Buprenorphine-Naloxone Maintenance and Lactation.

BACKGROUND: Breastfeeding among lactating people with opioid use disorder taking buprenorphine monotherapy is generally accepted, as low concentrations of buprenorphine and metabolites in human milk have been well-established. The use of buprenorphine-naloxone for pregnant and lactating people with opioid use disorder is expanding and there is no information available regarding the concentrations of naloxone and their metabolites in human milk to recommend the use of this combination medication during lactation. RESEARCH AIMS: To determine the concentrations of buprenorphine and naloxone and their primary metabolites in human milk, maternal plasma, and infant plasma, among lactating buprenorphine-naloxone maintained people and their infants. METHODS: Four lactating buprenorphine-naloxone maintained people provided plasma and human milk samples on Days 2, 3, 4, 14, and 30 postpartum. Infant plasma was obtained on Day 14. RESULTS: Concentrations of buprenorphine, norbuprenorphine and their glucuronide metabolites were present in maternal plasma and human milk at low concentrations, consistent with previous research in lactating buprenorphine monotherapy participants. Naloxone was not detected, or was detected at concentrations below the limit of quantification, in maternal plasma and in all except one human milk sample at Day 30. Naloxone was not detected or detected at concentrations below the limit of quantification in all infant plasma samples. CONCLUSION: Results support the use of buprenorphine-naloxone by lactating people who meet appropriate criteria for breastfeeding.

Quantification of psilocin in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC-MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7-200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification.

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens.

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC-MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-µg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010-2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC-MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 µg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.

Chiral separation and quantitation of methylphenidate, ethylphenidate, and ritalinic acid in blood using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC) is a technique that analyzes compounds that are temperature-labile, have moderately low weight, or are chiral compounds. Methylphenidate (MPH) is a chiral compound with two chiral centers. MPH has two chiral metabolites, ethylphenidate (EPH) and ritalinic acid (RA). MPH is sold as a racemic mixture. The d-enantiomer of threo-MPH is responsible for medicinal effects. Due to the differing effects of the enantiomers, it is important to analyze the enantiomers individually to better understand their effects. This method utilizes SFCand solid-phase extraction (SPE) to separate and analyze the enantiomers of MPH, EPH, and RA in postmortem blood. The objective of this method was to assess a unique approach with SFC for enantiomeric separation of MPH, EPH, and RA. A SPE method was developed and optimized to isolate the analytes in blood and validated as fit-for-purpose following international guidelines. The linear range for MPH and EPH was 0.25-25 and 10-1000 ng/mL for RA in blood. Bias was -8.6% to 0.8%, and precision was within 15.4% for all analytes. Following method validation, this technique was applied to the analysis of 49 authentic samples previously analyzed with an achiral method. Quantitative results for RA were comparable to achiral technique, whereas there was loss of MPH and EPH over time. The l:d enantiomer ratio was calculated, and MPH demonstrated greater abundance of the d-enantiomer. This is the first known method to separate and quantify the enantiomers of all three analytes utilizing SFC and SPE.

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 µL) were diluted with 900 µL 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.

Evaluation of Alcohol Markers in Urine and Oral Fluid after Regular and Hard Kombucha Consumption.

Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3 and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography-tandem mass spectrometry (LC-MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff [urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC-MS-MS in urine specimens within the first five voids from 83% of participants with median (range) concentrations of 240 (100-3,700) ng/mL for EtS and 830 (530-2,200) ng/mL for EtG. Neither marker was positive by IA nor LC-MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5-8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559-70,250) ng/mL for EtS and 763 (104-12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.

Analysis of methylphenidate, ethylphenidate, lisdexamfetamine, and amphetamine in oral fluid by liquid chromatography-tandem mass spectrometry.

Oral fluid is an alternative matrix that has proven to be useful for the detection of drugs. Oral fluid is easy to collect, noninvasive, and may indicate recent drug use. There are limited methods available that analyze cognitive stimulants in oral fluid. Cognitive stimulants are used to treat attention-deficit/hyperactivity disorder (ADHD), a neurological disorder that emerges from lack of dopamine in the brain. To combat this disorder, medications inhibit dopamine and norepinephrine reuptake by blocking transporters in the brain. Though commonly diagnosed in children, ADHD may extend beyond adolescence and abuse of medications in college students is not uncommon. The goal of this study was to develop and validate a quantitative method for methylphenidate, ethylphenidate, lisdexamfetamine, and amphetamine in oral fluid using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytes were isolated by solid-phase extraction and analyzed on an Agilent 1290 Infinity II Liquid Chromatograph coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The linear range was 0.5-100 ng/ml (except lisdexamfetamine at 5-500 ng/ml). Bias and between-run precision were acceptable (±11.0% bias and ±12.2%CV). No interferences or carryover were observed and dilution integrity was sustained. This validated method was applied to four authentic oral fluid samples collected with Quantisal® devices from college students. Lisdexamfetamine was quantified in one sample at 5.8 ng/ml while amphetamine was quantified in all four samples at 6.0-78.8 ng/ml. This is the first known quantitative method in oral fluid that includes these analytes using LC-MS/MS and may give rise to interpretive value in a forensic toxicology setting.

