Identification and simultaneous quantification of potential genotoxic impurities in first-line HIV drug dolutegravir sodium using fast ultrasonication-assisted extraction method coupled with GC-MS and in-silico toxicity assessment.

Dolutegravir (DLG) has become a distinctive first-line antiretroviral therapy for the treatment of HIV in most countries due to its affordability, high efficacy, and low drug-drug interactions. However, the evaluation of genotoxic impurities (GTIs) in DLG and their toxicity assessment has not been explored thoroughly. Thus, in this study, a simple, fast, and selective analytical methodology was developed for the identification and determination of 7 GTIs in the comprehensive, explicit route of synthesis for the dolutegravir sodium (DLG-Na) drug. A facile, fast ultrasonication-assisted liquid-liquid extraction procedure was adapted to isolate the GTIs in DLG-Na and then analyzed using the gas chromatography (GC)-electron impact (EI)/mass spectrometer (MS) quantification (using selective ion monitoring mode) technique. This EI-GC/MS method was validated as per the current requirements of ICH Q2 (R1) guidelines. Under optimal method conditions, excellent linearities were achieved with R between 0.9959 and 0.9995, and high sensitivity was obtained in terms of detection limits (LOD) between 0.15 to 0.63 µg/g, and quantification limits (LOQ) between 0.45 to 1.66 µg/g for the seven GTIs in DLG. The obtained recoveries ranged from 98.2 to 104.3 % at LOQ, 15 µg/g, and 18 µg/g concentration levels (maximum daily dose of 100 mg). This developed and validated method is rapid, easy to adopt, specific, sensitive, and accurate in estimating the seven GTIs in a relatively complex sodium matrix of the DLG-Na drug moiety. As a method application, two different manufactured samples of DLG-Na drug substances were analyzed for the fate of the GTIs and drug safety for the intended dosage applications. Moreover, an in-silico QSAR toxicity prediction assessment was carried out to prove scientifically the potential GTI nature of each impurity from the alerting functional groups.

Efficient and environmentally friendly spectrofluorimetric determination of cariprazine: An analytical and green chemistry approach.

Cariprazine represents a new generation of antipsychotic medication, characterized by its heightened affinity for the D3 receptor. It has recently obtained approval as an adjunctive treatment option for patients diagnosed with major depressive disorder. In this study, a novel approach utilizing fluorescence spectroscopy was developed to analyze cariprazine. The methodology involves the transformation of cariprazine into a fluorescent compound by means of chemical derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Following excitation at 470 nm, the fluorescent derivative displayed peak fluorescence emission at 550 nm. The factors influencing the derivatization process were optimized. Upon reaching the optimal reaction conditions, a linear correlation (r2 = 0.9995) was observed between the fluorescence intensity and concentrations of cariprazine ranging from 20 to 400 ng/ml. Detection and quantitation limits were determined to be 5.85 and 17.74 ng/ml, respectively. The approach was accurate and precise, with percent recovery values ranging from 98.14% to 99.91% and relative standard deviations of less than 2%. Application of the method to the analysis of cariprazine in bulk and commercial capsules forms yielded accurate results. Moreover, adherence to environmentally friendly analytical practices was evident through alignment with the principles of green analysis, as demonstrated by the analytical eco-scale, AGREE, and GAPI greenness assessment tools.

An ultra-fast ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of palbociclib in human liver microsomes: In silico study for metabolic lability, absorption, distribution, metabolism, and excretion features, and DEREK alerts screening.

Palbociclib (Ibrance; Pfizer) was approved for the management of metastatic breast cancer characterized by hormone receptor-positive/human epidermal growth factor receptor 2 negative status. The objective of this study was to create a fast, precise, environmentally friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry approach for quantifying palbociclib (PAB) in human liver microsomes with the application for assessing metabolic stability. The validation features were performed in agreement with the bioanalytical method validation standards outlined by the US Food and Drug Administration. The StarDrop software (WhichP450 and DEREK modules) was used in screening the metabolic lability and structural alerts of PAB. The separation of PAB and encorafenib (as an internal standard) was achieved on a C8 column, employing an isocratic mobile phase. The inter-day and intra-day accuracy and precision ranged from -6.00% to 4.64% and from -2.33% to 3.13%, respectively. The constructed calibration curve displayed a linearity in the range of 1-3000 ng/mL. The sensitivity of the established approach was proven by the lower limit of quantification of 0.73 ng/mL. The Analytical GREEness calculator results revealed the high level of greenness of the developed method. The PAB's metabolic stability (t1/2 of 18.5 min and a moderate clearance (Clint) of 44.8 mL/min/kg) suggests a high extraction ratio medication that matched the WhichP450 software results.

