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Quantification of seven novel synthetic opioids in blood using LC–MS/MS
Abstract
Purpose
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).
Methods
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.
Results
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.
Conclusions
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.
... Concerning biological specimens, blood [82,88,90,94] and plasma [80,83,92] are used due to their usually high availability, while urine [80,94] is a good option when metabolite identification is desired. On the other hand, hair is the best option to study or evaluate chronic consumption [79,87] and oral fluid has the advantage of determining mainly the parent compound [89]. ...
... Concerning recoveries, Smith et al. [92] obtained values between 86 and 91%, while slightly better results with recoveries between 86 and 98% were obtained by Palmquist et al. [83]. Also with a Cerex R Clin II cartridge and LC-MS/MS (ESI) analysis, Lowry et al. [88] developed a method for the identification of seven new compounds of this class in whole blood, obtaining LOD of 0.125 ng/ml for all analytes but one (W-18, 0.25 ng/ml) and LOQ of 0.25 ng/ml (except for W-18, 1 ng/ml). These results show good sensitivity using 500 μl of blood (an adequate sample volume taking into account what is usually sent to a laboratory), although lower volumes are possible, as reported by other authors. ...
... Nimetazepam (92) Phenazepam (93) Pyrazolam ( Cloniprazepam (2) Desalkyl flurazepam (2) Diclazepam (1) Etizolam (2) Meclonazepam (3) Nimetazepam (2) Norflunitrazepam (6) Phenazepam (2) n.s. Clonazolam (77) Cloniprazepam (85) Desalkyl flurazepam (88) Diclazepam (92) Etizolam (93) Meclonazepam (86) Nimetazepam (88) Norflunitrazepam (64) Phenazepam (86) [118] In the absence of the LOQ value, the lowest point of the calibration curve was considered. (5) Deschloroetizolam (2) Diclazepam (2) Etizolam (2) Flubromazepam (2.5) ...
One of the problems associated with the consumption of new psychoactive substances is that in most scenarios of acute toxicity the possibility of quick clinical action may be impaired because many screening methods are not responsive to them, and laboratories are not able to keep pace with the appearance of new substances. For these reasons, developing and validating new analytical methods is mandatory in order to efficiently face those problems, allowing laboratories to be one step ahead. The goal of this work is to perform a critical review regarding bionalytical methods that can be used for the determination of new psychoactive substances (phenylethylamines, cathinones, synthetic cannabinoids, opioids, benzodiazepines, etc), particularly concerning sample preparation techniques and associated analytical methods.
... Separation was achieved using our previously published method with limit of detection and quantification at 0.125 and 0.25-1 ng/mL, respectively [25]. Mobile phase A consisted of 5 mM ammonium formate with 0.05% formic acid in water and mobile phase B consisted of 0.1% formic acid in methanol. ...
... Analytical runs were deemed acceptable if parameters met validation parameters such as R 2 ! 0.99, calibrators and QC concentrations within AE 20% of target, acceptable ion ratios, etc [25]. ...
... Samples were extracted using a previously validated method [25]. An additional protein precipitation step was added to reduce solvent usage and time. ...
Recently, there has been an increase in overdose deaths due to novel synthetic opioids (NSO). Due to backlogs experienced by many forensic laboratories, it is important to understand drug stability in a variety of storage conditions. The objective of this study was to investigate the stability of AH-7921, U-47700, U-49900, U-50488, MT-45, W-15, and W-18 in blood at various temperatures over a 36-week period. NSO were generally stable over the 36-week period (66%-118%) at low and high concentrations when blood samples were stored in the refrigerator or freezer. Most analytes were stable for at least 2 weeks at room temperature (77%-120%). At the elevated temperature (35°C), analytes were generally stable for at least 14 days (75%-109%). This study has determined the stability of several NSO at various temperatures over a 36-week period. These results reflect the forensic significance of keeping samples stored at proper temperatures. Blood samples suspected to contain synthetic opioids should be stored refrigerated or frozen, when possible, in order to preserve analyte stability, especially at low concentrations.
... Due to their diffusion, the development of quick analytical methods able to identify these drugs either in seized specimens or in biological matrices is mandatory [7,8]. Recently, different strategies-mainly based on chromatography-have been proposed, including, among others, GC-MS [9], LC-HRMS [10], SPE/LC-ESI-MS-MS [11], LC-QTOF [12], LC-MS/MS [13][14][15][16][17][18][19][20], and UHPLC-MS/MS [21,22] (see Table 1). On the other hand, From a pharmacokinetic point of view, AMs are subject to phase I and phase II metabolism, but a significant portion of the administered drug can be found unmodified in urine and plasma, in concentrations ranging from 0.15 µg·mL −1 up to µg·mL −1 [4,7,8] depending on the dose taken-usually between 5 mg and over 10 mg-and on the consumer's metabolic profile. ...
... Due to their diffusion, the development of quick analytical methods able to identify these drugs either in seized specimens or in biological matrices is mandatory [7,8]. Recently, different strategies-mainly based on chromatography-have been proposed, including, among others, GC-MS [9], LC-HRMS [10], SPE/LC-ESI-MS-MS [11], LC-QTOF [12], LC-MS/MS [13][14][15][16][17][18][19][20], and UHPLC-MS/MS [21,22] (see Table 1). On the other hand, electrochemistry offers several advantages when compared to chromatographic methods, such as low-cost and portability, ease of operation, as well as good sensitivity [23], so that the interest in developing electrochemical methods able to quantify NSOs at low concentration (<50-100 µM) in biological matrices is growing [24,25]. ...
In the present work, we describe the electrochemical behaviour of two opioids structurally related to aroyl amides of forensic interest, namely U-47700 and AH-7921. The data obtained allowed for the mise-au-point of a voltammetric determination protocol, that relies on differential pulse voltammetry (DPV) at a glassy carbon electrode in ethanol/0.1 M lithium perchlorate/0.10 M 2,6-lutidine. To apply the method to the analysis of real samples (urines), a clean-up and a preconcentration strategy by solid phase extraction (SPE) using the adsorbent Florisil have been developed. LOQ of 0.2 μg·mL−1 in urine samples with an enrichment factor of 20 and linear range from 5 to 150 μg·mL−1 were obtained.
