35°C). The results showed minimal effect on stability at the elevated temperature during the first two weeks, indicating that these analytes would be stable in the event of improper transport/handling within this timeframe. A method was validated for the quantification of U-47700, N-desmethyl-U-47700, and N,N-didesmethyl-U-47700 in human and rat plasma. This method was applied to a pharmacokinetic study where rats were injected with 0 (saline), 0.3, 1.0 or 3.0 mg/kg U-47700. Blood samples were collected at 15, 30, 60, 120, 240, 480 min after injection for quantification of U-47700 and its metabolites. Pharmacodynamic effects were also assessed at the same time points. It was determined that doses of U-47700 had a positive correlation with the behaviors observed which further demonstrates the analgesic effects of this novel synthetic opioid. Finally, a metabolic study utilizing human liver microsomes was conducted to investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism. In vitro metabolites were verified in vivo by analyzing authentic case specimens. Additionally, the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA were identified by examining activity at the CB1 receptor. The EC50 at the CB1 receptor for 5F-MDMB-PICA and 5F-MDMB-PINACA were found to be comparable to each other and JWH-018. There were 22 metabolites identified for 5F-MDMB-PICA and 21 metabolites identified for 5F-MDMB-PINACA. These studies have sought to identify toxicological issues that could arise when detecting a new NPS while providing the necessary data to the forensic toxicology community to further understand the activity and prevalence of such compounds. The challenges that arise when faced with a newly emerged NPS may be detrimental for forensic analysis and thorough characterization of new compounds is necessary for proper identification and detection

Vertical Ground Reaction Forces During Stair Descent Transition for Individuals with Femoroacetabular Impingement and Osteoarthritis

Femoroacetabular impingement (FAI) is a pathological condition characterized by the misshapen junction between the femoral head and acetabular rim causing joint friction. Not all individuals with FAI experience symptoms associated with the condition. Previous research has determined differences between those with and without symptomatic FAI (FAI-s) and those with and without osteoarthritis (OA) during stair ascent (Hammond et al., 2017 and Hall et al., 2017, respectively). No studies focused on stair descent ground reaction forces (GRFs) between individuals with FAI and/or OA exist. PURPOSE: This study analyzed key vertical GRF values during stair descent transition in adults with FAI-s, asymptomatic FAI (FAI-a) and OA. METHODS: Individuals were recruited by an orthopedic surgeon who used radiographs to assign group membership (FAI-s n=10, FAI-a n=11, OA n=10). Each person descended a 3-step staircase onto a Kistler force plate embedded into the floor (1000 Hz). The stair to floor transition was analyzed for three trials for each foot contact. Peak vertical impact and active forces (Fz1 and Fz2, respectively) were normalized by BW and the three trial average was analyzed. Dependent and independent t-tests were used to compare transition feet and groups (p \u3c 0.05). RESULTS: There were no differences for any group when comparing transition feet for Fz1 or Fz2 values. Additionally, there were no differences between the feet associated with the affected and unaffected hips for the FAI-s group. When comparing the larger and smaller Fz1 values for each person, all groups had a significant difference (p \u3c 0.005 for all). The FAI-s group had smaller Fz1 values than FAI-a group for both the left and right foot transition (1.57 ± 0.34 BW vs. 1.81 ± 0.20 BW, p=0.035 and 1.62 ± 0.23 BW vs 1.81 ± 0.13 BW, p=0.019, respectively). There was a trend toward Fz1 values being smaller for FAI-s when compared to OA for both foot transitions (p \u3c 0.08). There were no differences between the FAI-a and OA groups for Fz1 values and no differences between Fz2 values between any of the groups for either transition foot. CONCLUSION: Individuals with symptomatic FAI transition from stairs to floor with smaller impact forces when compared to the other groups, perhaps to avoid painful hip conditions. All participants had asymmetrical impact transition forces

Heroin-Related Compounds and Metabolic Ratios in Postmortem Samples Using LC-MS-MS

