Authentic Urine Samples Research Articles

B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine

Abstract Background Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL. Methods The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives). Conclusions The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

Human phase-I metabolism of three synthetic cannabinoids bearing a cumyl moiety and a cyclobutyl methyl or norbornyl methyl tail: Cumyl-CBMEGACLONE, Cumyl-NBMEGACLONE, and Cumyl-NBMINACA.

Synthetic cannabinoid receptor agonists (SCRAs) continue to show high prevalence on the new psychoactive substances drug market. Around 2019-2020, new SCRAs bearing a cumyl moiety emerged: Cumyl-CBMEGACLONE and Cumyl-NBMEGACLONE, carrying a cyclobutyl methyl (CBM) and a norbornyl methyl moiety (NBM) attached to the γ-carbolinone core. These were followed by Cumyl-NBMINACA, the indazole carboxamide analog of Cumyl-NBMEGACLONE. The study aimed at evaluating the human phase-I metabolism of these compounds and at identifying suitable urinary markers to prove their consumption. After enzymatic hydrolysis, 14 authentic urine samples (eight for Cumyl-CBMEGACLONE, four for Cumyl-NBMEGACLONE, and two for Cumyl-NBMINACA) were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Results were compared with in vitro metabolites generated by pooled human liver microsomes incubation. Fifteen human phase-I metabolites were identified for Cumyl-CBMEGACLONE, nine for Cumyl-NBMEGACLONE, and thirteen for Cumyl-NBMINACA. The main in vivo metabolites were built by monohydroxylation, dihydroxylation, or trihydroxylation. The following urinary biomarkers are suggested for detecting the consumption of the investigated SCRAs: products of monohydroxylation at the CBM and at the core for Cumyl-CBMEGACLONE; two products of monohydroxylation at the norbonyl methyl tail for Cumyl-NBMEGACLONE; and metabolites built by dihydroxylation at the NBM substructure and by an additional hydroxylation at the cumyl moiety for Cumyl-NBMINACA.

Direct and selective alkylation of indazole-3-carboxylic acid for the preparation of synthetic cannabinoids and metabolites

The most common method for the synthesis of synthetic cannabinoids with an indazole core utilizes methyl indazole-3-carboxylate as a starting material. However, this method commonly suffers from poor selectivity and low yield. In the current work, a method using indazole-3-carboxylic acid as the starting material was developed and successfully applied in the synthesis of nine synthetic cannabinoids and six of their metabolites. The method provided selective alkylation at the N1-position and resulted in overall yields in the range of 51–96 %. Five of the synthetic cannabinoids have not been reported in the literature and were synthesized in a proactive attempt to predict future SCs. All of the synthesized metabolites have previously been encountered in either in vitro studies or authentic urine samples. Hence, the method proved to be useful for production of SC metabolites, which are relevant for forensic toxicology. All synthesized compounds were characterized with NMR and LC-QTOF-HRMS.

High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine

BackgroundGas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the δ13C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for δ13C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 °C) because at 200 °C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. ResultAn HT-LC-IRMS method capable of determining the δ13C values of four steroids (i.e., testosterone (T), 5α-androstane-3α,17β-diol (ααβ), 5β-androstane-3α,17β-diol (βαβ) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 ‰ (ααβ and βαβ) to 0.49 ‰ (T), and the detection limit was set at 10 ng mL−1 (T, ααβ+βαβ). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. SignificanceHT-LC-IRMS can be used to determine δ13C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.

Human metabolism of the semi-synthetic cannabinoids hexahydrocannabinol, hexahydrocannabiphorol and their acetates using hepatocytes and urine samples.