... Somewhat confusingly, the American Academy of Forensic Sciences (AAFS) Standards Board's 'Standard Practices for Method Validation in Forensic Toxicology' state that the 'calibration model shall not be evaluated simply via its correlation coefficient (r)', but that 'assessment of coefficient of determination (r 2 ) for linear models' is an 'appropriate alternative' among others (also cited are the residuals plot, analysis of variance (ANOVA) lack-of-fit test and significance of the second-order term of quadratic models) (3). The use of r or R 2 as a figure demonstrating linearity of the data in method validation has been widespread and persists to this day, see for example a sampling of recent publications (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). ...
... R 2 does not indicate if your dataset is linear; it assumes linearity R 2 is often presented as a means to assess linearity in method validation, i.e., to evaluate whether the data produced by the method under evaluation are linear. While different thresholds are quoted for 'acceptable linearity', an R 2 above 0.9 is often cited as a criterion (2,9,(14)(15)(16). Unfortunately, R 2 evaluates the strength of the association between the x and y variables assuming that the regression model used is the adequate one. ...
The coefficient of correlation (r) and the coefficient of determination (R2 or r2) have long been used in analytical chemistry, bioanalysis and forensic toxicology as figures demonstrating linearity of the calibration data in method validation. We clarify here what these two figures are and why they should not be used for this purpose in the context of model fitting for prediction. R2 evaluates whether the data are better explained by the regression model used than by no model at all (i.e., a flat line of slope = 0 and intercept $\bar y$), and to what degree. Hopefully, in the context of calibration curves, the fact that a linear regression better explains the data than no model at all should not be a point of contention.
Upon closer examination, a series of restrictions appear in the interpretation of these coefficients. They cannot indicate whether the dataset at hand is linear or not, because they assume that the regression model used is an adequate model for the data. For the same reason, they cannot disprove the existence of another functional relationship in the data. By definition, they are influenced by the variability of the data. The slope of the calibration curve will also change their value. Finally, when heteroscedastic data are analyzed, the coefficients will be influenced by calibration levels spacing within the dynamic range, unless a weighted version of the equations is used.
With these considerations in mind, we suggest to stop using r and R2 as figures of merit to demonstrate linearity of calibration curves in method validations. Of course, this does not preclude their use in other contexts. Alternative paths for evaluation of linearity and calibration model validity are summarily presented.
... There are several published methods for the confirmation of U-compounds in various biological matrices, with the majority of methods developed for blood, serum/plasma, and/or urine [25,27,[69][70][71][72][73]. Two major considerations when developing confirmation methods for U-compounds are sensitivity (or predicted concentration range) and isobaric species (different compounds with the same molecular formula). ...
... U-47700 was reported in 543 postmortem blood samples between October 2016 and September 2017, with a mean and median concentration of 143 ng/mL and 14 ng/mL, respectively (range: 0.20-3800 ng/mL). After the development of a seven-analyte panel that included U-47700, U-49900 and U-50488, 15 authentic postmortem blood samples were reported positive for U-47700, with a mean concentration of 214 ng/mL (range: 3.2-1448 ng/mL) [71]. ...
The recreational use of opioid drugs is a global threat to public health and safety. In particular, an epidemic of opioid overdose fatalities is being driven by illicitly manufactured fentanyl, while novel synthetic opioids (NSOs) are appearing on recreational drug markets as standalone products, adulterants in heroin, or ingredients in counterfeit drug preparations. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a prime example of a non-fentanyl NSO that is associated with numerous intoxications and fatalities. Here, we review the medicinal chemistry, preclinical pharmacology, clandestine availability, methods for detection, and forensic toxicology of U-47700 and its analogs. An up-to-date summary of the human cases involving U-47700 intoxication and death are described. The evidence demonstrates that U-47700 is a potent μ-opioid receptor agonist, which poses a serious risk for overdosing and death. However, most analogs of U-47700 appear to be less potent and have been detected infrequently in forensic specimens. U-47700 represents a classic example of how chemical entities from the medicinal chemistry or patent literature can be diverted for use in recreational drug markets. Lessons learned from the experiences with U-47700 can inform scientists, clinicians, and policymakers who are involved with responding to the spread and impact of NSOs.
... After the discovery of these demethylated metabolites, researchers began incorporating one or both metabolites into their scope of testing. Analytical methods for U-47700 built using LC-MS/MS-based systems achieved LODs and LOQs ranging from 0.005-0.1 μg/L and 0.05-0.5 μg/L, respectively[44,49,97].After the emergence and identification of non-fentanyl related NSOs in forensic casework, many methods were developed that detected multiple NSOs, including AH-7921 AH-8533, AH-8529, MT-45, U-47700, U-50488, and/or U-49900[21,25,53,55,62,98,99,100]. These methods analyzed traditional matrices such as whole blood, urine, and tissues, while two methods analyzed for the alternative matrices such as hair and oral fluid. ...
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.
... According to Lowry et al. [26], in fatalities involving U-47700, blood concentrations of this substance range from 0.090 to 1460 ng/mL. Among the cases presented in Table S1, blood concentrations of U-47700 in cases of fatal intoxication with this substance alone were: in femoral blood 525-1490 ng/mL [9,27], in cardiac blood 340-1347 [9,28]; peripheral blood 190 ng/mL [28] and 380 ng/mL in nonspecified blood [29]. ...
Purpose
We present a case of fatal intoxication with U-47700 in combination with other NPS ( N -ethylhexedrone, adinazolam, 4-chloro- N -isopropylcathinone (4-CIC), 4-chloromethcathinone (4-CMC) and sertraline) confirmed by identification and quantification in biological materials and evidences found at the scene in 2017 in Poland.
Methods
Blood and urine samples were extracted with ethyl acetate from alkaline medium (pH 9); powders/crystals were diluted with methanol. The analysis was carried out using ultra-high-performance liquid chromatography–tandem mass spectrometry. Validation criteria were evaluated for blood and urine at the concentrations of 10 and 100 ng/mL.
Results
The validation parameters of the method were within acceptable ranges. In the presented case, the determined concentrations of drugs were as follows, in blood: U-47700, 1470 ng/mL; N -ethylhexedrone, 58 ng/mL; adinazolam, 18 ng/mL; 4-CIC, 8.0 ng/mL; 4-CMC, 1.7 ng/mL; in urine: U-47700, 3940 ng/mL; N -ethylhexedrone, 147 ng/mL; adinazolam, 82 ng/mL; 4-CIC, 130 ng/mL; 4-CMC, 417 ng/mL. Sertraline (blood, 89 ng/mL; urine, 32 ng/mL) was also determined in both materials. The same substances were also found in 5 powders/crystals: U-47700 (12% by weight), N -ethylhexedrone (54%), adinazolam (14%), 4-CIC (23%), 4-CMC (26%). After 775 days of storage, biological samples at + 4 °C, the most stable substance was sertraline and the less, synthetic cathinones, especially 4-CIC and 4-CMC.
