Monohydroxy Metabolites Research Articles

Association between urinary polycyclic aromatic hydrocarbons and risk of metabolic associated fatty liver disease.

To investigate the effect of urinary PAHs on MAFLD. The study included 3,136 adults from the National Health and Nutrition Examination Survey (NHANES) conducted between 2009 and 2016. Among them, 1,056 participants were diagnosed with MAFLD and were designated as the case group. The analysis of the relationship between monohydroxy metabolites of seven PAHs in urine and MAFLD was carried out using logistic regression and Bayesian kernel regression (BKMR) models. In single-pollutant models, the concentration of 2-hydroxynaphthalene (2-OHNAP) was positively correlated with MAFLD (OR = 1.47, 95% CI 1.18, 1.84), whereas 3-hydroxyfluorene (3-OHFLU) and 1-hydroxypyrene (1-OHPYR) demonstrated a negative correlation with MAFLD (OR = 0.59, 95% CI 0.48 0.73; OR = 0.70, 95% CI 0.55, 0.89). Conversely, in multi-pollutant models, 2-OHNAP, 2-hydroxyfluorene (2-OHFLU), 2-hydroxyphenanthrene, and 3-hydroxyphenanthrene (2&3-OHPHE) displayed positive correlations with MAFLD (OR = 6.17, 95% CI 3.15, 12.07; OR = 2.59, 95% CI 1.37, 4.89). However, 3-OHFLU and 1-OHPYR continued to exhibit negative correlations with MAFLD (OR = 0.09, 95% CI 0.05, 0.15; OR = 0.62, 95% CI 0.43, 0.88). Notably, the BKMR analysis mixtures approach did not indicate a significant joint effect of multiple PAHs on MAFLD, but identified interactions between 3-OHFLU and 2-OHFLU, 1-OHPYR and 2-OHFLU, and 1-OHPYR and 3-OHFLU. No significant association was found between mixed PAHs exposure and the risk of MAFLD. However, interactions were observed between 3-OHFLU and 2-OHFLU. Both 2-OHFLU and 2&3-OHPHE exposure are significant risk factors for MAFLD, whereas 3-OHFLU is a key protective factor for the disease.

Identification and yield of metabolites of chlorinated paraffins incubated with chicken liver microsomes: Assessment of their potential to convert into metabolites

Chlorinated paraffins (CPs) are emerging environmental pollutants. Although metabolism has been shown to affect the differential accumulation of short-chain (SCCPs), medium-chain (MCCPs) and long-chain (LCCPs) CPs in birds, CP metabolites have rarely been reported and the extent to which they are formed is still unclear. In this study, single and mixed CP standards were incubated with chicken liver microsomes in vitro to study the generation of CP metabolites. Putative aldehyde/ketone and carboxylic acid metabolites identified by mass spectroscopy data were shown to be false positive results. Phase I metabolism of CPs first formed monohydroxylated ([M-Cl+OH]) and then dihydroxylated ([M-2Cl+2OH]) products. The yields of monohydroxylated metabolites of CPs decreased with increasing carbon chain length and chlorine content at the initial stage of reaction. Notably, the yield of monohydroxylated metabolites of SCCPs with 51.5% Cl content reached 21%, and that of 1,2,5,6,9,10-hexachlorodecane (C10H16Cl6) was as high as 71%. Thus, monohydroxy metabolites of CPs in birds should not be ignored, especially those of SCCPs. This study provides important data that could support improvements to the ecological/health risk assessment of CPs.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl using LC-QTOF-HRMS together with synthesized references.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5μM) with cryopreserved hepatocytes for up to 5h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the β position of the phenylpropanoyl moiety was highly favored; β-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

Associations between urinary monohydroxylated polycyclic aromatic hydrocarbons and unexplained recurrent spontaneous abortion and health risk assessment in a large case-control study in China