Research Progress on Detection of New Psychoactive Substance Piperazines in vivo.

Piperazines are a class of new psychoactive substances with hallucinogenic effects that affect the central nervous system by affecting the level of monoamine neurotransmitters. Abuse of piperazines will produce stimulating and hallucinogenic effects, accompanied by headache, dizziness, anxiety, insomnia, vomiting, chest pain, tachycardia, hypertension and other adverse reactions, and may even cause cardiovascular diseases and multiple organ failure and lead to death, seriously affecting human physical and mental health and public safety. The abuse of new psychoactive substance piperazines has attracted extensive attention from the international community. The study of its pharmacological toxicology and analytical methods has become a research hotspot in the field of forensic medicine. This paper reviews the in vivo processes, sample treatment and analytical methods of existing piperazines, in order to provide reference for forensic identification.

Investigation into the genotoxic impurity, 1-methyl-4-nitrosopiperazine, in rifampicin.

This study assessed the presence of the genotoxic impurity 1-methyl-4-nitrosopiperazine (MNP) in 27 batches of rifampicin capsules obtained from 11 manufacturers in China. While they were below the temporary limit of 5 ppm set by the US Food and Drug Administration, the observed levels (0.33-2.36 ppm) exceeded the acceptable threshold of 0.16 ppm. Building upon preliminary findings and degradation experiments, we concluded that MNP is a by-product of the oxidative degradation of rifampicin or is introduced via oxidation or nitrosation during the synthesis process involving 1-methyl-4-aminopiperazine. The pathways of MNP formation were confirmed in this study. Furthermore, we observed that the addition of antioxidants, sealed storage, and selection of dominant crystal forms can aid in controlling MNP levels.

Simultaneous quantification of five antiretrovirals in human tissues using ultra-high performance liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring at the sites of action.

Although antiretroviral therapy (ART) is highly effective for the treatment of HIV-1 infection to suppress virus in the blood, HIV persists in tissues. HIV persistence in the tissues is due to numerous factors, and one of those factors are antiretroviral (ARV) concentrations. ARV concentrations in tissues must be adequate to suppress HIV at the sites of action. While therapeutic drug monitoring in the plasma is well-known, drug monitoring in the tissues provides local assessments of adequate ARV exposure to prevent localized HIV resistance formation. Towards these efforts, we validated an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method in human tissues (cervical, rectal, and vaginal tissues) for the simultaneous quantification of five ARVs: bictegravir, cabotegravir, dolutegravir, doravirine, and raltegravir. For this assay, protein precipitation with acetonitrile with stable, isotopically-labeled internal standards followed by supernatant pre-concentration was performed. Analyte separation was accomplished using a multistep UPLC gradient mixture of 0.1 % formic acid in water (A) and acetonitrile (B) with a Waters Cortecs T3 (2.1x100 mm) column. The assay was extensively validated as per the United States Food and Drug Administration Bioanalytical Method Validation Guidance over a clinically observed range (0.05-50 ng/mL) with superb linearity (R2 > 0.99 across all ARVs). The assay run time was 8.5 min. This analytical method achieves appropriate performance of trueness (85.5-107.4 %), repeatability, and precision (CV < 15 %). Our method will be employed for the therapeutic monitoring of guideline-recommended ARVs in human tissues for monitoring therapeutic efficacy in HIV treatment and prevention research efforts.

Variable temperature-nuclear magnetic resonance experiment and high-resolution MS/MSn measurement of hydroxycarbodenafil, and its PDE5 inhibitory activity.

Hydroxycarbodenafil, an analogue of carbodenafil, was detected in a dietary supplement in China in 2020. However, previous reports have not identified some carbon signals from the piperazine ring in nuclear magnetic resonance (NMR) experiments. Because the compound contains an amide bond, the reaction was suggested to be characteristic of compounds with rotational isomers. Variable-temperature NMR is used to determine the rotational barrier between different conformations by changing the measurement temperature. Using this technique, we succeeded in obtaining the first distinct data, including the carbon signals of the piperazine ring in the NMR spectrum of hydroxycarbodenafil. We also confirmed that this technique could be applied to other carbodenafil analogues. Multi-stage mass spectrometry (MSn) measurements with a high-resolution mass spectrometer specific to the substructures were performed to develop a protocol for the structural determination of the carbodenafil analogues. In addition, hydroxycarbodenafil was analysed using X-ray crystallography, and its inhibitory activity against phosphodiesterase type 5 (PDE5) was measured. The IC50 value of the inhibitory activity of hydroxycarbodenafil for PDE5A1, a PDE5 isoform, of 2.9 nM was lower than the 4.5 nM for sildenafil, a positive control.

Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Electrochemical degradation of antibiotic enoxacin using a novel PbO2 electrode with a graphene nanoplatelets inter-layer: Characteristics, efficiency and mechanism.

A novel PbO2 electrode was fabricated by adding graphene nanoplatelets (GNP) inter-layer into ß-PbO2 active layer (called GNP-PbO2) and utilized to degradation of antibiotic enoxacin (ENO). The GNP-PbO2 electrode had a much rougher surface than the typical PbO2 electrode, with smaller crystal size and lower charge-transfer resistance at the electrode/electrolyte interface. Notably, the GNP inter-layer increased the oxygen evolution potential of PbO2 electrode (2.05 V vs. SCE), which was very beneficial to inhibit oxygen evolution and promote ·OH production. The relatively best operating parameters for ENO removal and energy efficiency were current density of 20 mA cm-2, initial pH of 7, initial ENO concentration of 100 mg L-1 and electrode distance of 4 cm. Furthermore, indirect radical oxidation was found to be the main way during electrolysis process. Based on the observed analysis of intermediate products, the main reaction pathways of ENO included hydroxylation, defluorination and piperazine ring-opening. Finally, combinating with the electro-oxidation capability, stability and safety evaluation, we can conclude that GNP-PbO2 is a promising anode for treatment of various organic pollutants in wastewater.

Detection of unsuspected cathinone and piperazine-type drugs in urine samples positive for methamphetamine and amphetamine collected in emergency departments. / Detección no sospechada de catinonas y piperacinas en pacientes consumidores de metanfetamina y anfetamina atendidos en servicios de urgencias hospitalarios.

OBJECTIVES: To detect the presence of unsuspected and/or undeclared cathinone and piperazine-type designer drugs in methamphetamine (METH) and amphetamine users treated in emergency departments, and to compare clinical and toxicologic profiles. MATERIAL AND METHODS: Retrospective observational study of emergency department patients treated for confirmed acute intoxication by recreational drugs (METH and amphetamines) between March 2019 and December 2020. We ordered high-performance liquid chromatography with tandem mass spectrometry to detect cathinones (methylone, fluoromethcathinone, mexedrone, fluoromethamphetamine, mephedrone, methylenedioxypyrovalerone) and synthetic piperazines (meta-chlorophenylpiperazine and trifluoromethylphenylpiperazine). Demographic, clinical, and toxicologic variables were analyzed with SPSS software (version 23). RESULTS: Thirty-nine patients were included: 24 (61.5%) had used METH and 15 (38.5%) an amphetamine. Synthetic cathinones were detected in samples from 11 patients (28.2%), 10 (90.9%) in the METH group and 1 (9.1%) in the amphetamine group (P = .028). The METH users had taken mephedrone (8 patients) or methylone (2 patients); the amphetamine user had taken mephedrone. None of the patients had declared use of a cathinone; nor was use suspected. The mean (SD) number of substances involved was higher among users of cathinones (3.5 [1.13] vs 2.5 [1.40] in those who took no cathinones; P = .036). Among the cathinone users, 90.9% were men, 90.9% had used METH, and 45.5% had practiced chemsex. HIV positivity was significantly associated with cathinone use (in 45.5% vs 10.7% of those not using cathinones; P = .028). All 5 of the patients who had taken cathinones and also practiced chemsex were HIV positive. Significantly more patients who had taken cathinones presented with anxiety (72.7% vs 21.43%; P = .007). No differences in clinical management were found. CONCLUSION: Detection of METH in intoxicated patients should raise suspicion of probable use of a synthetic cathinone. Patients in whom new psychoactive substances are detected should be kept under observation, and clinical protocols should include referring them to addiction treatment centers.