Conclusions
The described case of fatal intoxication with NPS presented postmortem concentrations of U-47700, 4-CMC, N -ethylhexedrone, adinazolam and 4-CIC for the first time in the literature. The paper also showed stability study of these substances stored at + 4 °C for 775 days.
... The above method was successful in detecting and quantifying 12 U-series compounds and 2 metabolites, including the isobaric pairs (U-48800/U-51754 and U-49900/isopropyl U-47700/propyl U-4700), which were all separated chromatographically, which allowed them to be quantitated without interference from each other. Successful chromatographic resolution for these compounds has not been previously reported (23). Further, this method was used to confirm the presence of U-48800, one of the short-lived alternatives to U-47700, in authentic blood specimens collected from death investigation casework. ...
We report a method for the detection and quantitation of 12 drugs and 2 metabolites in the same structural class as the illicit mu-opioid agonist U-47700 in human whole blood. These substances are either known or suspected to be present as potential novel opioids in illicit drug markets. The general class of these drugs was developed in pharmaceutical research programs in the 1970s, but these drugs have recently become of concern for overdoses and death in opioid users in the USA and internationally. The scope of analysis included the following compounds: methylenedioxy U-47700, ethylenedioxy U-47700, ethylenedioxy U-51754, U-69593, U-47931E (bromadoline), U-47700, U-48800, U-49900, U-51754, U-50488, propyl U-47700 and isopropyl U-47700. Additionally, two metabolites N,N-didesmethyl U-47700 and desmethyl U-47700 were also included in the scope. Drugs were extracted from human whole blood using solid-phase extraction, and the extracts were analyzed by liquid chromatography tandem mass spectrometry. The assay was validated with respect to bias, carryover, interference, within-run and between-run precision, and accuracy. Eight medicolegal death investigation cases that had screened positive for U-48800 by liquid chromatography time-of-flight mass spectrometry were successfully confirmed and quantified using this method. The mean and median concentrations of U-48800 in these cases were 2.5 (±2.1) and 1.8 ng/mL, respectively, with a range of concentrations of 0.27–6.2 ng/mL. Case history information including the presence of other drugs in combination are described and discussed.
Over the last decade, there has been a significant growth in the market and number of new psychoactive substances (NPS). One of the NPS groups that has grown rapidly in recent years, bringing a new set of problems, consists of new synthetic opioids. The extreme potency of these compounds poses a high risk of acute poisoning, as an overdose can cause respiratory depression. Most of the information regarding human pharmacokinetics of new opioids is based on toxicological case reports and the data on concentrations of new opioids in human blood are scarce. The interpretation of results usually requires a comparison to previously published cases; therefore, a referenced compilation of previously published concentration data would be useful.
The data were collected by searching the PubMed and Scopus databases and by using the Google search engine. All the available data from articles and reports that measured new opioid concentrations in plasma, serum, or whole blood were included in the data analysis.
The presented tables list the observed concentrations in fatal and nonfatal cases involving 37 novel synthetic opioids.
Blood levels of new opioids are extremely difficult to interpret. Low blood concentrations of these substances do not rule out acute poisoning as their high potency creates a risk of respiratory depression even at low doses. Opioid tolerance, frequent presence of other drugs, and additional diseases make it impossible to define concentration ranges, especially the minimum fatal concentrations. This report provides quick access to the source articles quantifying novel synthetic opioids.
Rationale:
The objective of this study was to develop, optimize, and validate a method for the determination and quantification of 17 hypoglycemic drugs in fingerprints using ultra-high-performance liquid chromatography-tandem hybrid triple quadrupole linear ion trap mass spectrometry (UHPLC-QTRAP-MS/MS). We also aimed to apply the present method to the fingerprints collected from patients with hyperglycemia.
Methods:
The scheduled multiple reaction monitoring-information dependent acquisition-enhanced product ion (SMRM-IDA-EPI) scanning mode was utilized. The chromatographic system consisted of an Acquity UHPLC® BEH C18 column (3.0×100 mm, 1.7μm) and a mobile phase of 0.01% (v/v) formic acid in water and methanol. Analytes were extracted via a precipitation protein procedure. The method was validated in accordance with the US food and drug administration (FDA) guidance and applied to the analysis of fingerprint deposits from subjects who have taken the drugs.
Results:
The limits of detection (LODs) and the lower limits of quantification (LLOQs) of 17 hypoglycemic drugs were 0.001 to 0.020 and 0.002 to 0.050 ng/fingerprint, respectively. The correlation coefficients (r) for the calibration curves were > 0.99 in the range of 0.050-50.000 ng/fingerprint. The matrix effect and recovery of 17 hypoglycemic drugs at three concentrations ranged from 81.1 to 117.3% and 80.0 to 109.6%, respectively. The validation data (intra- and inter-day combined) for accuracy ranged from 85.5 to 117.2%, the CV (%) data were ≤ 19.7%. All analytes were found to be stable stored in the autosampler (4°C) for 24 h. This validated method was successfully applied to detect hypoglycemic drugs in fingerprints from patients with hyperglycemia.
Conclusions:
A quantification method for hypoglycemic drugs in fingerprints was developed, optimized, and validated. This sensitive method could be used for drug monitoring and providing reference information in forensic investigations.