Polycyclic aromatic hydrocarbons (PAHs) are a group of environmental endocrine disruptors with known carcinogenic, reproductive, and developmental toxicity. Important knowledge gaps remain regarding the relationship between PAH exposure and unexplained recurrent spontaneous abortion (URSA). In the present study, twelve monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) were measured in the urine of 413 URSA cases and 434 controls. The main OH-PAHs measured in this study were monohydroxy metabolites of naphthalene, followed by fluorene and phenanthrene. After the creatinine correction, the median concentration of urinary OH-PAHs in the control group (17.4 μg/g Creatinine) was higher than that in the case group (14.2 μg/g Creatinine). There was no positive relationship between PAH exposure and URSA using binary logistic regression analysis. Among 847 Chinese women of childbearing age, residential environment, type of drinking water, and education level were the influencing factors of PAH exposure. The health risk assessment showed that over 98% of women had a carcinogenic risk with carcinogenic risk values above the acceptable level (10−6). Although this large-scale case-control study did not observe an association between PAH exposure and URSA, more attention should be paid to the high carcinogenic risk due to PAH exposure in women of reproductive age.

Associations between concentrations of serum α-klotho and selected urinary monohydroxy metabolites of polycyclic aromatic hydrocarbons: data for US adults aged 40-79years.

For the first time, the associations between urinary concentrations of oxidant polycyclic aromatic hydrocarbon (PAH) metabolites and serum concentrations of anti-oxidant α-klotho were estimated for US adults aged 40-79years. Multivariate regression models with α-klotho as dependent variable and one of the urinary metabolite of PAH as independent variables were fitted. In the absence of albuminuria and normal (eGFR > 90mL/min/1.73 m2) kidney function, 10% increases in concentrations of 2-hydroxynaphthalene, 9-hydroxyfluorene, and ∑PAH were associated with 0.25%, 0.32%, and 0.19% decreases in serum α-klotho concentrations. In the absence of albuminuria and near normal (60 < = eGFR < 90mL/min/1.73 m2) kidney function, 10% increases in concentrations of 1-hydroxynaphthalene, 9-hydroxyfluorene, 1-hydroxyphenanthrene, and ∑PAH were associated with 0.17%, 0.38%, 0.34%, and 0.18% decreases in serum α-klotho concentrations. To what degree, these mild decreases in α-klotho are a matter of concern, is a subject ripe for discussion and additional investigations. When kidney function was normal or near normal but albuminuria was present, the associations between α-klotho and different metabolites of PAH were, more or less, randomly positive or negative and none reached statistical significance. To conclude, exposure to polycyclic aromatic hydrocarbons may result in reduced concentrations of α-klotho, an antiaging protein.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists QMPSB and QMPCB (SGT-11) Including Isozyme Mapping and Esterase Activity.

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.

Characterising the exposure of Australian firefighters to polycyclic aromatic hydrocarbons generated in simulated compartment fires

Firefighters are exposed to a wide variety of chemicals including polycyclic aromatic hydrocarbons (PAHs) while attending fire scenes. The objective of this study was to understand the exposure of firefighters to PAHs when attending simulated compartment fires that consisted of either a diesel pan or particleboard fire. Firefighters remained in the compartment fires for 15min while using standard gear including self contained breathing apparatus (SCBA). Firefighters were able to remove firefighting clothing and shower within 10min of leaving the burn. Air samples were collected from inside the compartment during the fire. Twenty-six (26) firefighters participated in the study providing urine and skin wipe samples collected from the wrist and neck before and after either one of the burn types. The concentrations of PAHs were measured in skin wipes and air samples, while concentrations of monohydroxy metabolites of PAHs (OH-PAHs) were measured in urine. The concentrations of all PAHs were significantly higher (p<0.05) in the smoke layer of particleboard fires than in diesel pan fires. Correspondingly, the level of PAHs deposited on the wrists and necks of participants attending the particleboard fires was higher than those attending diesel pan fires. Urine samples from participants who attended diesel pan fires showed no significant difference (p>0.05) in the concentration of all OH-PAHs between pre-burn and post-burn. Samples from participants who attended particleboard fires, showed no significant difference (p>0.05) between 1-hydroxypyrene (1-OH-PYR) concentrations in urine pre- and post-burn. However, median concentrations of hydroxynaphthalenes (OH-NAPs), hydroxyfluorenes (OH-FLUs) and hydroxyphenanthrenes (OH-PHEs) increased significantly from 5.2, 0.44 and 0.88μgg-1 creatinine pre-burn to 12, 1.4 and 1.2μgg-1 creatinine post-burn, respectively. This suggests that in compartment burns with high concentrations of PAHs in the smoke layer, such as those created by the particleboard fires, exposure to PAHs can be observed though urinary OH-PAH metabolites. Overall, concentrations of urinary OH-PAHs were relatively low considering the potential exposure in these burns. This suggests protective equipment in combination with rapid removal of firefighting ensembles and showering are relatively effective in controlling exposure.

