Methionine329 in human serum albumin: A novel target for alkylation by sulfur mustard

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine³²⁹ in human serum albumin (HSA) as a novel target of SM forming a HETE‐methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet³²⁹(‐HETE)Phe, LGM(‐HETE)F, was detected after pepsin‐mediated proteolysis and subsequent analysis by microbore liquid chromatography–electrospray ionization–high‐resolution tandem‐mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(‐HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA‐derived biomarkers HETE‐CysPro and HETE‐CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met³²⁹ in vitro and in vivo was limited to 5 days making this modification a beneficial short‐time biomarker. Furthermore, it was found that the HETE‐methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N‐terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.     

Synthesis of 13C labeled sulfur and nitrogen mustard metabolites as mass spectrometry standards for monitoring and detecting chemical warfare agents

¹³C labeled (>M + 4) metabolites of nitrogen and sulfur‐based chemical warfare agent metabolites were prepared from readily available and ¹³C labeled commercial starting materials. The new chemical routes are efficient in the number of chemical steps, can be scaled to afford gram quantities, and occur in good yields on the basis of the ¹³C label. These labeled compounds are useful as internal standards in mass spectrometry for monitoring chemical warfare agents and their metabolites. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys¹⁰-residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.     

Characterization of hallucinogenic phenethylamines using high‐resolution mass spectrometry for non‐targeted screening purposes

Hallucinogenic phenethylamines such as 2,5‐dimethoxyphenethylamines (2C–X) and their N ‐(2‐methoxybenzyl) derivatives (25X–NBOMe) have seen an increase in novel analogues in recent years. These rapidly changing analogues make it difficult for laboratories to rely on traditional targeted screening methods to detect unknown new psychoactive substances (NPS). In this study, twelve 2C–X, six 2,5‐dimethoxyamphetamines (DOX), and fourteen 25X–NBOMe derivatives, including two deuterated derivatives (2C–B‐d ₆ and 25I–NBOMe‐d ₉), were analyzed using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF‐MS). Collision‐induced dissociation (CID) experiments were performed using collision energies set at 10, 20, and 40 eV. For 2C–X and DOX derivatives, common losses were observed including neutral and radical losses such as NH₃ (17.0265 Da), •CH₆N (32.0500 Da), C₂H₇N (45.0578 Da) and C₂H₉N (47.0735 Da). 2C–X derivatives displayed common product ions at m/z 164.0837 ([C₁₀H₁₂O₂]^+•), 149.0603 ([C₉H₉O₂]⁺), and 134.0732 ([C₉H₁₀O]^+•) while DOX derivatives had common product ions at m/z 178.0994 ([C₁₁H₁₄O₂]^+•), 163.0754 ([C₁₀H₁₁O₂]⁺), 147.0804 ([C₁₀H₁₁O]⁺), and 135.0810 ([C₉H₁₁O]⁺). 25X–NBOMe had characteristic product ions at m/z 121.0654 ([C₈H₉O]⁺) and 91.0548 ([C₇H₇]⁺) with minor common losses corresponding to 2‐methylanisole (C₈H₁₀O, 122.0732 Da), 2‐methoxybenzylamine (C₈H₁₁NO, 137.0847 Da), and •C₉H₁₄NO (152.1074 Da). Novel analogues of the selected classes can be detected by applying neutral loss filters (NLFs) and extracting the common product ions. Copyright © 2017 John Wiley & Sons, Ltd.     

Identification of recombinant human relaxin‐2 in equine plasma by liquid chromatography‐high resolution mass spectrometry

Relaxin (RLX) is a peptide hormone belonging to the relaxin‐like peptide family. Relaxin‐2 (RLX‐2), a heteromeric polypeptide consisting of an A‐chain (24 amino acids) and a B‐chain (29 amino acids) linked together by two inter‐chain disulfide bonds, is the main circulating RLX hormone in human. Due to its ability to dilate blood vessels surrounding the smooth muscles via induction of nitric oxide resulting in the increase of blood and oxygen supplies to the muscles, it may enhance athletic performance and is therefore banned in horseracing, equestrian competitions, and human sports. In order to control the abuse of rhRLX‐2, a definitive method is required to detect and confirm the presence of rhRLX‐2 in biological samples. This paper describes, for the first time, the detection and confirmation of rhRLX‐2 in equine plasma by liquid chromatography‐high resolution mass spectrometry (LC‐HRMS) after immunoaffinity extraction. rhRLX‐2 could be detected at less than 0.1 ng/ml, and confirmed at less than 0.2 ng/ml in plasma samples. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

A high‐throughput and broad‐spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography–high‐resolution mass spectrometry

A high‐throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide‐conjugated drug targets. This is followed by extraction using solid‐supported liquid extraction (SLE) and analysis using liquid chromatography–high‐resolution mass spectrometry (LC–HRMS). The entire set‐up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC–HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography–triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post‐race or out‐of‐competition urine samples collected from racehorses. This method was developed for pre‐race urine testing in Hong Kong; however, it is also suitable for testing post‐race or out‐of‐competition urine samples, especially when a quick total analysis time is desired.     

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.     

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.     

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.     

