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Cobalt, chromium, and nickel are used in orthopedic prostheses. They can be released, accumulate in many organs, and be toxic. The aim of this study is to evaluate the cytotoxicity of these metals on human hepatocytes and to improve our knowledge of their cellular toxicity mechanisms by metabolomic analysis. HepaRG cells were incubated for 48 h with increasing concentrations of metals to determine their IC

Fingermark patterns are one of the oldest means of biometric identification. During this last decade, the molecules that constitute the fingermark residue have gained interest among the forensic research community to gain additional intelligence regarding its donor profile including its gender, age, lifestyle or even its pathological state. In this work, the molecular composition of fingermarks have been studied to monitor the variability between donors and to explore its capacity to differentiate individuals using supervised multi-class classification models. Over one year, fingermarks from thirteen donors have been analysed by Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (n = 716) and mined by different machine learning approaches. We demonstrate the potential of the fingermark chemical composition to help differentiating individuals with an accuracy between 80% and 96% depending on the period of sample collection for each donor and size of the pool of donors. It would be premature at this stage to transpose the results of this research to real cases, however the conclusions of this study can provide a better understanding of the variations of the chemical composition of the fingermark residue in between individuals over long periods and help clarifying the notion of donorship.

Microsampling techniques became more popular in the last decades, and their use for common analyses such as trace element quantification by inductively coupled plasma mass spectrometry (ICP-MS) has been investigated. We decided to compare two of these techniques (dried blood spots and microtubes) to evaluate their potential for the analysis of 12 trace elements in human whole blood: aluminum (Al), total arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn). Signal contributions from blank filter paper and instability at room temperature for several elements in the dried blood spot samples restrained our enthusiasm for the use of this technique. Conversely, microtube samples presented low background contamination and good stability under different temperature conditions. Therefore, our results demonstrate that the use of microtubes is more suitable than dried blood spots for trace element quantification in human blood, both in research and routine analysis.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health. ispartof: TOXICOLOGY IN VITRO vol:81 ispartof: location:England status: published

BackgroundAccording to the World Health Organization, road traffic injuries lead to 1.3 million deaths each year and represent the first cause of death for young adults under 30 years old. The use of psychoactive substances, including alcohol, drugs and pharmaceuticals is a well-known risk factor for road traffic injuries. Our study aims to assess the prevalence of substances consumed by drivers in Western Switzerland. Such studies are capital to improve prevention and develop public awareness campaigns.MethodsTo assess the prevalence of psychoactive substances among drivers, roadside controls were performed in collaboration with local polices, using their classical sampling procedures to detect drivers under the influence of drugs or alcohol, over two time periods (P1: 2006-2008, P2: 2017-2020). When impaired-driving was not suspected by the police, minimal-invasive sampling strategies were performed on volunteer drivers, after a road safety survey. A posteriori analyses and statistical interpretation were then performed.ResultsAmongst the 1’605 drivers included in the study, 1’048 volunteers provided an oral fluid sample, whilst 299 provided a dried blood spot sample. The percentage of drivers testing positive to at least one substance which may impair driving ability was stable over time, with a rate of 10.5% of positivity measured over both periods. The proportion of medicinal drugs and drugs of abused showed a slight variation between both periods, with 7.6 % and 6.3 % of prescribed drugs and 3.6 % and 4.9 % of illicit drugs for P1 and P2 respectively. Regarding the consumption of illicit drugs, the highest percentage of positivity was measured in biological fluids of drivers under the age of 35, during nights and week-ends, periods which are considered particularly prone to fatal accidents for this age group. Disturbingly, the road safety survey highlighted that drivers’ perception of the risk of getting positively controlled whilst driving after drug consumption is low (3.3 on a 1-to-10 scale, N=299).ConclusionThe number of positive cases measured in voluntary drivers who passed the preliminary police check demonstrates the importance of systematic biofluid sampling strategies regarding driving under the influence of psychoactive substances. Although the number of fatal road accidents globally decreases overtime, the results of this study reveal the need for both better prevention and deterrent processes that could potentially reduce the risk of fatal road accidents associated with drug consumption.