A novel switchable solvent liquid-phase microextraction technique based on the solidification of floating organic droplets: HPLC-FLD analysis of polycyclic aromatic hydrocarbon monohydroxy metabolites in urine samples

A novel switchable solvent liquid-phase microextraction technique, based on the solidification of floating organic droplets (SS-LPME-SFO), was developed for the pretreatment of aqueous samples.

Electrospun core-shell nanofibers as an adsorbent for on-line micro-solid phase extraction of monohydroxy derivatives of polycyclic aromatic hydrocarbons from human urine, and their quantitation by LC-MS.

A method is described for automatted on-line micro-solid phase extraction using polydopamine-modified polystyrene/silica electrospun nanofibers as an adsorbent for monohydroxy metabolites of polycyclic aromatic hydrocarbons (OH-PAHs). They were then quantified by liquid chromatography-mass spectrometry. The sorbent has a network structure, high porosity, stability, hydrophilicity and good extraction capacity. Under optimal experimental conditions, the method has (a) wide linear ranges (0.03-25ng·mL-1 for 3-hydroxyphenanthrene and 1-hydroxypyrene; and 0.05-25ng·mL-1 for 2-naphthol), (b) good precision (with relative standard deviations from 2.0 to 12.4%), (c) highenrichment factors (24-112), (d) low limits of detection (2-21pg·mL-1), and (e) acceptable recoveries from spiked samples (79.1 to 108%). The method was successfully applied for the analysis of trace OH-PAHs in urine. Graphical abstractSchematic illustration of online micro-solid phase extraction using polydopamine-modified polystyrene/silica electrospun nanofibers as adsorbent for the enrichment and determination of monohydroxy metabolites of polycyclic aromatic hydrocarbons (OH-PAHs) prior to LC-MS analysis.

Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids

A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca2+-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner.

Atypical Pharmacodynamic Properties and Metabolic Profile of the Abused Synthetic Cannabinoid AB-PINACA: Potential Contribution to Pronounced Adverse Effects Relative to Δ9-THC

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ9-THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ9-THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo, producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ9-THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana.

Urinary polycyclic aromatic hydrocarbon metabolites among 3-year-old children from Krakow, Poland

