Characterization of in vitro and in vivo metabolism of leelamine using liquid chromatography-tandem mass spectrometry

1. Leelamine is a diterpene compound found in the bark of pine trees and has garnered considerable interest owing to its potent anticancer properties. The aim of the present study was to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

2. We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono-hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSⁿ fragmentation obtained by collision-induced dissociation using LC-MS/MS in HLMs.

3. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite, whereas CYP2C19, CYP1A1, and CYP3A4 contributed to some extent.

4. Moreover, we identified only one metabolite M1, in the urine, but none in the feces. In conclusion, leelamine was metabolized to a mono-hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.

     

Structural characterization of a degradation product of rocuronium using nanoelectrospray‐high resolution mass spectrometry

Rocuronium bromide is a non‐depolarizing neuromuscular blocking agent that causes rapid muscle relaxation after intravenous injection. Regulatory authorities for registration of pharmaceuticals for human use require the evaluation of the stability of active compounds under various stress conditions. Forced degradation of rocuronium bromide was performed under hydrolytic, thermal, photolytic, and oxidative settings. HPLC‐UV/vis analysis revealed an unknown degradation product under oxidative conditions (1% H₂O₂, reflux for 1 h). Investigation of the respective HPLC fraction by high resolution mass spectrometry indicated a formal loss of CH₂ and an addition of one oxygen atom to the intact drug molecule. Additional multistage mass spectrometric structural elucidation experiments aided by complementary information from analysis of the intact drug and known rocuronium‐related compounds showed that the morpholine moiety was unstable under oxidative stress. The data demonstrated that the morpholine ring was opened and transformed to an N‐ethanoyl‐formamide group. The structure was supported by appropriate mechanistic explanations. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo metabolism and detection of 3‐HO‐PCP,a synthetic phencyclidine,in human samples and pooled human hepatocytes using high resolution mass spectrometry

The new psychoactive substance (NPS) 3‐HO‐PCP, a phencyclidine (PCP) analog, was detected in a law enforcement seizure and in forensic samples in Denmark. Compared with PCP, 3‐HO‐PCP is known to be a more potent dissociative NPS, but no toxicokinetic investigations of 3‐HO‐PCP are yet available. Therefore, 3‐HO‐PCP was quantified in in vivo samples, and the following were investigated: plasma protein binding, in vitro and in vivo metabolites, and metabolic targets. All samples were separated by liquid chromatography and analyzed by mass spectrometry. The unbound fraction in plasma was determined as 0.72 ± 0.09. After in vitro incubation with pooled human hepatocytes, four metabolites were identified: a piperidine‐hydroxyl‐and piperidine ring opened N‐dealkyl‐COOH metabolite, and O‐glucuronidated‐ and O‐sulfate‐conjugated metabolites. In vivo, depending on the sample and sample preparation, fewer metabolites were detected, as the O‐sulfate‐conjugated metabolite was not detected. The N‐dealkylated‐COOH metabolite was the main metabolite in the deconjugated urine sample. in vivo analytical targets in blood and brain samples were 3‐HO‐PCP and the O‐glucuronidated metabolite, with 3‐HO‐PCP having the highest relative signal intensity. The drug levels of 3‐HO‐PCP quantified in blood were 0.013 and 0.095 mg/kg in a living and a deceased subject, respectively. The 3‐HO‐PCP concentrations in deconjugated urine in a sample from a living subject and in post‐mortem brain were 7.8 and 0.16 mg/kg, respectively. The post mortem results showed a 1.5‐fold higher concentration of 3‐HO‐PCP in the brain tissue than in the post mortem blood sample.     