Chronic inflammation that affects primarily metabolic organs, such as white adipose tissue (WAT), is considered as a major cause of human obesity-associated co-morbidities. However, the molecular mechanisms initiating this inflammation in WAT are poorly understood. By combining transcriptomics, ChIP-seq and modeling approaches, we studied the global early and late responses to a high-fat diet (HFD) in visceral (vWAT) and subcutaneous (scWAT) AT, the first being more prone to obesity-induced inflammation. HFD rapidly triggers proliferation of adipocyte precursors within vWAT. However, concomitant antiadipogenic signals limit vWAT hyperplastic expansion by interfering with the differentiation of proliferating adipocyte precursors. Conversely, in scWAT, residing beige adipocytes lose their oxidizing properties and allow storage of excessive fatty acids. This phase is followed by tissue hyperplastic growth and increased angiogenic signals, which further enable scWAT expansion without generating inflammation. Our data indicate that scWAT and vWAT differential ability to modulate adipocyte number and differentiation in response to obesogenic stimuli has a crucial impact on the different susceptibility to obesity-related inflammation of these adipose tissue depots. Electronic supplementary material The online version of this article (10.1007/s00018-020-03485-z) contains supplementary material, which is available to authorized users.

Cannabis is the most consumed drug of abuse, making it the primary target for identification and quantification in human whole blood regarding forensic and clinical toxicology analyses. Among biological matrices, blood is the reference for toxicological interpretation. A highly sensitive and selective liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS) was developed for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxytetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and cannabidiol (CBD). Those cannabinoids were extracted from 1 mL of whole blood by a simple liquid–liquid extraction (LLE) in acidic conditions. HRMS was performed on an Orbitrap-based instrument using its trapping capabilities and increased selectivity for parallel reaction monitoring (PRM) quantification in positive polarity with a negative polarity switching for THC-OH and THC-COOH. Although selected reaction monitoring (SRM) and PRM-targeted methods have similar performance in terms of linearity, dynamic range, precision and repeatability, Orbitrap-based PRM provides a higher specificity due to the use of high-resolution mode separating background ions from the targeted molecules. The method was fully validated according to guidelines set forth by the “Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP). Trueness was measured below 107% for all tested concentrations. Repeatability and intermediate precision were found to be lower than 12% while the assay was found to be linear in the concentration range of 0.4–20 ng/mL for THC, THC-OH and CBD and of 2–100 ng/mL for THC-COOH. Recovery (RE) and matrix effect (ME) ranged from 70.6 to 102.5% and from −40 to 6.6%, respectively. The validated method provides an efficient procedure for the simultaneous and rapid quantification of cannabinoids in PRM mode providing an alternative over classical SRM.

These past years, the chemical composition of fingermarks have attracted interest of researchers to meet multiple objectives like the determination of an individual's age, gender or lifestyle or the impact of some fingermark detection processes, to cite a few. These studies have highlighted the need to investigate the consistency of the fingermark composition over time. This research explores the evolution of the secretion residue composition of thirteen donors over one year, focusing on the intravariability. The dual use of Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI-MSI) and chemometrics provided valuable data regarding the evolution of composition over time as well as the consistency of presence of hundreds of compounds.

In the last decade, there have been many scientific developments regarding the use of mass spectrometry to analyse the composition of fingermarks. In this context, the development of a dedicated quality control test strip would benefit the forensic community by providing a way to assess the reproducibility of the measures as well as to perform inter-laboratory comparisons. To accomplish this goal, the use of a chemical printer offers the possibility of combining a visual template with artificial fingerprint secretions. The design of the quality control test strip as well as the preliminary assessment of its performance with fingermark detection reagents and matrix-assisted laser desorption-ionisation combined with mass spectrometry imaging (MALDI-MSI) are presented in this paper. The chosen template combines two geometric patterns intended to help assess the chemical analysis (full square) and imaging (lined square) capabilities of the instrument. The artificial secretion is composed of two distinct solutions: artificial sweat and artificial sebum. The printing reproducibility and chemical homogeneity of the quality control test strips were assessed in two ways: (1) using MALDI-MSI, the printed pattern was analysed and the m/z values compared to the reference list based on the artificial secretion composition, and (2) using two common fingermark detection techniques, the printed pattern was processed using an amino acid reagent (ninhydrin) and a lipid stain (Oil Red O). Overall, the results highlight the potential of a printed quality control test strip for the assessment of the quality of fingermark detection techniques as well as the possibility of performing quality monitoring of mass-spectrometry-based techniques over time.

Background and purpose Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography-high-resolution mass spectrometry-based metabolomic approach. Experimental approach Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key results In this study, we reported four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared to the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Furthermore, mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared to other volunteers. Conclusion and implications The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalised medicine.