Hexahydrocannabinol (HHC), hexahydrocannabiphorol (HHCP) and their acetates, HHC-O and HHCP-O, respectively, are emerging in Europe as alternatives to tetrahydrocannabinol (THC). This study aimed to elucidate the metabolic pathways of the semi-synthetic cannabinoids HHC, HHCP, HHC-O and HHCP-O from incubation with human hepatocytes. The metabolites of HHC were also identified in authentic urine samples. HHC, HHCP, HHC-O and HHCP-O were incubated with primary human hepatocytes for 1, 3 and 5 h. Authentic urine samples from cases screened positive for cannabis in blood using ELISA but confirmed negative were analysed both non-hydrolysed and hydrolysed for HHC metabolites. Potential metabolites were identified using ultra-high performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight mass spectrometer (QToF-MS). HHC and HHCP were primarily metabolised through monohydroxylation (monoOH), followed by oxidation to a carboxylic acid metabolite. HHC-O and HHCP-O were rapidly metabolised to HHC and HHCP, respectively. In authentic urine samples, 18 different metabolites were identified, and 99.3% of hydroxylated metabolites were glucuronidated. 11-OH-HHC, 5'OH-HHC and another metabolite with a monoOH on the side chain were the only metabolites present in all 16 urine samples. The metabolism of HHC and HHCP were similar, although the longer alkyl side chain of HHCP (heptyl) led to greater hydroxylation on the side chain than HHC (pentyl). The use of HHC and HHCP can be differentiated from the use of THC and other phytocannabinoids, but the use of the acetate analogues may not be differentiable from their non-acetate analogues.

Characterization of iso-LSD metabolism using human liver microsomes in comparison to LSD and its applicability as urinary biomarker for LSD consumption.

Urinalysis of lysergic acid diethylamide (LSD) poses a challenge due to its rapid metabolism, resulting in little to no LSD detectable in urine. Instead, its primary metabolite, 2-oxo-3-hydroxy-LSD, is predominantly detected. In this study, we observed several urine profiles with iso-LSD detected together with 2-oxo-3-hydroxy-LSD. Iso-LSD is derived from illicit preparation of LSD as a major contaminant, and it was detected at higher abundance than LSD and 2-oxo-3-hydroxy-LSD in certain urine samples. Therefore, the metabolism of iso-LSD and its potential as a viable urinary biomarker for confirming LSD consumption is of interest. For metabolism studies, LSD and iso-LSD were incubated in human liver microsomes (HLMs) at 0 min, 60 min and 120 min to characterize their metabolites using LC-QTOF-MS. For urinary analysis, 500 µL of urine samples underwent enzymatic hydrolysis and clean-up using supported-liquid extraction (SLE) prior to analysis by LC-QTOF-MS. From HLM incubation study of LSD, the metabolites detected were dihydroxy-LSD, 2-oxo-LSD, N-desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD with LSD levels decreasing significantly throughout all time points, consistent with the existing literatures. For HLM study of iso-LSD, metabolites eluting at retention times after the corresponding metabolites of LSD were detected, with iso-LSD levels showing only a slight decrease throughout all time points, due to a slower metabolism of iso-LSD compared to LSD. These findings corroborate with the urinalysis of 24 authentic urine samples, where iso-LSD with 2-oxo-3-hydroxy-LSD was detected in the absence of LSD. Based on our findings, iso-LSD is commonly detected in urine (18 out of 24 samples) sometimes with traces of possible 2-oxo-3-hydroxy-iso-LSD. The slower metabolism and high detection rate in urine make iso-LSD a viable urinary biomarker for confirming LSD consumption, especially in the absence of LSD and/or 2-oxo-3-hydroxy-LSD.

Detection of ADB-4en-PINACA metabolite in the authentic urine samples

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ADB-HEXINACA-a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB-HEXINACA, an SCRA containing a hexyl-substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-qToF-MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB-HEXINACA. Thirty-two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB-HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB-HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB-HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

In vitro, invivo metabolism and quantification of the novel synthetic opioid N-piperidinyl etonitazene (etonitazepipne).