An improved assessment of environmental risks to public water bodies requires screening a large number of micropollutants. This study reports the development of a novel target screening method based on solid-phase extraction (SPE), HPLC, and high-resolution Orbitrap MS for the analysis of micropollutants with diverse chemical properties. First, target compounds were screened for their detectability by Orbitrap MS. An optimized SPE cartridge and HPLC column maximized recovery and separated most target compounds. The sensitivity and repeatability of the method was validated by determining the detection limits and relative standard deviation (RSD). Eighty-four compounds with highly diverse properties were simultaneously detected with detection limits of 0.1–100 ng/L. Of these compounds, 52 were quantitated, with R² ≥ 0.99 by linearity analysis and SPE recovery ratios of ≥50%. The remaining 32 compounds were qualitatively detected, with R² < 0.99 or SPE recovery ratio of <50%. Satisfactory repeatability was obtained (RSD < 13.5%). This method was applied to the surveillance of the Arakawa River in Japan in 2019. Thirty-two compounds, including pesticides, surfactants, plasticizers, adhesives, and industrial solvents, were detected in the river. The measured concentrations of 13 compounds were compared with their predicted no effect concentrations (PNECs). Decanoic acid showed a higher concentration than the corresponding PNEC value, suggesting that its risk to the Arakawa water environment required further evaluation. The concentrations of dicyclohexylamine, 1,3-diphenylguanidine, and 2,4-dichlorophenoxyacetic acid were higher than their corresponding PNEC/10 values, demonstrating that these compounds were of higher priority than other compounds.
In many jurisdictions, public safety and public health entities are working together to enhance the timeliness and accuracy of the analytical characterization and toxicology testing of novel synthetic opioids. The improved sharing and early detection of these analytical data are intended to inform surveillance, interdiction efforts, patient intervention and treatment, all of which are critical to curbing the opioid epidemic. Forensic practitioners working to identify novel synthetic opioids struggle to provide timely results when encountering new or unknown substances, such as the fentanyl analogs. These compounds, which mimic heroin in pharmacologic effect but can be far more potent, are inconsistently present in chemical identification libraries, and are currently largely unavailable as reference materials for analytical comparison. Additionally, federal, state and local governments as well as nongovernmental organizations require potency, toxicity and potential-for-abuse data to evaluate the potential health risks of emerging drug threats. Subsequent scheduling efforts and criminal prosecutions also require these thorough drug characterization studies. Pilot programs have demonstrated that early communication of real-time drug toxicity and analytical data significantly impacts the successful response to emerging opioids. High-quality, real-time, national-level data on chemical composition, toxicological test data, drug toxicity and overdoses, and analysis of seized materials by law enforcement are needed to track drug trends. However, the USA still lacks a national system to coordinate and communicate toxicology, medical and medical examiner and coroner data with the broader medical and law enforcement communities. Opportunities to address these gaps as well as recent advancements collected through interagency efforts and technical workshops in the toxicology and Published by Oxford University Press 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.
During 1999‒2015, 568,699 persons died from drug overdoses in the United States.* Drug overdose deaths in the United States increased 11.4% from 2014 to 2015 resulting in 52,404 deaths in 2015, including 33,091 (63.1%) that involved an opioid. The largest rate increases from 2014 to 2015 occurred among deaths involving synthetic opioids other than methadone (synthetic opioids) (72.2%) (1). Because of demographic and geographic variations in overdose deaths involving different drugs (2,3),†CDC examined age-adjusted death rates for overdoses involving all opioids, opioid subcategories (i.e., prescription opioids, heroin, and synthetic opioids),§cocaine, and psychostimulants with abuse potential (psychostimulants) by demographics, urbanization levels, and in 31 states and the District of Columbia (DC). There were 63,632 drug overdose deaths in 2016; 42,249 (66.4%) involved an opioid.¶From 2015 to 2016, deaths increased across all drug categories examined. The largest overall rate increases occurred among deaths involving cocaine (52.4%) and synthetic opioids (100%), likely driven by illicitly manufactured fentanyl (IMF) (2,3). Increases were observed across demographics, urbanization levels, and states and DC. The opioid overdose epidemic in the United States continues to worsen. A multifaceted approach, with faster and more comprehensive surveillance, is needed to track emerging threats to prevent and respond to the overdose epidemic through naloxone availability, safe prescribing practices, harm-reduction services, linkage into treatment, and more collaboration between public health and public safety agencies.
Opioid overdose deaths quadrupled from 8,050 in 1999 to 33,091 in 2015 and accounted for 63% of drug overdose deaths in the United States in 2015. During 2010-2015, heroin overdose deaths quadrupled from 3,036 to 12,989 (1). Sharp increases in the supply of heroin and illicitly manufactured fentanyl (IMF) are likely contributing to increased deaths (2-6). CDC examined trends in unintentional and undetermined deaths involving heroin or synthetic opioids excluding methadone (i.e., synthetic opioids)* by the four U.S. Census regions during 2006-2015. Drug exhibits (i.e., drug products) obtained by law enforcement and reported to the Drug Enforcement Administration's (DEA's) National Forensic Laboratory Information System (NFLIS) that tested positive for heroin or fentanyl (i.e., drug reports) also were examined. All U.S. Census regions experienced substantial increases in deaths involving heroin from 2006 to 2015. Since 2010, the South and West experienced increases in heroin drug reports, whereas the Northeast and Midwest experienced steady increases during 2006-2015.(†) In the Northeast, Midwest, and South, deaths involving synthetic opioids and fentanyl drug reports increased considerably after 2013. These broad changes in the U.S. illicit drug market highlight the urgent need to track illicit drugs and enhance public health interventions targeting persons using or at high risk for using heroin or IMF.
Since 2013, the Miami-Dade County Medical Examiner Department has experienced an increase in the number of opioid-related deaths. The majority of cases coincided with the introduction of fentanyl into the local heroin supply. From 2014 to 2015, Miami-Dade County experienced a near 600% increase in fentanyl-related deaths, followed by an additional 200% increase in 2016. In 2015, two novel fentanyl analogs were identified in medical examiner cases: beta-hydroxythiofentanyl and acetyl fentanyl. In 2016, four additional fentanyl analogs emerged: para-fluoroisobutyryl fentanyl, butyryl fentanyl, furanyl fentanyl and carfentanil, as well as the synthetic opioid U-47700. In order to address this epidemic, a method was developed and validated to identify 44 opioid-related and analgesic compounds in postmortem samples using ultra high performance liquid chromatography ion trap mass spectrometry with MSn capabilities. The limit of detection for all compounds ranged from 0.1 to 5 ng/mL, with a majority having MS3 spectral fragmentation. Blood, urine, liver or brain specimens from ~500 postmortem cases were submitted for analysis based on case history and/or initial screening results. Of those cases, 375 were positive for illicit fentanyl and/or one or more fentanyl analogs. Due to the potency of these compounds, they were almost always included in the cause of death. Worth emphasizing and extremely alarming is the detection of carfentanil in 134 cases, 104 of which were initially missed by gas chromatography mass spectrometry. By incorporating this sensitive, highly specific, and evolving screening procedure into the workflow, the toxicology laboratory continues to effectively assist the medical examiners in determining the cause and manner of death of decedents in Miami-Dade County.