Background HIV-infected patients display an increased risk of cardiovascular events. HIV and antiretroviral therapy (ART) have both been suggested to increase this risk but their specific role remains unclear. Autoantibodies against apolipoprotein A1 (anti-apoA1 IgG) are also associated with cardiovascular risk. The aim of this study was to analyse anti-apoA1 IgG levels and inflammatory markers as well as the metabolomic profile in a cohort of HIV-free, ART-experienced and naïve HIV-infected patients. Methods 144 participants from a South African cohort were categorised into HIV-free (n=50), HIV-infected/ART-experienced (n=50) and HIV-infected/ART-naïve (n=44) groups. The levels of anti-apoA1 IgG were assessed by homemade ELISA assays. We performed targeted metabolomic analyses using standard operating procedures and measured inflammatory biomarkers using the Meso Scale Discovery® platform. Additionally, we conducted in vitro experiments on human aortic endothelial cells treated with anti-apoA1 IgG and assessed levels of adhesion molecules. Results Inflammatory markers (IFNg, IL-6, IL-10, TNFa, CRP all p While ART treatment significantly reduced IFNg, IL-10, TNFa, VCAM-1, anti-apoA1 IgG levels, no significant differences were observed in other biomarkers between ART-experienced and naïve participants. Treatment of human aortic endothelial cells with anti-apoA1 IgG (40μg/ml) induced a significant increase in these adhesion molecules compared to cells treated with control IgG (40μg/ml). Metabolomic analyses identified 257 endogenous circulating metabolites in plasma. Among these metabolites, we found that in HIV-free and infected patients 14 and 17 metabolites were significantly correlated with anti-apoA1 IgG levels, respectively. Among these 17 metabolites, 2 were positively correlated with the tryptophan pathway; kynurenine (r=0.292, p=0.0069) and 5-hydroxyindolacetate (r=0.316, p=0.0019). Interestingly, kynurenine was not significantly correlated with anti-apoA1 IgG levels in HIV-free patients. Conclusion HIV-infected patients displayed a more proatherogenic profile compared to HIV-free subjects. Anti-apoA1 IgG induced adhesion molecules expression. Metabolomics identified metabolites that were significantly associated with anti-apoA1 IgG levels and inflammatory biomarkers known to underlie atherosclerosis burden. Funding Acknowledgement Type of funding sources: None.

For the quantification of the pineal hormone melatonin and its metabolite, 6-hydroxymelatonin, in human overnight urine, a single accurate method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Urine samples were deconjugated using β-glucuronidase/arylsulfatase from Helix pomatia before solid phase extraction (SPE) purification. Chromatographic separation was performed using a reverse phase C18 column with a 7-minute gradient elution. Water was used as matrix to prepare the calibration standards, and deuterated analogues of melatonin and 6-hydroxymelatonin were used as internal standards. This newly developed method was validated in terms of linearity, accuracy, repeatability, intermediate precision, recovery, matrix effect, and stability according to the guidelines of the European Medicines Agency. The method was successfully applied to the analysis of overnight urine samples from 12 healthy volunteers, showing significant correlations of urinary melatonin and 6-hydroxymelatonin excretion rates with age. The urinary 6-hydroxymelatonin to melatonin ratio was also established and will be assessed in further studies as a potential endogenous metric of CYP1A2 activity.

Drug response is subject to an important within- and between-individual variability owing, mainly, to pharmacokinetic and pharmacodynamic factors. Pharmacokinetics includes metabolism by cytochrome P450 (CYP450), major enzymes of phase I reactions that are responsible for the biotransformation of around 60% of the currently approved drugs. CYP450 activity and/or expression are influenced by multiple intrinsic and extrinsic factors, such as drug-drug interactions or genetic polymorphisms. Present phenotyping strategies with xenobiotics used to assess CYP450 activity could be replaced by less invasive procedures using endogenous CYP450 biomarkers. In this work, we review existing knowledge on endobiotics and their ability to characterise variability of the CYP1A2, CYP2C19, CYP2D6 and CYP3A enzymes in humans. To date, it appears that there is a lack of clinical data for the majority of the endogenous compounds described in the literature or some important limitations to allow their use in clinical practice. Additional studies are needed to fill the gap or to identify new candidates, in particular through the use of metabolomics. The use of multivariate models is also a very promising approach to enhance prediction by combining several endogenous phenotyping metrics and other covariates.