Analysis of cis and trans 3‐methylfentanyl by liquid chromatography–high resolution mass spectrometry and findings in forensic toxicology casework

3‐methylfentanyl (3‐MF), N‐(3‐methyl‐1‐phenethyl‐4‐piperidyl)‐N‐phenyl‐propanamide, has reappeared on the US illicit drug market since its disappearance after a series of overdose deaths in 1988. 3‐MF presents an analytical challenge, due to presence of cis and trans stereoisomers, each with different potencies, and ultimately very low concentrations in the blood after use. A method was developed using liquid chromatography–time‐of‐flight–mass spectrometry for the analysis of (±)‐cis‐3‐MF and (±)‐trans‐3‐MF in blood specimens after solid phase extraction. The linear dynamic range of this method was 0.1–10 ng/mL. Blood samples from 25 postmortem cases and 2 human performance case involving 3‐MF were submitted for quantitative analysis. The mean and median concentration for the (±)‐cis‐3‐MF were 0.84 ng/mL (±0.81) and 0.67 ng/mL, respectively, range 0.14–3.43 ng/mL. The resulting (±)‐trans‐3‐MF mean concentration was 0.46 ng/mL (±0.38) and the median concentration was 0.37 ng/mL with a range of 0.11–1.90 ng/mL. The resulting (±)‐cis‐3‐MF and (±)‐trans‐3‐MF concentrations were summed to give the total amount of 3‐MF present in the case with the resulting average concentration at 1.28 ng/mL (±1.16), median at 1.01 ng/mL and range 0.18–5.18. As the estimated dose of this compound is approximately 0.1 mg–0.5 mg with the resulting concentrations in the sub‐nanogram range, it is necessary for forensic toxicology laboratories to obtain instruments sensitive enough to detect these substances in driving under the influence of drugs and postmortem cases. Quantitation of 3‐MF with separation of (±)‐cis and (±)‐trans‐3‐MF provides additional detail for more specific toxicological interpretation.     

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.     

Retrospective analysis for valproate screening targets with liquid chromatography–high resolution mass spectrometry with positive electrospray ionization: An omics‐based approach

Liquid chromatography coupled with high‐resolution mass spectrometry (LC–HRMS) is an important analytical tool in the systematic toxicological analysis performed in forensic toxicology. However, some important compounds, such as the antiepileptic drug valproate (valproic acid; VPA), cannot be directly detected with positive electrospray ionization (ESI⁺) due to poor ionization. Here we demonstrate an omics‐based retrospective analysis for the identification of indirect screening targets for VPA in whole blood with LC–ESI⁺–HRMS. Analysis was performed utilizing data acquired across four years from LC–ESI⁺–HRMS, with VPA results from a quantitative LC–MS/MS method. The combined data with VPA results were split into an exploration set (n = 68; 28% positive) and a test set (n = 37; 32% positive). Eight indirect targets for VPA were identified in the exploration set. The evaluation of these targets was confirmed with retrospective target analysis of the test set. Using a combination of two out of the eight indirect targets, we attained a sensitivity of 92% (n = 12; VPA concentration range: 4.4–29.7 mg/kg) and 100% specificity (n = 25) for VPA with LC–ESI⁺–HRMS. VPA screening targets were identified with retrospective data analysis and could be appended to the existing screening procedure. A sensitive and specific screening with LC–ESI⁺–HRMS was achieved with targets corresponding to the sodium adducts of C₇H₁₄O₃ and C₈H₁₄O₃. Three chromatographic resolved isomer peaks were observed for the latter, and the consistently most intense peak was tentatively identified as 3‐hydroxy‐4‐en‐VPA.     

A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

Target analysis using liquid chromatography–tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non‐target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid‐phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high‐resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%–131%), matrix effects (35%–237%), limites of detection (0.0002–100 ng/mL), and intra‐ and inter‐day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non‐target screening method is efficient for conducting anti‐doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.     

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 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.     

Targeting misuse of 2‐amino‐N‐ethyl‐1‐phenylbutane in urine samples: in vitro–in vivo correlation of metabolic profiles and development of LC‐TOF‐MS method

A phenyethylamine derivative, 2‐amino‐N‐ethyl‐1‐phenylbutane (2‐AEPB), has recently been detected in doping control and drugs‐of‐abuse samples, and identified as a non‐labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2‐AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2‐AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N‐oxidation was also observed (M3). In vitro‐in vivo correlation was studied in an excretion study with a single, oral dose of 2‐AEPB‐containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2‐AEPB and M1 were mainly found in free urinary fraction, whereas higher inter‐individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide‐ and sulpho‐conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2‐AEPB. Copyright © 2014 John Wiley & Sons, Ltd.     

Baicalin as a potentially promising drug for the management of sulfur mustard induced cutaneous complications: A review of molecular mechanisms

Sulfur mustard (SM) is a bifunctional alkylating agent with strong blistering, irritant, mutagenic and cytotoxic properties. SM has been widely deployed as a chemical warfare agent for over a century, leading to extensive casualties. Skin is among the first and most heavily damaged organs upon SM exposure. Unfortunately, a considerable fraction of SM-intoxicated patients are still suffering from chronic cutaneous complications. While these complications adversely affect patients’ quality of life, there is as yet no ideal treatment for them and therapeutic options are limited and mainly symptomatic. During recent decades, remarkable progress has been made in understanding molecular mechanisms underlying SM-induced dermatotoxicity and several intra- and extracellular targets have been identified. This review argues that baicalin, a bioactive flavonoid from the roots of Scutellaria spp., could counteract different molecular and biochemical abnormalities that mediate SM dermatotoxicity and could therefore be regarded as a promising therapeutic option for the management of SM-induced cutaneous lesions.     

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.     

Development and validation of a simple online‐SPE method coupled to high‐resolution mass spectrometry for the analysis of stanozolol‐N‐glucuronides in urine samples

Stanozolol is still the most commonly used illicit anabolic‐androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of great interest in the field of antidoping analysis. In this work, a very simple, fast, and highly sensitive online solid‐phase extraction method coupled with liquid chromatography–high‐resolution tandem mass spectrometry (HPLC‐HRMSMS) for the analysis of stanozolol‐N‐glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/mL, high accuracy (87.1%–102.1%), precision (3.1%–7.8%), and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry‐over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1′N‐ and 2′N‐stanozolol‐glucuronide in human urine for the first time due to the access to reference material.