In this case report, we present an evaluation of the distribution of postmortem concentrations of 3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) in a fatality attributed principally to the drug. A man who had a history of drug abuse was found unresponsive and not breathing on his bed. Drug paraphernalia, indicating drug insufflation, was located in the decedent's room. Toxicology screening tests in peripheral blood initially identified U-47700 using an alkaline drug screen with gas chromatography-mass spectrometry (GC-MS) following solid-phase extraction. It was subsequently confirmed and quantitated by GC-MS-specific ion monitoring analysis following liquid-liquid extraction. The U-47700 peripheral blood concentration was quantitated at 190 ng/mL compared to the central blood concentration of 340 ng/mL. The liver concentration was 1,700 ng/g, the vitreous was 170 ng/mL, the urine was 360 ng/mL and the gastric contained only a trace amount (<1 mg). Other drugs detected in peripheral blood were alprazolam (0.12 mg/L), nordiazepam (<0.05 mg/L), doxylamine (0.30 mg/L), diphenhydramine (0.14 mg/L), ibuprofen (2.4 mg/L), salicylic acid (<20 mg/L) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (2.4 ng/mL). The cause of death was certified as acute U-47700 and alprazolam abuse, and the manner of death was certified as accident.
AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a designer opioid with ∼80% of morphine's µ-agonist activity. Over a 6-month
period, we encountered nine deaths where AH-7921 was involved and detected in blood from the deceased. Shortly after the last
death, on August 1 2013, AH-7921 was scheduled as a narcotic and largely disappeared from the illicit market in Sweden. AH-7921
was measured by a selective liquid chromatography–MS-MS method and the concentrations of AH-7921 ranged from 0.03 to 0.99
µg/g blood. Six of our cases had other drugs of abuse on board and most had other medications such as benzodiazepines, antidepressants
and analgesics. However, the other medicinal drugs encountered were present in postmortem therapeutic concentrations and unlikely
to have contributed to death. In addition to the parent compound, we identified six possible metabolites where two N-demethylated dominated and four mono-hydroxylated were found in trace amounts in the blood. In conclusion, deaths with AH-7921
seem to occur both at low and high concentrations, probably a result of different tolerance to the drug. Hence, it is reasonable
to assume that no sharp dividing line exists between lethal and non-lethal concentrations. Further, poly-drug use did not
seem to be a major contributing factor for the fatal outcome.
The interactive effects of mu and kappa opioid receptor agonists on respiratory function were investigated following their i.c.v. injection into conscious rats. The highly selective mu receptor agonist [D-Ala2,N-Methyl-Phe4,Gly-ol] enkephalin (DAMGO; 1.2-10 nmol) and the relatively selective mu agonist morphine (20 and 30 nmol) significantly decreased arterial pH and PO2, and increased arterial PCO2 and blood pressure. Morphine and a low dose of DAMGO (1.2 nmol) also significantly elevated respiratory rate. Heart rate was decreased by DAMGO and, depending upon dose, was either decreased (20 nmol) or increased (30 nmol) by morphine. The selective kappa opioid agonist U-50,488H (200 nmol i.c.v.), which by itself had no significant effect on either respiration or cardiovascular function, dose-dependently antagonized the acidotic, hypoxemic and hypercapnic effects of both DAMGO (2.5 nmol) and morphine (30 nmol). Furthermore, these mu antagonistic properties of U-50,488H were blocked completely after pretreatment with 25 nmol of the highly selective kappa opioid antagonist nor-binaltorphimine. These results indicate that the antagonism of mu opioid respiratory depressant effects by U-50,488H is kappa opioid receptor mediated.
The opioid epidemic has become a national health emergency in the USA. While heroin and prescription opioid abuse is not uncommon, synthetic opioid use has risen dramatically, creating a public safety concern. Like traditional opioids, novel synthetic opioids are abused due to their analgesic and euphoric effects. Some adverse side effects include respiratory distress, nausea and decreased consciousness. Synthetic opioids have emerged into the illicit and online drug market, including AH-7921, MT-45, U-series and W-series. Though originally developed by pharmaceutical companies, these substances are not well studied in humans and comprehensive analytical methods for detecting and quantifying these opioids are limited. Oral fluid is a useful biological matrix for determining recent drug use, does not require a trained medical professional, and can be collected under direct observation, deterring adulteration. The purpose of this research was to develop and validate a comprehensive analytical method for the detection and quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18 and W-15 in oral fluid collected via Quantisal. This was achieved by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The limits of detection and quantitation were 5 ng/mL and 10 ng/mL, respectively. Linearity was observed between 10 and 500 ng/mL (R2 ≥ 0.9959). Bias and imprecision were <±11.1%. Matrix effects ranged from -21.1 to 13.7%. No carryover was detected following injection of the highest calibrator. All analytes were stable (within ±15% change from baseline) under all tested conditions (24 h at room temperature, 72 h at 4°C, and in the autosampler for 60 h at 4°C).
Recently, it has been documented that there has been a rise in synthetic opioid abuse. Synthetic opioids are compounds that were created to act as agonists for the opioid receptors. Like synthetic cannabinoids, most of these compounds were created by research groups or pharmaceutical companies in an attempt to find compounds that have medicinal use. Synthetic opioids have severe health implications when abused that can include hospitalization and death. Due to the high potency and the low dose required to produce the desired effects for these compounds, it was hypothesized that they may not be detectable in human performance case samples. However, this report documents a male driver who was involved in a single-vehicle incident. First responders treated the subject with naloxone as opioid drug impairment was suspected and he was transported to the local emergency room. The subject consented to a blood draw for a driving under the influence (DUI) investigation. Initial routine testing identified alprazolam at 55 ng/mL and fentanyl at less than 0.5 ng/mL. Further testing using a validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay, confirmed the presence of carfentanil, furanyl fentanyl, para-fluoroisobutyryl fentanyl, U-47700 and its metabolite. To the author's knowledge, this is the first report of a DUI cases where carfentanil, U-47700 and other synthetic opioids were confirmed and described in a human performance blood sample. This case demonstrates the need to supplement routine toxicological analyses with a sensitive methodology that can detect synthetic opioids in human performance cases where opioid use may be implicated.