Metabolites of 5F‐AKB‐48, a synthetic cannabinoid receptor agonist,identified in human urine and liver microsomal preparations using liquid chromatography high‐resolution mass spectrometry

New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N‐?(1‐Adamant?yl)‐?1‐?(5‐?fluoropentyl)‐?1H‐?indazole‐?3‐?carboxamide (5F‐AKB‐48), also known as 5F‐APINACA, was identified as an adulterant in herbal products. This compound deviates from earlier JHW‐type synthetic cannabinoids by having an indazole ring connected to an adamantyl group via a carboxamide linkage. Synthetic cannabinoids are completely metabolized, and identification of the metabolites is thus crucial when using urine as the sample matrix. Using an authentic urine sample and high‐resolution accurate‐mass Fourier transform Orbitrap mass spectrometry, we identified 16 phase‐I metabolites of 5F‐AKB‐48. The modifications included mono‐, di‐, and trihydroxylation on the adamantyl ring alone or in combination with hydroxylation on the N‐fluoropentylindazole moiety, dealkylation of the N‐fluoropentyl side chain, and oxidative loss of fluorine as well as combinations thereof. The results were compared to human liver microsomal (HLM) incubations, which predominantly showed time‐dependent formation of mono‐, di‐, and trihydroxylated metabolites having the hydroxyl groups on the adamantyl ring. The results presented here may be used to select metabolites specific of 5F‐AKB‐48 for use in clinical and forensic screening. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic characterization of AH‐7921, a synthetic opioid designer drug: in vitro metabolic stability assessment and metabolite identification,evaluation of in silico prediction,and in vivo confirmation

AH‐7921 (3,4‐dichloro‐N‐[(1‐dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non‐fatal and fatal intoxications. To comprehensively study AH‐7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH‐7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH‐7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time‐of‐flight high‐resolution mass spectrometry (MS). High‐resolution full scan MS and information‐dependent acquisition MS/MS data were analyzed with MetabolitePilot? (SCIEX) using multiple data processing algorithms. The presence of AH‐7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite? (Molecular Discovery). AH‐7921 in vitro half‐life was 13.5 ± 0.4 min. We identified 12 AH‐7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di‐demethylated, N‐hydroxylated and glucuronidated, eluted after AH‐7921 and was the most abundant metabolite in non‐hydrolyzed urine. MetaSite? correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di‐desmethyl AH‐7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH‐7921 intake. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro metabolism of the novel synthetic opioid agonist cyclopropylfentanyl and subsequent confirmation in authentic human samples using liquid chromatography‐high resolution mass spectrometry

Novel synthetic opioids (NSOs) are a class of novel psychoactive substances (NPS) that are growing in popularity and presenting a significant public health risk. Included in this class are derivatives of the highly potent analgesic, fentanyl. Cyclopropylfentanyl (CycP‐F) was first reported to the EU Early Warning System in August 2017, and was subsequently linked to more than 100 deaths in the US alone. Limited pharmacological, pharmacokinetic or toxicological data is available for many emerging NSOs; however we can expect novel fentanyl analogues to present limited detection windows, short onset, narrow therapeutic indices and the potential for very high potency. Knowledge of the metabolism of these drugs is essential for the identification of analytical targets for their detection. Therefore in vitro metabolites of CycP‐F were produced using human liver microsomal incubations. Metabolites formed were elucidated using liquid chromatography‐high resolution accurate mass analysis (LC‐HRAM). Identified metabolites were added to our accurate mass screening database for NPS which was utilised for subsequent screening analysis. CycP‐F and metabolites were identified in two human blood case samples. Eleven metabolites were identified in vitro, with the major metabolites produced via N‐dealkylation, monohydroxylation and N‐oxidation. Analysis of the positive case samples identified four in vivo metabolites, all of which were observed in vitro. The major metabolite identified in vitro and in vivo was the N‐dealkylated nor‐metabolite; two further mono‐hydroxylated and one dihydroxylated metabolite were detected in vivo.     