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-3ß-D-glucuronide (NOMG), oxymorphone-3ß-D-glucuronide (NOMG), noroxymorphone (NOM), oxymorphone (OM), 6α-oxycodol (αOCL), 6ß-oxycodol (ßOCL), 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 (Tmax) in the urine was achieved within 1.5 h for OC and within 3 h for NOC and OMG. Maximum concentration (Cmax) 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, αOCL, ßOCL, 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.

Determination of morphine, codeine, and thebaine concentrations from poppy seed tea using magnetic carbon nanotubes facilitated dispersive micro-solid phase extraction and GC-MS analysis.

With tightening enforcement and restrictions amid the opioid epidemic, poppy seed tea is consumed as an alternative to mitigate the withdrawal symptoms or as a home remedy to relieve pain and stress. Previously published studies suggested the potential danger of consuming tea brewed with a moderate to a large amount of poppy seed. In this study, the effects of small quantity and repeat brewing on opiate concentrations were evaluated. A dispersive-micro solid phase extraction facilitated by magnetic carbon nanotubes (Mag-CNTs/d-µSPE) was developed, optimized, successfully validated, and applied to ten poppy seed tea samples using gas chromatography-mass spectrometry (GC-MS) analysis. A total of ten poppy seed samples were evaluated in this work. Two grams of bulk poppy seeds were brewed with 6 mL of heated and acidified DI water three times. The brewed tea samples were subjected to the validated Mag-CNTs/d-µSPE/GC-MS analysis. The total mean opiate concentrations obtained from three brews were 1.1-1926, 20.2-311, and 9.0-100 mg/kg for morphine, codeine, and thebaine, respectively. The total opiate yields obtained from the small quantity brewing, i.e., 6 g seed in 18 mL tea, in this study may provide minimal analgesic and euphoric effects. Over 80% of the total opiate yield was extracted in the first brew with acidified deionized water from the 10 min brewing period, and opiate yields from the second and third brew were minimal. However, potential overdose could occur for some tea samples when scaled up to the starter quantity of seed suggested for new users.

Quantification of fentanyl analogs in oral fluid using LC-QTOF-MS.

Oral fluid is a valuable alternative matrix for forensic toxicologists due to ease of observed collection, limited biohazardous exposure, and indications of recent drug use. Limited information is available for fentanyl analog prevalence, interpretation, or analysis in oral fluid. With increasing numbers of fentanyl-related driving under the influence of drug (DUID) cases appearing in the United States, the development of detection methods is critical. The purpose of the present study was to develop and validate a quantitative method for fentanyl analogs in oral fluid (collected via Quantisal™) using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Validation resulted in limits of detection and quantification ranging from 0.5 to 1 ng/mL. Established linear range was 1-100 ng/mL for all analytes, except acetyl fentanyl at 0.5-100 ng/mL (R2  > 0.994). Within- and between-run precision and bias were considered acceptable with maximum values of ±15.2%CV and ±14.1%, respectively. Matrix effects exhibited ionization enhancement for all analytes with intensified enhancement at a low concentration (9.3-47.4%). No interferences or carryover was observed. Fentanyl analogs were stable in processed extracts stored in the autosampler (4° C) for 48h. The validated method was used to quantify fentanyl analogs in authentic oral fluid samples (n=17) from probationers/parolees. Fentanyl and 4-ANPP concentrations were 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively.

Short- and Long-Term Stability of Methylphenidate and Its Metabolites in Blood.