Polycyclic aromatic hydrocarbons (PAHs) are widespread in the environment and can adversely affect human health. The aim of the present study is to describe the level of PAHs exposure in children living in Kraków, one of the most polluted cities in Poland, and to determine the relationship of urinary biomarkers with environmental PAHsexposure.Urinary monohydroxy metabolites (OH-PAHs) of 20 PAHs were assessed in 218 three-year old children, of which only 10 were present in nearly all the samples: monohydroxy metabolites of naphthalene, fluorene, phenantrene and pyrene. Of the metabolites analyzed, hydroxynaphthalenes were predominant and constituted almost 73% of total excreted OH-PAHs, while 1-OH-PYRene was the least abundant (2.3% of total OH-PAHs). All measured urinary OH-PAHs were statistically significantly correlated with each other (R = 0.165–0.880) but the highest correlation coefficients with other individual OH-PAHs and with total OH-PAHs were observed for 2-OH-FLUOR.Children exposed at home to environmental tobacco smoke (ETS) had higher concentrations of fluorene and pyrene urinary metabolites compared to those without ETS exposure; and those exposed to gas-based appliances used for cooking or heating water had higher levels of fluorene and phenanthrene metabolites than children not exposed. The use of coal, wood or oil for heating was associated with elevated levels of 1-OH-PYRene. Urinary PAHs metabolites only modestly reflect high molecular weight carcinogenic PAHs exposures such as those monitored in air in the present study. None of the measured PAHs metabolites was correlated with airborne PM2.5 and only two were slightly correlated with measured higher molecular mass airborne PAHs.The average concentrations of these specific metabolites in Polish children were much higher than observed in other pediatric populations living in developed countries. Our findings suggest that to capture various sources of PAHs, in addition to 1-OH-PYRene, biomonitoring of PAHs exposure should include 2-OH-NAP and 2-OH-FLUOR.

Effect of antiepileptic drug comedication on lamotrigine concentrations.

AimTo estimate the effect size of concomitant antiepileptic therapy on the concentrations of lamotrigine, a drug often prescribed in combination with other antiepileptic drugs (AED), which can act as enzyme inducers or inhibitors.MethodsA total of 304 patients with epilepsy, aged 18-70 years, were divided into a lamotrigine monotherapy group and groups receiving lamotrigine with AEDs that act as enzyme inducers, enzyme inhibitors, or both. We compared lamotrigine monotherapy serum concentrations with those where lamotrigine was administered with a metabolic inhibitor valproate, metabolic inducers carbamazepine, oxcarbazepine, phenobarbital, phenytoin, or topiramate, and both an inducer and an inhibitor.ResultsComparison of trough lamotrigine monotherapy concentrations and lamotrigine polytherapy concentrations showed an almost similar median concentration in case of drug-inducers, and higher lamotrigine concentration in case of comedication with valproate as an inhibitor. A significant difference was confirmed after dose correction (P < 0.001). Significant positive correlations of lamotrigine trough serum concentrations with valproate were observed before and after the dose correction (r = 0.480, P < 0.001 and r = 0.561, P < 0.001, respectively). Positive correlations between the dose-corrected lamotrigine trough concentration and carbamazepine (r = 0.439; P < 0.001) or monohydroxy metabolite of oxcarbazepine (MHD) (r = 0.675; P < 0.001) were also significant.ConclusionHigher valproate levels resulted in higher inhibition potency and higher lamotrigine levels. Increased dose-corrected concentrations of inducers carbamazepine and MHD, after the process of induction was finished, did not lower lamotrigine concentrations. These findings can be of clinical significance for optimal AED dosing.

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry.

Over the past ~8years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.

In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl.

New fentanyl analogs have recently emerged as new psychoactive substances and have caused numerous fatalities worldwide. To determine if the new analogs follow the same metabolic pathways elucidated for fentanyl and known fentanyl analogs, we performed in vitro and in vivo metabolite identification studies for acetylfentanyl, acrylfentanyl, 4-fluoro-isobutyrylfentanyl, and furanylfentanyl. All compounds were incubated at 10μM with pooled human hepatocytes for up to 5h. For each compound, four or five authentic human urine samples from autopsy cases with and without enzymatic hydrolysis were analyzed. Data acquisition was performed in data-dependent acquisition mode during liquid chromatography high-resolution mass spectrometry analyses. Data was analyzed (1) manually based on predicted biotransformations and (2) with MetaSense software using data-driven search algorithms. Acetylfentanyl, acrylfentanyl, and 4-fluoro-isobutyrylfentanyl were predominantly metabolized by N-dealkylation, cleaving off the phenethyl moiety, monohydroxylation at the ethyl linker and piperidine ring, as well as hydroxylation/methoxylation at the phenyl ring. In contrast, furanylfentanyl's major metabolites were generated by amide hydrolysis and dihydrodiol formation, while the nor-metabolite was minor or not detected in case samples at all. In general, in vitro results matched the in vivo findings well, showing identical biotransformations in each system. Phase II conjugation was observed, particularly for acetylfentanyl. Based on our results, we suggest the following specific and abundant metabolites as analytical targets in urine: a hydroxymethoxy and monohydroxylated metabolite for acetylfentanyl, a monohydroxy and dihydroxy metabolite for acrylfentanyl, two monohydroxy metabolites and a hydroxymethoxy metabolite for 4-fluoro-isobutyrylfentanyl, and a dihydrodiol metabolite and the amide hydrolysis metabolite for furanylfentanyl.