N-piperidinyl etonitazene (etonitazepipne) is a newly synthesized opioid related to the 2-benzylbenzimidazole analog class. Etonitazepipne has been formally notified and placed under intensive monitoring in Europe in January 2022. Nitazenes have high affinity at µ-opioid receptor (MOR). Etonitazepipne, specifically shows a EC50 of 2.49 nM, suggesting about 50 times higher potency combined with higher efficacy compared to morphine. Antinociceptive potency l ('hot plate test' with rats) was 192-fold greater than that of morphine. Here we report on a post-mortem case involving etonitazepipne and its quantification using a standard addition method (SAM) through liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, characterization and identification of phase I human metabolites using invitro assay based on pooled human liver microsomes (pHLM) was performed along with the analysis of authentic urine samples by means of high-performance liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The concentration of etonitazepipne in post-mortem blood and urine was 8.3 and 11 ng/mL, respectively. SAM was validated by assessing the following parameters: intraday and interday repeatability, matrix effect and recovery rate in post-mortem blood. A total of 20 and 14 metabolites were identified after pHLM incubation and urine analysis, respectively. Most pronounced invitro and invivo transformations were O-deethylation, hydroxylation, ketone reduction, and combinations thereof. Considering small traces of the parent drug often found in real cases, the identification of metabolic biomarkers is crucial to identify exposure to this drug. O-deethylated, oxidated metabolites, and combination thereof are proposed as urinary biomarkers along with the parent compound.

Development of new matrix reference materials for quantitative urine analysis in drug tests.

Accurate quantitative analyses require standardized methods to control and improve the analytical process in the laboratory. The availability of urine reference materials (RMs) may offer a feasible option to improve the accuracy of urine analysis and to control matrix effects. This paper presents the complete process of the development of matrix RMs in urine, including sample preparation, homogeneity, and stability studies, as well as uncertainty assessment. A freeze-drying process was developed, and freeze-dried human and pig urine samples were prepared and verified to have comparable homogeneity to liquid samples and higher stability than liquid human, pig, and artificial urine samples at 4℃ or room temperature and under extreme conditions. A total of 21 authentic urine samples from August 2022 were measured with freeze-dried RMs and spiked urine samples, and the reliability of the quantification of the RMs was compared. The freeze-dried human urine matrix RM appeared to be an excellent tool for daily quality control, as it showed high stability and gave the most consistent results with spiked samples.

Copper(II) phthalocyanine as an electrocatalytic electrode for cathodic detection of urinary tryptophan.

Herein, we introduce a novel method for tryptophan detection via a reduction reaction facilitated by its interaction with a copper(II) phthalocyanine (CuPc) electrocatalytic electrode. This method addresses challenges associated with the susceptibility of the oxidation response to interference from various species when measuring tryptophan in bodily fluids. The reduction currents exhibit a linear increase with tryptophan concentrations in two ranges: 0.0013-0.10 mM and 0.10-1.20 mM, with the sensitivities of 14.7 ± 0.5 μA mM-1 and 3.5 ± 0.1 μA mM-1, respectively. The limit of detection (LOD, 3SB/m) is determined to be 0.39 μM. The sensor exhibits excellent reproducibility, with the relative standard deviation of <5%. Application of the sensor to authentic urine samples yields a % recovery of 101 ± 4%.

B-330 Homogeneous Enzyme Immunoassay for Hydrocodone

Abstract Background Hydrocodone is a semi-synthetic derivative of codeine and produces opioid-like effects similar to morphine. Hydrocodone (Trade names: Vicodin, Lortab, Hycodan, Vicoprofen) is one of the most frequently prescribed opioids in the U.S. as an antitussive (cough suppressant) and narcotic analgesic agent for the treatment of moderate to severe pain. Its ease of prescription and opioid-like effects has led to widespread drug diversion and abuse. The two commercially available homogeneous immunoassays for the detection of hydrocodone in urine at a cutoff of 300 ng/mL suffer undesirable cross-reactivity to oxycodone and morphine at concentrations below 25 000 ng/mL. ARK Diagnostics has developed the ARK Hydrocodone Assay to detect hydrocodone at a cutoff of 300 ng/mL with no cross-reactivity to oxycodone, morphine, and codeine at concentrations below 100 000 ng/mL. Methods The ARK™ Hydrocodone Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff of 300 ng/mL and semi-quantitative range up to 800 ng/mL. The performance of this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, spiked recovery, specificity, Histogram Overlap Analysis of ±25% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 3.9% to 9.9%CV. Spiked recovery of hydrocodone ranged from 86.2% (720 ng/mL) to 102.9% (240 ng/mL). The active metabolite, hydromorphone, showed an approximate equivalence to the 300 ng/mL hydrocodone cutoff at 299 ng/mL (100.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 96.9% (98 positives and 128 negatives). Conclusion The ARKTM Hydrocodone Assay measures hydrocodone and its metabolite hydromorphone in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