OBJETIVO: Determinar la incidencia de catinonas y piperazinas, no sospechadas y/o declaradas en consumidores de metanfetamina (MANF) y anfetamina (ANF) atendidos en servicios de urgencias hospitalarios (SUH) y comparar los perfiles clínicos y toxicológicos. METODO: Estudio retrospectivo de pacientes con intoxicación aguda por drogas recreativas con MANF y ANF confirmadas analíticamente atendidos en 3 SUH entre marzo de 2019 y diciembre de 2020. Se detectaron por HPLC-MS/MS las catinonas [metilona, fluorometcatinona, mecedrona, fluorometanfetamina, mefedrona, metilendioxipirovalerona (MDPV)] y las piperazinas sintéticas [meta-clorofenilpiperazina (mCPP), trifluorometilfenilpiperazina (TFMPP)]. RESULTADOS: Se incluyeron 39 pacientes: 24 (61,5%) en el grupo MANF y 15 (38,5%) en el ANF. En 11 (28,2%), se detectaron catinonas sintéticas (grupo CAT), 10 en el grupo MANF (8 mefedrona, 2 metilona) y 1 en el grupo ANF (1 mefedrona) (90,9% vs 9,1%; p = 0,028). Ninguno de los pacientes declaró consumo de catinonas. El número de drogas implicadas en la intoxicación fue superior en el grupo CAT (3,5 [1,13] vs 2,5 [1,40]; p = 0,036). El perfil clínico del grupo CAT fue: varón (90,9%), consumidor de MANF (90,9%) y usuario de chemsex (45,5%). El diagnóstico de VIH se asoció significativamente al grupo CAT (45,5% vs 10,7%; p = 0,028). Los pacientes del grupo CAT presentaron mayor ansiedad (72,7% vs 21,4%; p = 0,007). No se hallaron diferencias en su manejo clínico. CONCLUSIONES: La detección de MANF debería considerarse un dato de sospecha de consumo de catinonas sintéticas, y en esos casos debería contemplarse la detección de nuevas sustancias psicoactivas de abuso.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7-111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.

Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for Attending to Task-Relevant Information.

In a changing environment, organisms need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats completed an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic disruption of the anterior cingulate cortex (Experiment 1) as well as selective disruption of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but facilitated 2 sets of extradimensional-shifts. This pattern of results signals the loss of a corticothalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate medial prefrontal system that promotes the converse pattern, that is, switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these 2 systems regulates cognitive flexibility and choice.

LC-MS/MS Estimation of the Anti-Cancer Agent Tandutinib Levels in Human Liver Microsomes: Metabolic Stability Evaluation Assay.

PURPOSE: Tandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML). MATERIALS AND METHODS: In silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column). RESULTS: The established LC-MS/MS method exhibited linearity (5-500 ng/mL) with r2 ≥0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 µL/min/mg) and in vitro half-life (29.0 min) values. CONCLUSION: TND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.

Concentrations of aripiprazole and dehydroaripiprazole in hair segments from deceased individuals with mental disorders.

Segmental hair analysis provides information regarding previous long-term drug exposure, which is useful in the evaluation of cause of death for individuals with mental disorders. The aim was to analyze postmortem concentrations of the antipsychotic drug aripiprazole and its active metabolite dehydroaripiprazole in hair segments from individuals with known aripiprazole intake. Hair samples were collected during autopsy. Each sample was segmented into one to six 1cm segments, depending on the length of the hair shaft. Pulverized hair was extracted and analyzed using a previously published ultra-high-performance liquid chromatography-tandem mass spectrometric method. The 10th-90th percentile of aripiprazole concentrations in all hair segments (n=78) from 17 individuals were 0.024ng/mg-11ng/mg with a median of 2.3ng/mg, and the 10th-90th percentile concentrations of dehydroaripiprazole were 0.020ng/mg-11ng/mg, with a median of 2.6ng/mg, in all hair segments (n=71). The metabolite-to-parent drug ratios ranged from 0.21 to 1.5, with a median of 0.72. The administered doses were calculated for each individual based on aripiprazole prescription data and pharmacy pickups, giving dose estimates of 1mg-32mg daily. A positive significant correlation was observed between concentrations in hair and blood, whereas no trends were observed between the concentrations in hair and the estimated doses. Besides aripiprazole, other antipsychotic drugs were found in several hair segments, indicating a high degree of polypharmacy among all subjects. The present study establishes concentrations of aripiprazole and dehydroaripiprazole in hair segments from 17 deceased individuals with long-term aripiprazole use. In addition, hair analysis demonstrates the possibility of evaluating polypharmacy.

Mass balance, metabolic disposition, and pharmacokinetics of [14C]ensartinib, a novel potent anaplastic lymphoma kinase (ALK) inhibitor, in healthy subjects following oral administration.