U-47700 was developed by the Upjohn Co. in the 1970s as part of their search for a selective μ-opioid agonist with similar potency as morphine. U-47700 has re-emerged recently in the illicit drug market and is easily and cheaply obtained via the internet as well as on the street, many times falsely sold as another drug. Several fatalities from U-47700 have been reported in scientific literature, often in combination with other intoxicants. This case report describes the first death in south-central Kansas resulting solely from U-47700 intoxication: a 26-year-old white male found dead in his bedroom with apparent drug paraphernalia. Autopsy findings were consistent with opioid overdose, but toxicological examination, utilizing immunoassay and instrumental techniques, was negative for opioids. U-47700 was detected in a comprehensive alkaloid screen by GC/MS and GC-NPD, and quantitation was performed using GC-NPD on a variety of specimens to provide a full tissue distribution. Quantitation of U-47700 in this individual revealed the following: heart blood 0.26 mg/L, femoral blood 0.40 mg/L, vitreous fluid 0.09 mg/L, brain 0.38 mg/kg, liver 0.28 mg/kg and urine 4.6 mg/L.
In this study, two fatalities associated with the synthetic opioids AH-7921 and MT-45 are reported. Within the last few years, both compounds have emerged on the recreational drug market and are sold as “research chemicals” on the internet. In the first case, a 22-year-old woman was found dead in the bedroom of her apartment by two of her friends. A plastic bag labeled “AH-7921” was found in the apartment and the two friends stated that the deceased had consumed AH-7921 prior to her death. The woman was a known drug addict. In the second case, a 24-year-old man was found dead in his room by his mother. The deceased was sitting on a chair in front of his desk slumped over. Several bags of white powder labeled “MT-45”, “Methoxmetamine” and “Methoxphenidine” were found in his room.
Toxicological analyses of femoral blood, heart blood, liver, pericardial fluid, urine, vitreous humor and stomach content of the deceased were performed using liquid chromatography–quadrupole-time-of-flight mass spectrometry (LC–QTOF-MS). Time-of-flight mass spectrometry was carried out on an LC-Triple TOF 5600 system (AB Sciex) with electrospray ionization operated in positive mode. In the first case, additional hair analysis was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and LC–QTOF-MS.
In both cases, the relevant synthetic opioid could be detected in all analyzed samples. The concentration of AH-7921 was determined to be 450 μg/L in femoral blood. MT-45 was present at a concentration of 2900 μg/L in femoral blood. Besides methoxmetamine which could qualitatively be detected in femoral blood, urine and stomach content no methoxphenidine was found. In summary, deaths of the young individuals could be, by exclusion of other causes of death, attributed to the consumption of an overdose of AH-7921 and MT-45, respectively.
U-47700 is a synthetic opioid analgesic and a potent, short acting structural isomer of the earlier opioid AH-7921 that has recently invaded the drug arena in Europe and the Unites States. Although the drug was synthesized and patented in the 1970s, it was first identified in October 2014, as a powder sample that was seized by Swedish Customs. Sweden formally notified the European Union Early Warning System in January 2015. Animal studies proved that U-47700 is a strong μ-opioid receptor agonist and has a morphine-like analgesic action, being 7.5 times higher than morphine. The drug has a much lower affinity for the κ-opioid receptor. This newly appearing psychoactive substance has already led to more than 25 confirmed fatalities associated with U-47700 in Europe and the United States and to six non-fatal intoxications reported in the United States. The aim of this review is to summarize the current knowledge about this drug, regarding its chemistry, synthesis, pharmacology, toxicology and metabolism, as well as its international legal status. The existing analytical methods for the determination of U-47700 in biological samples are also presented. Published or reported U-47700 related cases, fatalities or intoxications, and self reports from drug users are reviewed.
Following series of synthetic cannabinoid and synthetic cathinone derivatives, the illicit drug market has begun to see increased incidence of synthetic opioids including fentanyl and its derivatives, and other chemically unrelated opioid agonists including AH-7921 and MT-45. Among the most frequently encountered compounds in postmortem casework have been furanyl fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylfuran-2-carboxamide, Fu-F) and U-47700 (trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide). Both drugs have been reported to be present in the heroin supply and to be gaining popularity among recreational opioid users, but were initially developed by pharmaceutical companies in the 1970s as candidates for development as potential analgesic therapeutic agents. A method was developed and validated for the analysis of U-47700, U-50488 and furanyl fentanyl in blood specimens. A total of 20 postmortem cases, initially believed to be heroin or other opioid-related drug overdoses, were submitted for quantitative analysis. The analytical range for U-47770 and U-50488 was 1-500 and 1-100 ng/mL for furanyl fentanyl. The limit of detection was 0.5 ng/mL for all compounds. Within the scope of the method, U-47700 was the only confirmed drug in 11 of the cases, 5 cases were confirmed for both U-47700 and furanyl fentanyl, and 3 cases were confirmed only for furanyl fentanyl. The mean and median blood concentrations for U-47700 were 253 ng/mL (±150) and 247 ng/mL, respectively, range 17-490 ng/mL. The mean and median blood concentrations for furanyl fentanyl were 26 ng/mL (±28) and 12.9 ng/mL, respectively, range 2.5-76 ng/mL. Given the widespread geographical distribution and increase in prevalence in postmortem casework, toxicology testing should be expanded to include testing for "designer opioids" in cases with histories consistent with opioid overdose but with no traditional opioids present or insufficient quantities to account for death.
Context:
The opioid epidemic has included use of traditional drugs and recently newer synthetics. It is critically important to recognize and identify these new drugs both clinically and through appropriately designed toxicology testing. There is little available information on a synthetic gaining popularity, U-47700.
Case details:
A 23-year-old female presented after using "U4" by nasal insufflation and injection. She was cyanotic with respiratory depression and responded to naloxone in the field. She was found to have non cardiogenic pulmonary edema and hemoptysis which improved with BiPAP. Urine and serum samples were analyzed using mass spectrometry, confirming 3,4-dichloro-N-[(1R,2R)-2-(dimethylamino)cyclohexyl]-N-methylbenzamide or U-47700. The sample was further analyzed elucidating metabolism specifics. Drug and metabolite concentrations were subsequently measured in both serum and urine. The parent compound of U-47700 was detected at 394 ng/mL and 228 ng/mL in serum and urine, respectively. Metabolites detected in appreciable amounts included the desmethyl (1964 ng/mL in urine), bisdesmethyl (618 ng/mL), desmethyl hydroxy (447 ng/mL), and bisdesmethyl hydroxy forms (247 ng/mL) of U-47700.