Simultaneous detection of 87 synthetic cannabinoids and their metabolites in urine samples by UHPLC-MS/MS and application to 109 authentic forensic cases

Synthetic cannabinoids (SCs) have emerged on the designer drug market at an alarming rate in recent years. Consequently, the current methods for detecting SCs and their metabolites in urine require further optimization. An efficient and rapid quantitative UHPLC-MS/MS method was developed for the detection of 87 SCs and their metabolites in urine. Urine (0.1 mL) was incubated for 30 min at 55 °C by adding 10 μL β-glucuronidase and then processed by acetonitrile protein precipitation. A Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm,1.8 µm) was used for chromatographic separation. Mobile phase A was 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile in water, and mobile phase B was acetonitrile. The limits of detection (LOD) ranged from 0.02 ng/mL to 2 ng/mL and the limits of quantitation (LOQ) ranged from 0.05 ng/mL to 5 ng/mL. The accuracy was in the range of 88.6% to 112%. The relative standard deviations of the intraday and interday imprecisions were in the range of 1.09% to 18.7%, and the recoveries were in the range of 16.4% to 102% and the matrix effect ranged from 70.4% to 114%. After full validation, the method showed good selectivity, accuracy, precision, linearity, and stability within the calibration range. It's simple, rapid and covers a variety of analytes in forensic cases. It was successfully applied to urine samples from 109 suspected SCs users. The most frequently metabolites detected in authentic urine samples in this study were MDMB-4en-PINACA butanoic acid metabolite and ADB-BUTINACA acid metabolite.

Methylene blue molecularly imprinted polymer for melatonin determination in urine and saliva samples.

A highly sensitive and rapid electrochemical sensor was developed for detecting melatonin using a molecularly imprinted polymer (MIP) with methylene blue as the functional monomer and melatonin as the template. The MIP was synthesized via a simple electropolymerization process that did not require an initiating reagent. The sensor demonstrated good selectivity for melatonin against common interferences such as lactate, cytosine, cytidine, urea, ascorbic acid, creatine, creatinine, serotonin, and tryptophan. Melatonin detection was achieved ata potential of 0.60V vs. Ag/AgCl with a sensitivity of 138.8 ± 4.7 µA µM‒1 in the linear range 0.097 - 200µM and a limit of detection of 29nM (3SB/m). The sensor exhibited excellent reproducibility and repeatability for both within (intra) and between (inter) electrodes (%RSD < 3% for n = 3). The sensor was applied to authentic urine and saliva samples with recoveries of 103 ± 1% and 102 ± 1%, respectively.

Pharmacological profile, phase I metabolism, and excretion time profile of the new synthetic cathinone 3,4-Pr-PipVP.