Methylphenidate (MPH) is a medication used to combat attention-deficit/hyperactivity disorder by speeding up brain activity. MPH has two chiral centers; however, d-threo-MPH is responsible for its effects. Few studies have analyzed MPH and its metabolites, ritalinic acid (RA) and ethylphenidate (EPH), in blood. Stability studies are crucial in a forensic setting to provide insight on ideal storage conditions and analysis time. In this study, d,l-MPH, d,l-EPH and RA were analyzed at two concentrations (15 and 150 ng/mL) over 5 months at room temperature (∼25°C), refrigerated (4°C), frozen (-20°C) and elevated (35°C) temperatures. Analytes were analyzed using a validated liquid chromatography--mass spectrometry method. RA concentrations increased 53% at 25°C after 24 h, while d- and l-MPH concentrations dropped 18.1 and 20.6%, respectively. Additionally, d- and l-EPH concentrations decreased 22.3 and 28.8%, respectively. All analytes were stable at 4°C for 1 week (±17% change). At -20°C, all analytes were stable for 5 months. At 35°C, l-EPH remained stable for 24 h (14.4% loss) at the high concentration, while RA increased 244%. Losses of 64.1, 68.7 and 27.2% were observed for d- MPH, l-MPH and d-EPH, respectively. Due to this, a follow-up study was designed to assess the breakdown of MPH. The short-term experiment assessed d,l-MPH at two concentrations for 1 month in the same conditions. As MPH decreased, RA concentrations rose. At 25°C, it took 2 weeks for MPH to metabolize completely into RA. In refrigerated and frozen temperatures, MPH did not completely metabolize to RA. In elevated temperatures, MPH broke down to RA within 2 weeks. Due to this, it was concluded that d,l-MPH breaks down in the blood to its metabolite RA and may make data interpretation difficult if samples are not properly stored. The optimal storage for these analytes is recommended at -20°C.

Long-Term Stability of 13 Fentanyl Analogs in Blood.

Fentanyl analogs continue to play a major role in proliferating the opioid epidemic in the USA. With high rates of overdose deaths, forensic laboratories experience backlogs, which may lead to false-negative results due to drug instability. To address this issue, a quantitative method was validated for fentanyl analogs (3-methylfentanyl, 4-anilino-N-phenethylpiperidine (4-ANPP), 4-fluoro-isobutyrylfentanyl (4-FIBF), acetylfentanyl, acrylfentanyl, butyrylfentanyl, carfentanil, cyclopropylfentanyl, fentanyl, furanylfentanyl, methoxyacetylfentanyl, p-fluorofentanyl and valerylfentanyl) in blood using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) and used to assess long-term stability under various temperature conditions (-20°C, 4°C, ∼25°C and 35°C) for 9 months. Authentic specimens were also analyzed 6 months apart for applicability to postmortem blood. Method validation resulted in calibration ranges of 1-100 ng/mL and limits of detection of 0.5 ng/mL. Precision and bias were acceptable (within ±7.2% coefficient of variation (CV) and ±15.2%, respectively). Matrix effects exhibited ion enhancement for all analytes, except carfentanil and 4-ANPP in low-quality control (>25%). For long-term stability, fentanyl analogs (except acrylfentanyl) remained stable under room temperature and refrigerated conditions at low and high concentrations (81.3-112.5% target) for 9 months. While most fentanyl analogs remained stable frozen, degradation was observed after 2 weeks (four freeze/thaw cycles). At elevated temperatures, most analytes were stable for 1 week (74.2-112.6% target). Acrylfentanyl was unstable after 24 h under elevated (70% loss) and room temperatures (53-60% loss), 48-72 h when refrigerated (28-40% loss) and 4 weeks when frozen (22% loss). In authentic bloods (n = 7), initial furanylfentanyl (FuF) and 4-ANPP concentrations were 1.1-3.6 and 1.4-6.4 ng/mL, respectively. Percentage loss of FuF and 4-ANPP over 6 months were 16.3-37.4% and 0.2-26.8%, respectively. Samples suspected to contain fentanyl analogs are recommended to be stored refrigerated or frozen with limited freeze/thaw cycles. Due to instability, in the event of an acrylfentanyl overdose, samples should be analyzed immediately or stored frozen with analysis within 1 month.

A sensitive LC-MS/MS method for the quantitation of oxycodone, noroxycodone, 6α-oxycodol, 6ß-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, 6ß-oxycodol, 6α-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, 6α-oxycodol and 6ß-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, 6α-oxycodol, and 6ß-oxycodol were detectable up to 9 h, while noroxymorphone and noroxycodone were still detected at 24 h.

Maternal Poppy Seed Tea Ingestion and Ensuing Neonatal Abstinence Syndrome.

The incidence of neonatal abstinence syndrome has been rising in the USA. Nonpharmacological treatments resulting in similar withdrawal states in the newborn have also been described. We report an infant with neonatal abstinence syndrome born to a mother with daily poppy seed tea ingestion for the self-treatment of nausea. A sample of poppy seed tea was replicated using the mother's self-reported recipe. The sample was analyzed using liquid chromatography tandem mass spectrometry. This recipe produced a result of approximately 7.8 mg of morphine per serving which she reported to have drank 5-6 days per week, for an estimated 7 months during the course of her pregnancy.