Detection of metabolites of the new synthetic cannabinoid CUMYL‐4CN‐BINACA in authentic urine samples and human liver microsomes using high‐resolution mass spectrometry

CUMYL‐4CN‐BINACA(1‐(4‐cyanobutyl)‐N‐(2‐phenylpropan‐2‐yl)‐1H–indazole‐3‐carboxamide) is a recently introduced indazole‐3‐carboxamide‐type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL‐4CN‐BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 μM CUMYL‐4CN‐BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography–high‐resolution mass spectrometry (LC–HRMS). Less than 21% of the CUMYL‐4CN‐BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL‐4CN‐BINACA N‐butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL‐4CN‐BINACA was not detected; CUMYL‐4CN‐BINACA N‐butanoic acid (M16) was major metabolite after β‐glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL‐4CN‐BINACA N‐butanoic acid), M8 and M11 (hydroxylcumyl CUMYL‐4CN‐BINACA) as urinary marker metabolites to confirm CUMYL‐4CN‐BINACA intake.     

In vitro identification of metabolites of verapamil in rat liver microsomes

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS^n), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.     

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Flubromazolam is a triazole benzodiazepine with high potency and long‐lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high‐resolution mass spectrometry (LC‐HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)‐enzymes. To confirm those metabolites detected in vitro , authentic samples obtained from two forensic cases were also analyzed by LC‐HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL_H ) using well‐stirred and parallel‐tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate‐fortified pHLM with or without uridine 5'‐diphosphoglucuronic acid and incubations with CYP‐enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α‐ and/or 4‐position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α‐hydroxy‐flubromazolam and its corresponding glucuronide were detected in vivo together with the N ‐glucuronide of flubromazolam. The predicted CL _H of flubromazolam using the well‐stirred and parallel‐tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein‐binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α‐hydroxy‐flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.     

High resolution accurate mass screening of prohibited substances in equine plasma using liquid chromatography – Orbitrap mass spectrometry

A recent trend in the use of high resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi‐drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine blood samples would be desirable. This paper presents the development of a liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) screening method for equine plasma samples to cover over 320 prohibited substances in a single analytical run. Plasma samples were diluted and processed by solid‐phase extraction. The extracts were then analyzed with LC‐HRMS in full‐scan positive electrospray ionization mode. A mass resolution of 60 000 was employed. Benzyldimethylphenylammonium was used as an internal lock mass. Drug targets were identified by retention time and accurate mass, with a mass tolerance window of ±3 ppm. Over 320 drug targets could be detected in a 13‐min run. Validation data including sensitivity, specificity, extraction recovery and precision are presented. As the method employs full‐scan mass spectrometry, an unlimited number of drug targets can theoretically be incorporated. Moreover, the HRAMS data acquired can be re‐processed retrospectively to search for drugs which have not been targeted at the time of analysis. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro Phase I metabolism of indazole carboxamide synthetic cannabinoid MDMB‐CHMINACA via human liver microsome incubation and high‐resolution mass spectrometry

Synthetic cannabinoids have proliferated over the last decade and have become a major public health and analytical challenge, critically impacting the clinical and forensic communities. Indazole carboxamide class synthetic cannabinoids have been particularly rampant, and exhibit severe toxic effects upon consumption due to their high binding affinity and potency at the cannabinoid receptors (CB₁ and CB₂). MDMB‐CHMINACA, methyl 2‐[1‐(cyclohexylmethyl)‐1H‐indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate, a compound of this chemical class, has been identified in forensic casework and is structurally related to several other synthetic cannabinoids. This study presents the first extensive report on the Phase I metabolic profile of MDMB‐CHMINACA, a potent synthetic cannabinoid. The in vitro metabolism of MDMB‐CHMINACA was determined via incubation with human liver microsomes and high‐resolution mass spectrometry. The accurate masses of precursor and fragments, mass error (ppm), and chemical formula were obtained for each metabolite. Twenty‐seven metabolites were identified, encompassing twelve metabolite types. The major biotransformations observed were hydroxylation and ester hydrolysis. Hydroxylations were located predominantly on the cyclohexylmethyl (CHM) moiety. Ester hydrolysis was followed by additional biotransformations, including dehydrogenation; mono‐ and dihydroxylation and ketone formation, each with dehydrogenation. Minor metabolites were identified and reported. The authors propose that CHM‐monohydroxylated metabolites specific to MDMB‐CHMINACA are the most suitable candidates for implementation into bioanalytical assays to demonstrate consumption of this synthetic cannabinoid. Due to the structural similarity of MDMB‐CHMINACA and currently trending synthetic cannabinoids whose metabolic profiles have not been reported, the results of this study can be used as a guide to predict their metabolic pathways.     