1-(2,3-Dihydro-1H-inden-5-yl)-2-(piperidin-1-yl)pentan-1-one (3,4-Pr-PipVP), a novel synthetic cathinone (SCat), was first identified in 2022 in Germany. The product was marketed as 1-(bicyclo[4.2.0]octa-1,3,5-trien-3-yl)-2-(pyrrolidin-1-yl)pentan-1-one (3,4-EtPV), a substance not covered by the German New Psychoactive Substances Act (NpSG). Although originally intended to be an exploratory new synthetic cathinone containing the novel bicyclo[4.2.0]octatrienyl function, the compound was subsequently confirmed to contain an indanyl ring system scheduled under generic legislation like the NpSG. However, it is one of only a few marketed SCats carrying a piperidine ring. Inhibition experiments involving norepinephrine, dopamine, and serotonin transporters showed that 3,4-Pr-PipVP was a low potency blocker at all three monoamine transporters compared to related substances such as MDPV. Additionally, pharmacokinetic data were collected from pooled human liver microsomes incubations and from the analysis of authentic urine samples received after oral administration of 5mg 3,4-Pr-PipVP hydrochloride. Phase I metabolites were tentatively identified in vitro and in vivo using liquid chromatography-time-of-flight mass spectrometry. Main metabolites were formed by metabolic reduction of the carbonyl function with and without additional hydroxylations at the propylene bridge of the molecule. Keto-reduced H2 -3,4-Pr-PipVP and H2 -piperidine-OH-3,4-Pr-PipVP as well as aryl-OH-3,4-Pr-PipVP, and indanyl-OH-piperidine-OH-3,4-Pr-PipVP are suggested as most suitable biomarkers for the detection of 3,4-Pr-PipVP since they were detected for much longer than the parent compound. 3,4-Pr-PipVP could be detected for up to 21 h whereas its metabolites were detectable for up to about 4 days.

Development and validation of a liquid chromatography–tandem mass spectrometry method of a panel of commonly prescribed benzodiazepines in urine samples

AbstractBenzodiazepines are one of the most prescribed for therapeutic purposes and the most abused drugs. Therefore, liquid chromatography–tandem mass spectrometry–based methods that analyze several benzodiazepines simultaneously are required. In this study, we developed a simple, full validated in‐house method in a single‐triple quadrupole platform SCIEX QTRAP 4500 system for the simultaneous detection and quantification of the 10 most prescribed benzodiazepines within a 10‐min run time. The linear range was 12.5–500 ng/mL. All limits of detection were below 4.03 ng/mL and limits of quantitation below 12.22 ng/mL, except clobazam (7.27 and 22.04 ng/mL, respectively). Both the limit of detection and quantitation were lowest for diazepam (0.95 and 2.89 ng/mL, respectively). The method was evaluated for accuracy, precision, carry‐over, recovery, stability, and matrix effects, and all validation studies met internationally acceptable criteria. The applicability of the method was proven by analyzing authentic urine samples, internal, and third‐party external quality samples. This precise, accurate, shorter total run time and ease of urine sample analysis method is applicable in detecting most prescribed benzodiazepines and their metabolites even in glucuronide forms.

Application of Dried Urine Spots for Non-Targeted Quadrupole Time-of-Flight Drug Screening.

The use of dried urine spots (DUS) can simplify sample handling, shipment and storage when compared to liquid urine samples. To prepare DUS, a small amount of urine is pipetted on a filter paper card. The subsequent drying of the specimen can prevent the post-sampling formation or degradation of substances (e.g., caused by bacteria). To evaluate the potential of DUS screening, 17 authentic urine samples, containing a broad range of substances, were extracted and analyzed on a Sciex TripleTOF® 5600+ System using a non-targeted screening and library searching approach. The screening results were compared to the analysis of the same urine sample in liquid form, using the same high-resolution liquid chromatography--quadrupole time-of-flight mass spectrometry method. More than 65 different legal and illegal drugs were successfully identified within the investigated 17 urine samples using the DUS screening approach. When compared to the analysis of liquid urine, the following compounds could not be identified: 1x ecgonine methyl ester, 1x nicotine, 1x promazine and 1x 11-nor-9-carboxy-∆9-tetrahydrocannabinol. Overall, 95.2% of the target substances that have been detected in liquid urine were identified correctly using the DUS approach. In conclusion, DUS screening offers a simple, cost-effective and easier sample handling alternative to the traditional use of liquid urine and provides the detection of the most important substances for forensic requirements. Furthermore, the DUS sample preparation can be fully automated (sample documentation, internal standard application and extraction).

Advances in urinary biomarker research of synthetic cannabinoids.