Looking at flubromazolam metabolism from four different angles: Metabolite profiling in human liver microsomes, human hepatocytes, mice and authentic human urine samples with liquid chromatography high-resolution mass spectrometry

Flubromazolam is a triazolam benzodiazepine that recently emerged as a new psychoactive substance. Since metabolism data are scarce and good analytical targets besides the parent are unknown, we investigated flubromazolam metabolism in vitro and in vivo. 10μmol/L flubromazolam was incubated with human liver microsomes for 1h and with cryopreserved human hepatocytes for 5h. Mice were administered 0.5 or 1.0mg flubromazolam/kg body weight intraperitoneally, urine was collected for 24h. All samples, together with six authentic forensic human case specimens, were analyzed (with or without hydrolysis, in case it was urine) by UHPLC–HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF. Data mining was performed manually and with MassMetasite software (Molecular Discovery).A total of nine metabolites were found, all generated by hydroxylation and/or glucuronidation. Besides O-glucuronidation, flubromazolam formed an N+-glucuronide. Flubromazolam was not metabolized extensively in vitro, as only two monohydroxy metabolites were detected in low intensity in hepatocytes. In the mice samples, seven metabolites were identified, which mostly matched the metabolites in the human samples. However, less flubromazolam N+-glucuronide and an additional hydroxy metabolite were observed. The six human urine specimens showed different extent of metabolism: some samples had an intense flubromazolam peak next to a minute signal for a monohydroxy metabolite, others showed the whole variety of hydroxylated and glucuronidated metabolites. Overall, the most abundant metabolite was a monohydroxy metabolite, which we propose as α-hydroxyflubromazolam based on MSMS fragmentation. These metabolism data will assist in interpretation and analytical method development.

Validation of a tandem mass spectrometry method using combined extraction of 37 oxylipins and 14 endocannabinoid-related compounds including prostamides from biological matrices

There is a clinical need for more relevant coverage of bioactive lipids using smaller sample volumes. Therefore, we have validated a tandem mass spectrometry method for combined solid phase extraction of 37 compounds in the oxylipin (OxL) and 14 in the endocannabinoid (eCB) metabolome, as well as prostamides. The limits of quantification (LOQ) for compounds in the eCB metabolome were in the range 0.5–1000fg on column, intraday accuracy and precision ranges (%) were 83–125 and 0.3–17, respectively, and interday accuracy and precision ranges (%) were 80–119 and 1.2–20, respectively, dependent upon the compound and the concentration studied. Corresponding values for OxL were 0.5fg–4.2pg on column (LOQ), 85–115% (inter- and intraday accuracy) and <5% (precision). The combined extraction method was successfully applied to tissues, cell extracts, human plasma and milk samples. A deeper study of levels in elk, pig and cow brain, as well as cow heart and liver revealed tissue and species-specific elevation of eicosanoids: arachidonate diols, 20-HETE and 12(S)-HEPE (cow liver), LTB4 (cow brain), and monohydroxy metabolites (HETEs), epoxides and 5-oxo-ETE in elk brain, which might be caused by factors of stress and/or post-mortem reactions in the tissues.