Analysis of postmortem samples with the presence of morphine can sometimes be challenging to interpret. Tolerance complicates interpretation of intoxications and causes of death due to overlap in therapeutic and fatal concentrations. Determination of metabolites and metabolic ratios can potentially differentiate between abstinence, continuous administration, and perhaps time of administration. The purpose of this study was to (a) develop and validate a method for quantitation of morphine-3 beta-D-glucuronide, morphine-6 beta-D-glucuronide, normorphine, codeine-6 beta-D-glucuronide, norcodeine, codeine, 6-acetylmorphine, and ethylmorphine in urine using liquid chromatography-tandem mass spectrometry; (b) apply the method to opiate related deaths; (c) compare metabolic ratios in urine in different causes of death (CoD) and after different drug intakes and (d) compare heroin intoxications in rapid and delayed deaths. Validation parameters such as precision, bias, matrix effects, stability, process efficiency, and dilution integrity were assessed and deemed acceptable. Lower limits of quantitation ranged from 0.01-0.2 mu g/mL for all analytes. Autopsy cases (n=135) with paired blood and urine samples were analyzed. Cases were divided into three groups based on CoD; opiate intoxication, intoxication with other drugs than opiates, and other CoD. The cases were classified by intake: codeine (n=42), heroin (n=36), morphine (n=49), and ethylmorphine (n=3). Five cases were classified as mixed intakes and excluded. Heroin intoxications (n=35) were divided into rapid (n=15) or delayed (n=20) deaths. Parent drug groups were compared using metabolic ratio morphine-3 beta-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p <= 0.0001). Urine and blood concentrations, and metabolic ratios in rapid and delayed heroin intoxications were compared and determined a significant difference for morphine (p=0.001), codeine (p=0.009), 6-acetylmorphine (p=0.02) in urine, and morphine (p=0.02) in blood, but there was no significant difference (p=0.9) between metabolic ratios. Morphine-3 beta-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications

The Quantification of Oxycodone and Its Phase I and II Metabolites in Urine

The purpose of this research was to develop and validate an analytical method for the detection and quantification of noroxymorphone-3 beta-D-glucuronide (NOMG), oxymorphone-3 beta-D-glucuronide (NOMG), noroxymorphone (NOM), oxymorphone (OM), 6 alpha-oxycodol (alpha OCL), 6 beta-oxycodol (beta OCL), noroxycodone (NOC) and oxycodone (OC) in urine by liquid chromatography tandem mass spectrometry to be used in a human study. The method was validated according to the Academy Standards Board Standard Practices for Method Development in Forensic Toxicology. The method was then applied to a single-dose pilot study of a subject. Urine samples were collected from the subject after ingesting 10-mg OC as an immediate-release tablet. Additionally, urine specimens (n = 15) that had previously been confirmed positive for OC were analyzed using the validated method. The calibration range for NOMG and OMG was 0.05-10 µg/mL; for all other analytes, it was 0.015-10 µg/mL. Validation parameters such as bias, precision, carryover and dilution integrity, all met the validation criteria. After the method was validated, urine samples from the first subject in the controlled dose study were analyzed. It was observed that OC, NOC and OMG contained the highest concentrations and were present in either the 0.5 or 1 h void. NOC and OMG were detected until the 48 h collection, while OC was detectable till the 24 h collection. Time to reach maximum concentration (T-max) in the urine was achieved within 1.5 h for OC and within 3 h for NOC and OMG. Maximum concentration (C-max) in the urine for OC, NOC and OMG was 3.15, 2.0 and 1.56 µg/mg, respectively. OC concentrations in authentic urines ranged from 0.015 to 12 µg/mL. Ranges for NOMG and OMG were 0.054-9.7 µg/mL and 0.14-67 µg/mL, respectively. A comprehensive method for the quantification of NOMG, OMG, NOM, OM, alpha OCL, beta OCL, NOC and OC in urine was optimized and met the validation criteria. The concentrations of NOMG and OMG presented in this study provide the details needed in the forensic community to better comprehend OC pharmacokinetics.Funding Agencies: Strategic Research Area in Forensic Sciences at the University of Linköping, Sweden [304399, 304774]</p