New psychoactive substances (NPS) are chemical compounds designed to mimic the action of existing illicit recreational drugs. Synthetic cannabinoids (SCs) are a subclass of NPS which bind to the cannabinoid receptors, CB1 and CB2, and mimic the action of cannabis. SCs have dominated recent NPS seizure reports worldwide. While urine is the most common matrix for drug-of-abuse testing, SCs undergo extensive Phase I and Phase II metabolism, resulting in almost undetectable parent compounds in urine samples. Therefore, the major urinary metabolites of SCs are usually investigated as surrogate biomarkers to identify their consumption. Since seized urine samples after consuming novel SCs may be unavailable in a timely manner, human hepatocytes, human liver microsomes and human transporter overexpressed cell lines are physiologically-relevant in vitro systems for performing metabolite identification, metabolic stability, reaction phenotyping and transporter experiments to establish the disposition of SC and its metabolites. Coupling these in vitro experiments with in vivo verification using limited authentic urine samples, such a two-pronged approach has proven to be effective in establishing urinary metabolites as biomarkers for rapidly emerging SCs.

Portable Prussian Blue-Based Sensor for Bacterial Detection in Urine

Bacterial infections can affect the skin, lungs, blood, and brain, and are among the leading causes of mortality globally. Early infection detection is critical in diagnosis and treatment but is a time- and work-consuming process taking several days, creating a hitherto unmet need to develop simple, rapid, and accurate methods for bacterial detection at the point of care. The most frequent type of bacterial infection is infection of the urinary tract. Here, we present a wireless-enabled, portable, potentiometric sensor for E. coli. E. coli was chosen as a model bacterium since it is the most common cause of urinary tract infections. The sensing principle is based on reduction of Prussian blue by the metabolic activity of the bacteria, detected by monitoring the potential of the sensor, transferring the sensor signal via Bluetooth, and recording the output on a laptop or a mobile phone. In sensing of bacteria in an artificial urine medium, E. coli was detected in ~4 h (237 ± 19 min; n = 4) and in less than 0.5 h (21 ± 7 min, n = 3) using initial E. coli concentrations of ~103 and 105 cells mL-1, respectively, which is under or on the limit for classification of a urinary tract infection. Detection of E. coli was also demonstrated in authentic urine samples with bacteria concentration as low as 104 cells mL-1, with a similar response recorded between urine samples collected from different volunteers as well as from morning and afternoon urine samples.

Human phase-I metabolism and prevalence of two synthetic cannabinoids bearing an ethyl ester moiety: 5F-EDMB-PICA and EDMB-PINACA.

Around 2017, with the appearance of 5F-EDMB-PINACA, synthetic cannabinoids (SCs) carrying an ethyl ester moiety at the linked group started spreading on the market of new psychoactive substances (NPS). In 2020 and 2021, the indole analog of 5F-EDMB-PINACA (5F-EDMB-PICA) and the non-fluorinated analog of this compound (EDMB-PINACA) were analytically characterized. Here, we present suitable urinary markers to prove the consumption of these two ethyl analogs. Ten authentic urine samples for each compound were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Anticipated phase-I metabolites detected in urine samples were confirmed in vitro by applying a pooled human liver microsomes (pHLM) assay. Prevalence data were obtained from urines collected for abstinence control and submitted to a screening method for SC metabolites. Ten phase-I metabolites of 5F-EDMB-PICA and 18 of EDMB-PINACA were detected by LC-qToF-MS analysis of authentic urine specimens. The main in-vivo metabolites were built by ester hydrolysis, often coupled to further metabolic processes. Investigation of phase-I biotransformation led to the identification of ester hydrolysis, monohydroxylation, and defluorination products as the most suitable urinary biomarkers for 5F-EDMB-PICA. Metabolites formed by ester hydrolysis coupled to ketone formation and by monohydroxylation are suggested for the detection of EDMB-PINACA. From October 1, 2020 to February 1, 2022, among positive urine samples, 5.4% and 10.1% tested positive 5F-EDMB-PICA and EDMB-PINACA, respectively. Due to common metabolites shared among structurally related SCs, the unequivocal detection of their consumption remains challenging for forensic laboratories and requires sensitive methods to monitor multiple metabolites, ideally including highly specific species.