The characterization of isobaric product ions of fentanyl using multi‐stage mass spectrometry,high‐resolution mass spectrometry and isotopic labeling

This study uses a combination of multi‐stage mass spectrometry (MSⁿ), accurate mass measurements – with high‐resolution mass spectrometry (HRMS) – and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4‐ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4‐ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.     

Metabolic profiling of deschloro-N-ethyl-ketamine and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry

The aim of this study was to identify new markers of deschloro-N-ethyl-ketamine (O-PCE), a ketamine analogue that has been involved in acute intoxications with severe outcomes including death and whose metabolism has never been studied before. In vitro study after 2-h incubation with pooled human liver microsomes (HLMs) cross-checked by the analysis of urine and hair from a 43-year-old O-PCE user (male) were performed by liquid chromatography–high resolution mass spectrometry (LC-HRMS). Acquired data were processed by the Compound Discoverer® software, and a full metabolic profile of O-PCE was proposed. In total, 15 metabolites were identified, 10 were detected in vitro (HLMs) and confirmed in vivo (urine and/or hair), two were present only in HLMs, and the remaining three metabolites were identified only in biological specimens. While O-PCE was no longer detected in urine, nine metabolites were identified allowing to increase its detection window. In descending order of metabolites abundance, we suggest using 2-en-PCA-N-Glu (34%, first), M3 (16%, second), O-PCA-N-Glu (15.4%, third), OH-O-PCE (15%, fourth) and OH-PCE (11.9%, fifth) as target metabolites to increase the detection window of O-PCE in urine. In hair, nine metabolites were identified. OH-PCA was the major compound (78%) with a relevant metabolite to parent drug ratio (=6) showing its good integration into hair and making it the best marker for long-term monitoring of O-PCE exposure.     

Detection of LongR3-IGF-I,Des(1-3)-IGF-I,and R3-IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes

Insulin-like growth factor-I (IGF-I) and its analogs LongR³-IGF-I, Des(1-3)-IGF-I, and R³-IGF-I are prohibited substances in sport. Although they were never approved for use in humans, they are readily available as black market products for bodybuilding and can be used to enhance physical performance. This study's aims were to validate a fast and sensitive detection method for IGF-I analogs and to evaluate their detectability after intramuscular administration in rats. The sample preparation consisted of an immunopurification on MSIA™ microcolumns using a polyclonal anti-human-IGF-I antibody. The target substances were then directly analyzed by nano-liquid chromatography coupled with high-resolution mass spectrometry. Abundant signs of lower quality, oxidized peptide forms were found in black market products, justifying the need to monitor at least both the native and mono-oxidized forms. The analytical performance of this method (linearity, carry over, detection limits, precision, specificity, recovery, and matrix effect) was studied by spiking the analogs into human serum. Following a single intramuscular administration (100 μg/kg) in rats, detection was evaluated up to 36 h after injection. While unchanged Des(1-3)-IGF-I and R³-IGF-I were detected until 24 h after administration, LongR³-IGF-I disappeared rapidly after 4 h. Des(1)-LongR³-IGF-I, a new N-terminal Long-R³-IGF-I degradation product, was detected in addition to Des(1-10)-LongR³-IGF-I and Des(1-11)-LongR³⁻IGF-I: the latter was detected up to 16 h. The same products were found after in vitro incubation of the analogs in human whole blood, suggesting that observations in rats may be extrapolated to humans and that the validated method may be applicable to antidoping testing.     