Pharmacodynamics and pharmacokinetics of the novel synthetic opioid, U-47700, in male rats.

Novel synthetic opioids are appearing in recreational drug markets worldwide as adulterants in heroin or ingredients in counterfeit analgesic medications. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47700) is an example of a non-fentanyl synthetic opioid linked to overdose deaths. Here, we examined the pharmacodynamics and pharmacokinetics of U-47700 in rats. Male Sprague-Dawley rats were fitted with intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. injections of U-47700 HCl (0.3, 1.0 or 3.0 mg/kg) or saline, and blood samples (0.3 mL) were withdrawn via i.v. catheters at 15, 30, 60, 120, 240, 480 min post-injection. Pharmacodynamic effects were assessed at each blood withdrawal, and plasma was assayed for U-47700 and its metabolites by liquid chromatography tandem mass spectrometry. U-47700 induced dose-related increases in hot plate latency (ED50 = 0.5 mg/kg) and catalepsy (ED50 = 1.7 mg/kg), while the 3.0 mg/kg dose also caused hypothermia. Plasma levels of U-47700 rose linearly as dose increased, with maximal concentration (Cmax) achieved by 15-38 min. Cmax values for N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 were delayed but reached levels in the same range as the parent compound. Pharmacodynamic effects were correlated with plasma U-47700 and its N-desmethyl metabolite. Using radioligand binding assays, U-47700 displayed high affinity for µ-opioid receptors (Ki = 11.1 nM) whereas metabolites were more than 18-fold weaker. Our data reveal that U-47700 induces typical µ-opioid effects which are related to plasma concentrations of the parent compound. Given its high potency, U-47700 poses substantial risk to humans who are inadvertently exposed to the drug.

Quantification of Furanylfentanyl and its Metabolites in Human and Rat Plasma Using LC-MS-MS.

Fentanyl analogs (novel and traditional) continue to impact the ever-growing opioid epidemic. Furanylfentanyl (FuF) is one analog equipotent to fentanyl that has documented involvement in thousands of intoxication and fatality cases around the world. Due to its prevalence, toxicologists need to improve detection and understanding of this analog. A method for the quantification of FuF and its metabolites (4-ANPP, furanyl norfentanyl (FuNorF)) in a small volume (100 µL) of human plasma by LC-MS-MS was developed and validated according to ANSI/ASB Standard. The method was cross validated in rat plasma for a future pharmacokinetic (PK)/pharmacodynamic (PD) study. In human plasma, calibration ranges were 0.025-25 ng/mL (FuF and 4-ANPP) and 0.5-25 ng/mL (FuNorF). Limits of detection were 0.0125 ng/mL (FuF and 4-ANPP) and 0.25 ng/mL (FuNorF). Lower limits of quantification coincided with lowest calibrator concentrations of 0.025 ng/mL (FuF and 4-ANPP) and 0.5 ng/mL (FuNorF). Precision and bias values were determined to be acceptable for all analytes. Matrix effects were acceptable for all analytes (-8.6-25.0%), except FuNorF with suppression >25%. Extraction recoveries ranged from 84.5 to 98.1%. No carryover or endogenous interferences were observed. Qualitative interferences with 4-ANPP were observed from some n-acyl substituted fentanyl analogs predicted to be low-concentration standard impurities. Analytes were stable under all conditions and dilution integrity was sustained. The method was successfully cross validated in rat plasma with acceptable bias (-7.4-8.4%), precision (within-run < 19%CV and between-run < 12.6%CV), matrix effects (-9.3-17.2%, except FuNorF with >25% suppression), recoveries (79.2-94.5%) and dilution integrity (1/2 and 1/10).

5F-MDMB-PICA metabolite identification and cannabinoid receptor activity.