PURPOSE: Ensartinib is a novel, potent and highly selective inhibitor of anaplastic lymphoma kinase (ALK) that has promising clinical activity and low toxicity in patients with ALK-positive non-small cell lung cancer. This study was conducted to investigate the pharmacokinetics, metabolism and excretion of ensartinib following a single 200 mg/100 µCi oral dose of radiolabeled ensartinib to healthy subjects. METHODS: Six healthy male subjects were enrolled and administrated an oral suspension in a fasted state. Blood, urine and feces were collected. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of ensartinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and characterization. RESULTS: The mean total recovery was 101.21% of the radiolabeled dose with 91.00% and 10.21% excreted in feces and urine, respectively. Unchanged ensartinib was the predominant drug-related component in urine and feces, representing 4.39% and 38.12% of the administered dose, respectively. Unchanged ensartinib and its metabolite M465 were the major circulating components, accounting for the same 27.45% of the plasma total radioactivity (AUC0-24h pool), while other circulating metabolites were minor, accounting for less than 10%. Mean Cmax, AUC0-∞, T1/2 and Tmax values for ensartinib in plasma were 185 ng/mL, 3827 h ng/mL, 18.3 h and 3.25 h, respectively. The total radioactivity in plasma was cleared with terminal half-life of 27.2 h. Treatment with ensartinib was well tolerated, and no serious adverse events were reported. CONCLUSION: It was well tolerated in the six healthy male subjects following a single oral administration of 200 mg/100 µCi dose of ensartinib. Besides unchanged ensartinib, metabolite of M465 was the predominant circulating drug-related component. The drug was primarily eliminated in feces. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03804541.

Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C.

Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxins have been linked to sporadic cases of toxicosis in dogs, cattle, and humans, leading some to consider ROQC as a biomarker of PEN-A. Reported here are the development of a monoclonal antibody (mAb) and associated competitive enzyme-linked immunosorbent assay (ELISA) for the screening of ROQC in extracts of nuts (nut "milks"), and dog serum. The ELISA was sensitive for ROQC, with a level of 0.117 ng ml-1 inhibiting colour development by 50% (IC50), a limit of detection of 0.026 ng ml-1, and a dynamic range (IC20 to IC80) of 0.038 to 0.289 ng ml-1 in buffer. The assay was tolerant to significant levels of methanol. Recoveries from 4 types of nut milks spiked over the range of 0.25 to 2 ng ml-1 were in the range of 83.5% to 116%. A small survey of commercial nut "milks" and "creamers" indicated 4 of 35 samples contained ROQC at levels so low that they are unlikely to be significant to human health (<0.6 ng ml-1). The assay was also applied to canine serum. Recoveries from serum spiked over the range of 0.2 to 5 ng ml-1 ranged from 98.1% to 123%. The results suggest the ELISA can be applied to the screening of food products, such as nut extracts, as well as for the screening of serum from dogs suspected to be suffering from mycotoxin-induced tremors.

The phenomenon of para-Fluorophenylpiperazine (pFPP) in combination with the synthetic cannabinoid AMB-FUBINACA in seized plant material in New Zealand.

New psychoactive substances have emerged as a vast and diverse group of illicit drugs over the past decade, with synthetic cannabinoids comprising the largest of the categories. Commonly, a single synthetic cannabinoid is applied to plant material, creating a product that is designed to be smoked by the user. The clandestine preparation process can result in an unevenly distributed product, with varying concentration within and between plant materials. This investigation describes the novel co-detection of the synthetic cannabinoid AMB-FUBINACA, with the piperazine para-fluorophenylpiperazine (pFPP), in a number of plant material samples analysed in New Zealand in 2017. Of 157 samples of plant material containing AMB-FUBINACA, pFPP was detected in 55 of them. A range of pFPP concentrations was observed between the plant material samples, as well as intra-batch variation. The presence of both drugs may be designed to enhance, prolong or balance the psychoactive effects caused from smoking the plant material. However the intended purpose has not been verified. This is the first reported combination of a synthetic cannabinoid and a piperazine in plant material.

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products.

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06-5.30 ng/mL and a limit of quantification of 0.18-16.50 ng/mL, high linearity (R2 > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.

Detection and quantification of synthetic cathinones and selected piperazines in hair by LC-MS/MS.

New psychoactive substances (NPS)-such as synthetic cathinones and piperazines-are defined as substances designed to replicate the effects of traditional illegal drugs, including cocaine, ecstasy and amphetamines. These substances are known to potentially be much more potent than their analogs. In the past, there were many poisonings and deaths associated with NPS. Because of this, NPS identification and quantification have become more important in forensic toxicology. The present work aimed to develop, validate and apply a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of detecting 35 synthetic cathinones and piperazines in hair samples. All target analytes were resolved in a 12 min run time and identified based on the quantifier ion, at least one product ion and the retention time. Depending on the analyte, the calibration curves were linear over a maximal range of 0.01-3 ng/mg. The limits of detection and quantification were within the ranges of 0.006-0.052 ng/mg and 0.008-0.095 ng/mg, respectively. The precision, bias and matrix effect were all within acceptable GTFCh thresholds and the method was free from interferences. The validated method was successfully used to identify synthetic cathinones and piperazines in authentic hair samples (n = 40) from forensic cases, demonstrating its suitability for the screening and quantification of a wide number of new stimulants in hair specimens.