Addiction is a complex brain disease, whose molecular and cellular underpinnings remain largely unknown. Repeated drug use induces long-term plasticity in various brain areas that is considered to potentially trigger further compulsive use. Converging evidence has revealed that memory and addiction share both neural circuitry and molecular mechanisms. While many studies investigated the mesocorticolimbic system, the hippocampus has become a region of interest. Matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) presents a unique ability to map the toxicodynamic changes associated to drugs of abuse. In this study, the acute effect of cocaine in rat brain was investigated with or without co-administration of alcohol. Brain tissue sections for each condition were analyzed using a MALDI-MSI in negative polarity after 9-aminoacridine (9-AA) deposition on the tissue surface. Multivariate analysis including principal component analysis (PCA) and spatial shrunken centroids have been performed to differentiate the metabolic profiles in hippocampus according to the type of administration. This study offers promising results showing the potential of MSI for investigating the metabolomic changes in the brain associated with drug consumption. It may bring new insights into the study of toxicodynamic responses of cocaine consumption in reward-learning related regions.

To date, a considerable amount of research has been devoted to the study of fingermark composition to serve many goals, such as the assessment of variability between donors, the impact of detection sequences, the understanding of detection mechanisms, and the determination of the donor's gender, age or lifestyle. These developments raise the question of the degrees of variability and consistency over time, characterizing the secretion residue between individuals or for a given donor. In this preliminary study, the use of Matrix-Assisted Laser Desorption/Ionization combined with Mass Spectrometry and Imaging (MALDI-MSI) was investigated to explore the intra- and intervariability of the secretion residue composition. The combined use of MALDI-MSI with chemometrics tools permitted to sort fingermarks according to their type (i.e., eccrine, sebum-rich, or natural), to emphasize a short-term consistency of composition for a given donor, and to differentiate fingermarks from four individuals. These results support the use of MALDI-MSI to investigate to which extent the fingermark composition may vary or remain consistent over time. This research will be pursued by considering a larger set of donors and an extended period of time. Ultimately, it is expected to assess how chemometric analysis of secretion residue composition could benefit research and investigative purposes linked to fingermarks.

The number of people affected by Type 2 diabetes mellitus (T2DM) is close to half a billion and is on a sharp rise, representing a major and growing public health burden. Given its mild initial symptoms, T2DM is often diagnosed several years after its onset, leaving half of diabetic individuals undiagnosed. While several classical clinical and genetic biomarkers have been identified, improving early diagnosis by exploring other kinds of omics data remains crucial. In this study, we have combined longitudinal data from two population-based cohorts CoLaus and DESIR (comprising in total 493 incident cases vs. 1360 controls) to identify new or confirm previously implicated metabolomic biomarkers predicting T2DM incidence more than 5 years ahead of clinical diagnosis. Our longitudinal data have shown robust evidence for valine, leucine, carnitine and glutamic acid being predictive of future conversion to T2DM. We confirmed the causality of such association for leucine by 2-sample Mendelian randomisation (MR) based on independent data. Our MR approach further identified new metabolites potentially playing a causal role on T2D, including betaine, lysine and mannose. Interestingly, for valine and leucine a strong reverse causal effect was detected, indicating that the genetic predisposition to T2DM may trigger early changes of these metabolites, which appear well-before any clinical symptoms. In addition, our study revealed a reverse causal effect of metabolites such as glutamic acid and alanine. Collectively, these findings indicate that molecular traits linked to the genetic basis of T2DM may be particularly promising early biomarkers.

Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6–12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX—a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

The paper describes the first three deaths reported in Europe involved in isotonitazene consumption, a potent benzimidazole derivate opioid consumed in the recreational drug scene. Isotonitazene powder and purity determination was performed on the sample collected in the first death scene by NMR, HRMS, GC-FTIR, ATR-FTIR and GC-MS. Isotonitazene purity was determined by GC-MS analysis and proton NMR, and was defined to be above 95 % and 98 %, respectively. Quantification of isotonitazene in biological samples was performed using a targeted analysis based on SPE extraction and ultra-high performance liquid chromatography tandem mass spectrometry. The isotonitazene median concentration in femoral whole blood was 1.20ng/mL. Isotonitazene concentration in hair was similar or even lower compared to that seen in fentanyl abusers. Isotonitazene distribution in tissues converges in the brain, lungs and heart, respectively. Surprisingly, isotonitazene concentration in liver is the lowest measured for all tissues and fluids analyzed. Based on circumstantial evidence, autopsy findings and the results of the toxicological analysis, the medical examiner concluded that the cause of all three deaths was an acute intoxication with isotonitazene. Since isotonitazene toxic concentration levels are very low, the consumption of this new psychoactive drug is a real hazard for human health.