Discussion:
U-47700 is a potent μ-opioid receptor agonist and has recently been used recreationally, contributing to hospitalizations and likely deaths in the community. This is a case report describing an exposure to U-47700 with subsequent laboratory analysis. Based upon this one case, parent U-47700 appear to be an appropriate marker of use in a serum sample. However, demethylated metabolites appear dominant as urinary markers of U-47700 use.
In 2013 and 2014, more than 700 deaths were attributed to fentanyl and fentanyl analogues in the United States. Of recent concern is the cluster of unintentional fentanyl overdoses because of tablets thought to be "Norco" purchased on the street in Northern California. U-47700 (trans-3,4-dichloro-N-[2-(dimethyl-amino)cyclohexyl]-N-methylbenz-amide) is a nonfentanyl-based synthetic opioid with 7.5 times the binding affinity of morphine to μ-opioid. We report a case of fentanyl and U-47700 intoxication from what was thought to be illicitly purchased Norco. A 41-year-old woman presented to the emergency department (ED) for altered mental status shortly after ingesting 3 beige Norco pills bearing a Watson imprint. She had pinpoint pupils and respiratory depression, which reversed after 0.4 mg naloxone administration intravenously. She had complete recovery and was discharged from the ED after a 4-hour observation period. Serum testing with liquid chromatography-quadrupole time-of-flight mass spectrometry (LC 1260 QTOF/MS 6550; Agilent, Santa Clara, CA) confirmed the presence of the medications the patient reported receiving, and additionally fentanyl (15.2 ng/mL) and U-47700 (7.6 ng/mL). In this case report, street Norco purchased in Central California resulted in altered mental status requiring naloxone reversal because of fentanyl and the novel synthetic opioid U-47700. Because these compounds are not detected by routine urine drug testing and physical examination findings are similar to those of a traditional opioid toxidrome, emergency providers should use the patient's history and other circumstantial details to aid in diagnosis. In cases with suspicion of opioid or opioid analogue cause, we recommend that emergency providers contact their local poison control center, medical toxicologist, or public health department to aid in the investigation.
Background: In the last decade there has been a worldwide surge in the recreational abuse of novel psychoactive substances, particularly amphetamine derivatives and synthetic cannabinoids. Synthetic opioids such as AH-7921, MT-45, and U-47700, with structures distinct from those ever used therapeutically or described recreationally, have also recently emerged.
Case details: We report a patient who suffered respiratory failure and depressed level of consciousness after recreationally using a novel synthetic opioid labeled U-47700. A single dose of naloxone administered by paramedics completely reversed his opioid poisoning. Comprehensive laboratory analysis confirmed the presence of a novel synthetic opioid and excluded other drugs. The drug used appeared to have caused a false positive benzodiazepine result on the initial urine drugs of abuse panel.
Conclusion: The case we describe of toxicity from the synthetic opioid labeled U-47700 highlights the emerging trend of novel synthetic opioid abuse.
Background:
Novel substances often referred to as "designer drugs" have emerged as drugs of abuse, and recognition of these is difficult as routine blood and urine screening tests do not detect these agents. U-47700 is a synthetic selective μ-opioid agonist that can be bought online for as little as $40 per gram. We report two patients presenting after insufflation of U-47700, with subsequent confirmation of this substance in urine samples.
Case details:
A 26-year-old man and 24-year-old woman insufflated a substance they believed to be "synthetic cocaine." The man was found down with cyanosis and agonal respirations. He was intubated and taken to hospital where he recovered well with supportive care. The woman presented with anxiety, tremors and drowsiness and was admitted for observation. Urine samples from both patients were analyzed using GC/MS/MS and LC/QToF, and U-47700 was isolated in both cases. No other opioids were detected.
Discussion:
These cases are concerning because U-47700 is a relatively new agent that is easy to obtain over the internet and has the potential to cause significant morbidity and mortality.
The search for synthetic opioids as alternatives to opium-based derivatives has provided an important impulse to drug development around the globe. An important goal in the systematic evaluation of new drug candidates is the identification of compounds that provide a more favorable side-effect profile, which includes reduced dependence-producing properties and abuse liability. A rich source of information about these research efforts can be found in the scientific literature. However, the exploration of these important discoveries has also been increasingly mined by largescale producers of these materials, which are then offered for sale. These so-called ‘research chemicals’ or new psychoactive substances (NPS)[1] have created challenges to policy makers, clinicians, and law enforcement around the world.[2]
Recent examples of synthetic opioids that emerged as NPS on the market, and which were associated with severe cases of adverse effects, include 3,4-dichloro-N-
{[1-(dimethylamino)cyclohexyl]methyl}benzamide (AH-7921), 1-cyclohexyl-4-(1,2-
diphenylethyl)piperazines (MT-45) and N-phenyl-N-[1-(2-phenylethyl)piperidin-4-
yl]acetamide (acetylfentanyl), respectively (Figure 1). Following the recommendation provided by the World Health Organization’s Expert Committee on Drug Dependence
(ECDD),[3] AH-7921 was placed in Schedule I of the 1961 Single Convention, as amended by the 1972 Protocol in 2015.[4] Furthermore, ECDD’s recommendation to place MT-45 into Schedule I and acetylfentanyl in Schedules I and IV of the same Convention[5] have been recently confirmed by the Commission on Narcotic Drugs.[6]
-Dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) (Figure
1) has recently emerged on the market and can be purchased from various Internet retailers and is a structural isomer of AH-7921 (Figure 1). The preparation of U-
47700 and other derivatives was disclosed by the Upjohn Company in the 1970s[7] followed by the recognition that U-47700 showed increased analgesic properties and morphine-like behavioural features in mice compared to morphine itself.[8,9] The presence of two chiral centres gives rise to a cis- and trans- racemic mixture with the trans-form being advertised for sale. Binding studies also revealed that U-47700 displayed an appreciable selectivity for the μ-opioid receptor over the −opioid receptor.[10,11] A variety of cyclohexyl trans-1,2-diamines have been found to be potent analgesics and the vicinal 1,2-diamine pattern has provided access to a large range of substances with diverse biological activities.[12-14]
Since U-47700 did not progress to clinical trials, there is no direct clinical information pertaining to its effects. Keeping in mind the various limitations that may be associated with descriptions obtained from self-reporting users, its effects have been described with various positive and negative symptoms but appeared to be essentially comparable to other opioids. Specifically, euphoria was reported in individuals, sometimes being short-lived, as well as general lift in mood with these desired effects being experienced in waves. The negative effects were also opioid based, including nausea with some users describing respiratory depression. For some users, U-47700 had a shorter duration of action and the urge to keep re-dosing was stated as being very high.