Quantification of selected monohydroxy metabolites of polycyclic aromatic hydrocarbons in human urine

An analytical method was developed to quantitatively determine selected monohydroxy metabolites of polycyclic aromatic hydrocarbons (PAHs) in human urine. The procedure included enzymatic hydrolysis to cleave the conjugated metabolites, solid-phase microextraction enrichment, and gas chromatography-mass spectrometry analysis. The method proved to be sensitive enough to detect the selected PAH metabolites in human urine.

Effects on cell proliferation capacity and genome stability in periphery lymphocytes of occupational diesel exhaust exposed workers

To investigate the cell proliferation and genome stability in workers with occupational exposure to diesel exhaust (DE). In 2012, 117 DE-exposed workers and 106 control workers were recruited by cluster sampling in this study. The demographic data were obtained by questionnaire survey. The airborne fine particle and enriched polycyclic aromatic hydrocarbons (PAHs) at different workplaces were collected and analyzed. The concentrations of main PAHs monohydroxy metabolites in the urine were determined by high performance liquid chromatography-mass spectrometry (LC-MS), which could reflect the internal exposure level of DE. The cell proliferation capacity and genome stability in the periphery lymphocytes of workers were evaluated by cytokinesis-block micronucleus (CBMN) cytome assay. The concentrations (median (P5-P95)) of total PAHs monohydroxy metabolites in the urine of exposed group and control group were 12.96 (4.73-28.10), 4.76 (0.90-15.00) µg/L, respectively, and the exposed group was higher than that of controls (Z = -8.77, P < 0.001). The nuclear division index (NDI) of exposed group and control group was 1.68±0.13, 1.85±0.16, respectively, and the NDI of exposed group showed significantly decreased (t = 8.86, P < 0.001), while the genome instability index calculated by micronucleus, nuclear bridges and nuclear buds, of exposed group and control group was 13.27±6.26, 4.83±3.38, respectively, and the exposed group had statistically significant increase (Z = -10.08, P < 0.001). The tertiles of total PAHs monohydroxy metabolites in the urine were categorized into low, medium and high groups (<5.96, 5.96-12.46, >12.46 µg/L). With the NDI decreased, 1.81±0.17, 1.79±0.17, 1.68±0.14 (F = 13.14, P < 0.001), genome instability index began to increase 5.80±4.15, 9.97±7.14, 11.99±6.61 (/1 000), respectively (χ(2) = 36.74, P < 0.001). With the increase of total PAHs monohydroxy metabolites level in corresponding groups. In addition, the NDI was negatively correlated with the frequencies of micronucleus, nuclear bridges, nuclear buds and genome instability index, respectively, and the difference was statistically significant (P < 0.001). DE exposure lead to inhibition of cell proliferation capacity and increase genome instability in the peripheral lymphocytes of occupational-exposed population, providing important clues and evidence for early biomarkers monitoring.

Study on the phase I metabolism of novel synthetic cannabinoids, APICA and its fluorinated analogue.

The data are reported for an in vitro metabolism study of two novel synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and its fluorinated analog N-(1-adamantyl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-APICA, STS-135), which are active ingredients of smoking mixtures sold in Russia since 2012. The cannabinoids were isolated from herbal mixtures using preparative liquid chromatography and then incubated with human liver microsomes (HLMs). The formed metabolites were characterized by liquid chromatography - triple quadrupole mass spectrometry and high-resolution mass spectrometry with electrospray ionization in positive ion mode. It was found that HLMs produce mono-, di-, and trihydroxylated metabolites, as well as N-desalkyl metabolites, which can be further hydroxylated; the amide bond resisted the metabolic cleavage. For 5F-APICA, a series of oxidative defluorination products formed as well. For in vivo confirmation of the formed in vitro metabolites, spot urine samples from drug users were analyzed with the created method. It was shown that for the detection of APICA abuse, the preferred metabolites are the di- and tri-hydroxylated species, while in case of 5F-APICA, a monohydroxy metabolite is a better target. The N-despentyl (desfluoropentyl) hydroxyadamantyl metabolite also provides good retrospectivity to confirm the administration of any of these cannabinoids.