Urinary Pharmacokinetics of Immediate and Controlled Release Oxycodone and its Phase I and II Metabolites Using LC-MS-MS

Oxycodone (OC) is a schedule II semisynthetic opioid in the USA that is prescribed for its analgesic effects and has a high potential for abuse. Prescriptions for OC vary based on the dosage and formulation, immediate release (IR) and controlled release (CR). Monitoring OC metabolites is beneficial for forensic casework. The limited studies that involve pharmacokinetics of the urinary excretion of OC metabolites leave a knowledge gap regarding the excretion of conjugated and minor metabolites, pharmacokinetic differences by formulation, and the impact of CYP2D6 activity on the metabolism and excretion of OC. The objectives of this study were to compare urinary excretion of phase I and II metabolites by formulation and investigate if ratio changes over time could be used to predict the time of intake. Subjects (n = 7) received a single 10 mg IR tablet of Oxycodone Actavis. A few weeks later the same subjects received a single 10 mg CR tablet of Oxycodone Actavis. During each setting, urine was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 12, 14, 24, 48 and 72 h. Urine samples (100 mu L) were diluted with 900 mu L internal standard mixture and analyzed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD using a previously validated method. The CYP2D6 phenotypes were categorized as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Comparisons between IR and CR were performed using two-tailed paired t-test at a significance level of P = 0.05. The metabolite ratios showed a general increase over time. Four metabolite to parent ratios were used to predict the time of intake showing that predictions were best at the early time points.Funding Agencies: Strategic Research Area in Forensic Sciences at University of Linköping, Sweden [304399, 304774]</p

A sensitive LC-MS/MS method for the quantitation of oxycodone, noroxycodone, 6 alpha-oxycodol, 6 beta-oxycodol, oxymorphone, and noroxymorphone in human blood

The objective of this study was to develop and validate a highly sensitive method for the detection of oxycodone, noroxycodone, 6?-oxycodol, 6?-oxycodol, oxymorphone, and noroxymorphone in blood by liquid chromatography tandem mass spectrometry. The analytes were extracted from blood (0.5 mL) using Bond Elut Certify Solid Phase Extraction columns, evaporated to dryness and reconstituted before analysis was performed on an Acquity UPLC? I-class coupled to a Waters Xevo TQD. Academy Standards Board Standard Practices for Method Development in Forensic Toxicology were used for the validation of this method. The limit of quantitation for all analytes was established at 0.5 ng/mL. Calibration range for noroxymorphone, oxymorphone, 6?-oxycodol and 6?-oxycodol was 0.5?25 ng/mL and 0.5?100 ng/mL for noroxycodone and oxycodone. Precision (2.90?17.3%) and bias studies resulted in a ?15% deviation. There were no interferences observed from internal standard, matrix, or common drugs of abuse. Stability of all analytes at two concentrations at 24, 48, and 72 h in the autosampler did not exceed ?20% difference from the initial T0. Dilution integrity at a ten-fold dilution was acceptable as analyte concentrations ranged between (?18%) of the target concentration. Once validated, the method was used in a pilot dosing study of one male subject after taking a 10 mg immediate release tablet of oxycodone. Blood samples were collected at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 9, and 24 h after ingestion. Oxycodone and noroxycodone both reached Tmax at 1.5 h and had Cmax values of 25.9 and 12.8 ng/mL, respectively. Oxycodone, 6?-oxycodol, and 6?-oxycodol were detectable up to 9 h, while noroxymorphone and noroxycodone were still detected at 24 h.Funding Agencies|Strategic Research Area in Forensic Sciences at University of Linkoping, Sweden [304399, 304774]</p