Suitability evaluation of new endogenous biomarkers for the identification of nitrite‐based urine adulteration in mass spectrometry methods

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5‐hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO₂). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO₂ concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and ? 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography–high resolution mass spectrometry (LC–HRMS). Receiver operating characteristics (ROC) analysis was applied for cut‐off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4–5). Storage at ?20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO₂ and are ready for implementation into routine MS procedures.     

In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist,taranabant, in rats and monkeys

1. The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹⁴C]taranabant, the majority of the radioactivity was excreted within 72?h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile.

2. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey.

3. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

     

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty‐four peptides were extracted using mixed‐mode solid‐phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10–50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100–400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS‐NH₂, YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re‐analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.     

25C‐NBOMe and 25I‐NBOMe metabolite studies in human hepatocytes,in vivo mouse and human urine with high‐resolution mass spectrometry

25C‐NBOMe and 25I‐NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I‐NBOMe, whereas 25C‐NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 μM 25C‐NBOMe and 25I‐NBOMe; samples were analyzed by liquid chromatography high‐resolution mass spectrometry (LC‐HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC‐HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C‐NBOMe and 25I‐NBOMe were predominantly metabolized by O‐demethylation, followed by O‐di‐demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C‐NBOMe urine targets are 5’‐desmethyl 25C‐NBOMe, 25C‐NBOMe and 5‐hydroxy 25C‐NBOMe, and for 25I‐NBOMe 2’ and 5’‐desmethyl 25I‐NBOMe and hydroxy 25I‐NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.     

Identifying metabolites of diphenidol by liquid chromatography-quadrupole/orbitrap mass spectrometry using rat liver microsomes,human blood,and urine samples

In recent years, diphenidol [1,1-diphenyl-4-piperidino-1-butanol] has been one of the drugs that appears in suicide cases, but there are few research data on its metabolic pathways and main metabolites. Metabolite identification plays a key role in drug safety assessment and clinical application. In this study, in vivo and in vitro samples were analyzed with ultra-high-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. Structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, 10 Phase I metabolites and 5 glucuronated Phase II metabolites were found in a blood sample and a urine sample from authentic cases. Three other Phase I metabolites were identified in the rat liver microsomes incubation solution. The results showed that the main metabolic pathways of diphenidol in the human body include hydroxylation, oxidation, dehydration, N-dealkylation, methylation, and conjugation with glucuronic acid. This study preliminarily clarified the metabolic pathways and main metabolites of diphenidol. For the development of new methods for the identification of diphenidol consumption, we recommend using M2-2 as a marker of diphenidol entering the body. The results of this study provide a theoretical basis for the pharmacokinetics and forensic scientific research of diphenidol.     

Direct analysis in real time ‐ high resolution mass spectrometry (DART‐HRMS): a high throughput strategy for identification and quantification of anabolic steroid esters

High throughput screening is essential for doping, forensic, and food safety laboratories. While hyphenated chromatography‐mass spectrometry (MS) remains the approach of choice, recent ambient MS techniques, such as direct analysis in real time (DART), offer more rapid and more versatile strategies and thus gain in popularity. In this study, the potential of DART hyphenated with Orbitrap‐MS for fast identification and quantification of 21 anabolic steroid esters has been evaluated. Direct analysis in high resolution scan mode allowed steroid esters screening by accurate mass measurement (Resolution = 60 000 and mass error < 3 ppm). Steroid esters identification was further supported by collision‐induced dissociation (CID) experiments through the generation of two additional ions. Moreover, the use of labelled internal standards allowed quantitative data to be recovered based on isotopic dilution approach. Linearity (R² > 0.99), dynamic range (from 1 to 1000 ng mL^‐1), bias (<10%), sensitivity (1 ng mL^‐1), repeatability and reproducibility (RSD < 20%) were evaluated as similar to those obtained with hyphenated chromatography‐mass spectrometry techniques. This innovative high throughput approach was successfully applied for the characterization of oily commercial preparations, and thus fits the needs of the competent authorities in the fight against forbidden or counterfeited substances. Copyright © 2014 John Wiley & Sons, Ltd.