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High‐density lipoprotein (HDL) has been proposed to improve β‐cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine‐1‐phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic β‐cells and to determine its mechanisms. Primary cultures of β‐cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL‐S1P) isolated from T2DM patients was analyzed and correlated to the HDL‐induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose‐stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL‐S1P was strongly correlated to its pro‐secretory capacity (r = 0.633, p = 0.005), HDL‐cholesterol and apolipoprotein AI levels were not. HDL‐induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in β‐cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL‐S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra‐ and extra‐cellular sources of S1P independently of an effect on insulin biosynthesis genes., HDL improves insulin secretion by beta cells S1P plays a role in HDL‐induced insulin secretion HDL increases intracellular S1P content.

In this study, we assessed the potential use of the 1β-hydroxy-deoxycholic acid (1β-OH-DCA) to deoxycholic acid (DCA) urinary metabolic ratio (UMR) as a CYP3A metric in ten male healthy volunteers. Midazolam (MDZ) 1 mg was administered orally at three sessions: alone (control session), after pre-treatment with fluvoxamine 50 mg (12 h and 2 h prior to MDZ administration), and voriconazole 400 mg (2 h before MDZ administration) (inhibition session), and after a 7-day pre-treatment with the inducer rifampicin 600 mg (induction session). The 1β-OH-DCA/DCA UMR was measured at each session, and correlations with MDZ metrics were established. At baseline, the 1β-OH-DCA/DCA UMR correlated significantly with oral MDZ clearance (r = 0.652, p = 0.041) and Cmax (r = −0.652, p = 0.041). In addition, the modulation of CYP3A was reflected in the 1β-OH-DCA/DCA UMR after the intake of rifampicin (induction ratio = 11.4, p &lt, 0.01). During the inhibition session, a non-significant 22% decrease in 1β-OH-DCA/DCA was observed (p = 0.275). This result could be explained by the short duration of CYP3A inhibitors intake fixed in our clinical trial. Additional studies, particularly involving CYP3A inhibition for a longer period and larger sample sizes, are needed to confirm the 1β-OH-DCA/DCA metric as a suitable CYP3A biomarker.

This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of r s = 0.9978 for R-methadone and r s = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results. Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation.

Background and Purpose: Individualized assessment of the activity of cytochrome P450 2D6 (CYP2D6), a highly variable drug-metabolizing enzyme, is performed through phenotyping during which a probe drug is administered to measure the enzyme’s activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-invasive tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine in clinical practice. This study focuses on the identification of endogenous markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS)-based metabolomics approach. Experimental Approach: Data from a control session were compared to data from an inhibition session. Before the latter, healthy volunteers (extensive and ultrarapid metabolizers) received a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key Results: In CYP2D6 extensive and ultrarapid metabolizers (n = 37), mean relative intensities of five features were significantly reduced during the inhibition session compared to the control session (fold changes ≤ 0.67, FDR-adjusted P < 0.0001). Furthermore, mean relative intensities of these candidates were significantly higher in the CYP2D6 extensive-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6) (fold changes ≤ 0.67, P < 0.0001). Conclusion and Implications: The applied untargeted metabolomics strategy was able to identify five CYP2D6 endogenous metabolites, a promising discovery for non-invasive phenotyping and personalised medicine.

Redundant robots can provide improved kinematic performances compared to non-redundant robots, becoming suitable to perform complex tasks in several areas of application. Improved manipulability or dexterity and a larger workspace are some of the advantages of using redundant robots. This paper aims to present the design and development of an 8-DoF redundant robotic platform for medical applications. The platform is composed of a 7-DoF anthropomorphic cobot, i.e. Franka Emika, whose base is coupled to a 1-DoF linear axis. The aim of the linear axis is to enlarge the workspace of the robot. Robot-assisted Doppler sonography is presented as an example of medical application, where the robot, employed as probe-holder, is able to traverse the whole patient’s body during the exam.