A 30-year old man was found dead in his home after inhaling fumes of a powder burned on aluminum foil. Blood and urine were taken by the medical examiner during the external body examination and submitted to the laboratory for a comprehensive systematic toxicological analysis. A toxic fentanyl level of 10.9 μg/L was measured in the subclavian blood. Police investigation revealed that the man searched the internet for information on new psychotropic substances, among others including U-47700. A powder found in the victims home was transferred to the laboratory for analysis, in which trace amounts of fentanyl (0.0035% m/m) and U-47700 (0.0012% m/m) were identified by gas chromatography mass spectrometry.
MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) is just one of the many novel psychoactive substances (NPS) to have reached
the recreational drug market in the twenty-first century; it is however, one of the first designer opioids to achieve some
degree of popularity, in a market currently dominated by synthetic cannabinoids and designer stimulants. A single fatality
involving MT-45 and etizolam is described. A method for the quantitation of MT-45 in whole blood using liquid chromatography–tandem
mass spectrometry was developed and validated. The linear range was determined to be 1.0–100 ng/mL with a detection limit
of 1.0 ng/mL, and the method met the requirements for acceptable linearity, precision and accuracy. After analyzing the sample
on dilution and by standard addition, the concentration of MT-45 in the decedent's blood was determined to be 520 ng/mL, consistent
with other concentrations of MT-45 reported in drug-related fatalities. Etizolam was present at a concentration of 35 ng/mL.
This case illustrates the importance of considering non-traditional drugs in unexplained apparent drug-related deaths.
AH-7921 is a synthetic μ-opioid agonist, approximately equipotent with morphine. We report the death of two young individuals after ingestion of AH-7921 in combination with other psychoactive drugs. In the first case a young man died shortly after ingesting Internet drugs. Toxicological analysis of post mortem peripheral blood revealed AH-7921 (0.43mg/L), 2-FMA (0.0069mg/L) and 3-MMC (0.0021mg/L) as well as codeine (0.42mg/L), codeine-6-glucuronide (0.77mg/L) and acetaminophen (18.7mg/L). The second case involved a young female found dead at home. The only positive finding at medicolegal autopsy was needle marks. Toxicological analysis revealed AH-7921 (0.33mg/L), methoxetamine (MXE) (0.064mg/L), etizolam (0.27mg/L), phenazepam (1.33mg/L), 7-aminonitrazepam (0.043mg/L), diazepam (0.046mg/L), nordiazepam (0.073mg/L), and oxazepam (0.018mg/L) in blood. In both cases intoxication with AH-7921 in combination with other psychoactive drugs was considered to be the cause of death.
A case is presented of a 19-year-old white male who was found dead in bed by a friend. While no anatomic cause of death was observed at autopsy, toxicological analysis of his blood identified AH-7921, a synthetic opioid. AH-7921 was isolated by liquid-liquid extraction into n-butyl chloride from alkalinized samples. Extracts were analyzed and quantified by gas chromatography mass spectrometry in selected ion monitoring mode. The heart blood had an AH-7921 concentration of 3.9 mg/L and the peripheral blood concentration was 9.1 mg/L. In addition to the blood, all submitted postmortem specimens including urine, liver, kidney, spleen, heart, lung, brain, bile and stomach content were quantified. The following concentrations of AH-7921 were reported: 6.0 mg/L in urine, 26 mg/kg in liver, 7.2 mg/kg in kidney, 8.0 mg/kg in spleen, 5.1 mg/kg in heart, 21 mg/kg in lung, 7.7 mg/kg in brain, 17 mg/L in bile and 120 mg/125 mL in the stomach content. The medical examiner reported that the cause of death was opioid intoxication and the manner of death was accident.
In recent years, high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection has been demonstrated to be a powerful technique for the quantitative determination of drugs and metabolites in biological fluids. However, the common and early perception that utilization of HPLC-MS/MS practically guarantees selectivity is being challenged by a number of reported examples of lack of selectivity due to ion suppression or enhancement caused by the sample matrix and interferences from metabolites. In light of these serious method liabilities, questions about how to develop and validate reliable HPLC-MS/MS methods, especially for supporting long-term human pharmacokinetic studies, are being raised. The central issue is what experiments, in addition to the validation data usually provided for the conventional bioanalytical methods, need to be conducted to confirm HPLC-MS/MS assay selectivity and reliability. The current regulatory requirements include the need for the assessment and elimination of the matrix effect in the bioanalytical methods, but the experimental procedures necessary to assess the matrix effect are not detailed. Practical, experimental approaches for studying, identifying, and eliminating the effect of matrix on the results of quantitative analyses by HPLC-MS/MS are described in this paper. Using as an example a set of validation experiments performed for one of our investigational new drug candidates, the concepts of the quantitative assessment of the "absolute" versus "relative" matrix effect are introduced. In addition, experiments for the determination of, the "true" recovery of analytes using HPLC-MS/MS are described eliminating the uncertainty about the effect of matrix on the determination of this commonly measured method parameter. Determination of the matrix effect allows the assessment of the reliability and selectivity of an existing HPLC-MS/MS method. If the results of these studies are not satisfactory, the parameters determined may provide a guide to what changes in the method need to be made to improve assay selectivity. In addition, a direct comparison of the extent of the matrix effect using two different interfaces (a heated nebulizer, HN, and ion spray, ISP) under otherwise the same sample preparation and chromatographic conditions was made. It was demonstrated that, for the investigational drug under study, the matrix effect was clearly observed when ISP interface was utilized but it was absent when the HN interface was employed.
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Furanyl fentanyl, butyryl fentanyl, and U-47700 in blood and urine of drivers arrested for driving under the influence of drugs (DUID)
- R L Hartman
- M E Salamone
- D J Nemeth
- T M Gallagher
- J S Fellows
- L M Kadlec
- J M Beno
- RL Hartman