According to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), there were 179 different synthetic cannabinoids reported as of 2017. In the USA, 5F-MDMB-PINACA, or 5F-ADB, accounted for 28% of cannabinoid seizures 2016-2018. The synthetic cannabinoid, 5F-MDMB-PICA, is structurally similar to 5F-MDMB-PINACA with an indole group replacing the indazole. Limited data exist from in vivo or in vitro metabolic studies of these synthetic cannabinoids, so potential metabolites to identify use may be missed. The goals of this study were to (a) investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism utilizing human hepatocytes; (b) to verify in vitro metabolites by analyzing authentic case specimens; and (c) to identify the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA by examining activity at the CB1 receptor. Biotransformations found in this study included phase I transformations and phase II transformations. A total of 22 5F-MDMB-PICA metabolites (A1 to A22) were identified. From hepatocyte incubations and urine samples, 21 metabolites (B1 to B21) were identified with 3 compounds unique to urine specimens for 5F-MDMB-PINACA. Phase II glucuronides were identified in 5F-MDMB-PICA (n = 3) and 5F-MDMB-PINACA (n = 5). For both compounds, ester hydrolysis and ester hydrolysis in combination with oxidative defluorination were the most prevalent metabolites produced in vitro. Additionally, the conversion of ester hydrolysis with oxidative defluorination to pentanoic acid for the first time was identified for 5F-MDMB-PICA. Therefore, these metabolites would be potentially good biomarkers for screening urine of suspected intoxication of 5F-MDMB-PICA or 5F-MDMB-PINACA. Both 5F-MDMB-PICA and 5F-MDMB-PINACA were acting as full agonists at the CB1 receptor with higher efficacy and similar potency as JWH-018.

Oral Fluid and Drug Impairment: Pairing Toxicology with Drug Recognition Expert Observations.

According to the Governors Highway Safety Association, drugs are detected more frequently in fatally injured drivers than alcohol. Due to the variety of drugs (prescribed and/or illicit) and their various physiological effects on the body, it is difficult for law enforcement to detect/prosecute drug impairment. While blood and urine are typical biological specimens used to test for drugs, oral fluid is an attractive alternative matrix. Drugs are incorporated into oral fluid by oral contamination (chewing or smoking) or from the bloodstream. Oral fluid is non-invasive and easy to collect without the need for a trained professional to obtain the sample, unlike urine or blood. This study analyzes paired oral fluid and urine with drug recognition expert (DRE) observations. Authentic oral fluid samples (n = 20) were collected via Quantisal™ devices from arrestees under an institutional review board-approved protocol. Urine samples (n = 18) were collected with EZ-SCREEN® cups that presumptively screened for Δ9-tetrahydrocannabinol (cannabinoids), opiates, methamphetamine, cocaine, methadone, phencyclidine, amphetamine, benzodiazepines and oxycodone. Impairment observations (n = 18) were recorded from officers undergoing DRE certification. Oral fluid samples were screened using an Agilent Technologies 1290 Infinity liquid chromatograph (LC) coupled to an Agilent Technologies 6530 Accurate Mass Time-of-Flight mass spectrometer (MS). Personal compound and database libraries were produced in-house containing 64 drugs of abuse. An Agilent 1290 Infinity LC system equipped with an Agilent 6470 Triple Quadrupole MS was used for quantification of buprenorphine, heroin markers (6-acetylmorphine, morphine) and synthetic opioids. Subjects were 23-54 years old; 11 (55%) were male and 9 (45%) were female. Evaluator opinion of drug class was confirmed in oral fluid 90% of time and in urine 85% of the time in reference to scope of testing by the LC-MS methods employed (excludes cannabis and central nervous system depressants). Data indicate that oral fluid may be a viable source for confirming driving under the influence of drugs.

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.

Data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS.

Recently, fentanyl analogs account for significant number of opioid deaths in the United States. Routine forensic analyses are often unable to detect and differentiate these analogs due to low concentrations and presence of structural isomers. A data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid was developed and validated using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Data were acquired using Time of Flight (TOF) and All Ions Fragmentation (AIF) modes. The limits of detection (LOD) in blood were 0.1-1.0 ng/mL and 0.1-1.0 ng/mL in TOF and AIF modes, respectively. In oral fluid, the LODs were 0.25 ng/mL and 0.25-2.5 ng/mL in TOF and AIF modes, respectively. Matrix effects in blood were acceptable for most analytes (1-14.4%), while the nor-metabolites exhibited ion suppression >25%. Matrix effects in oral fluid were -11.7 to 13.3%. Stability was assessed after 24 h in the autosampler (4 °C) and refrigerator (4 °C). Processed blood and oral fluid samples were considered stable with -14.6 to 4.6% and -10.1 to 2.3% bias, respectively. For refrigerated stability, bias was -23.3 to 8.2% (blood) and -20.1 to 20.0% (oral fluid). Remifentanil exhibited >20% loss in both matrices. For proof of applicability, postmortem blood (n = 30) and oral fluid samples (n = 20) were analyzed. As a result, six fentanyl analogs were detected in the blood samples with furanyl fentanyl and 4-ANPP being the most prevalent. No fentanyl analogs were detected in the oral fluid samples. This study presents a validated screening technique for fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS with low limits of detection.