Discovery of a Novel Series of Imidazopyrazine Derivatives as Potent SHP2 Allosteric Inhibitors.

Protein tyrosine phosphatase SHP2 is an oncogenic protein that can regulate different cytokine receptor and receptor tyrosine kinase signaling pathways. We report here the identification of a novel series of SHP2 allosteric inhibitors having an imidazopyrazine 6,5-fused heterocyclic system as the central scaffold that displays good potency in enzymatic and cellular assays. SAR studies led to the identification of compound 8, a highly potent SHP2 allosteric inhibitor. X-ray studies showed novel stabilizing interactions with respect to known SHP2 inhibitors. Subsequent optimization allowed us to identify analogue 10, which possesses excellent potency and a promising PK profile in rodents.

Fatty acid acylated peptide therapeutics: discovery of omega-n oxidation of the lipid chain as a novel metabolic pathway in preclinical species.

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

T2Bacteria and T2Resistance Assays in Critically Ill Patients with Sepsis or Septic Shock: A Descriptive Experience.

The use of rapid molecular tests may anticipate the identification of causative agents and resistance determinants in the blood of critically ill patients with sepsis. From April to December 2021, all intensive care unit patients with sepsis or septic shock who were tested with the T2Bacteria and T2Resistance assays were included in a retrospective, single center study. The primary descriptive endpoints were results of rapid molecular tests and concomitant blood cultures. Overall, 38 combinations of T2Bacteria and T2Resistance tests were performed. One or more causative agent(s) were identified by the T2Bacteria assay in 26% of episodes (10/38), whereas negative and invalid results were obtained in 66% (25/38) and 8% (3/38) of episodes, respectively. The same pathogen detected by the T2Bacteria test grew from blood cultures in 30% of cases (3/10). One or more determinant(s) of resistance were identified by the T2Resistance assay in 11% of episodes (4/38). Changes in therapy based on T2Bacteria and/or T2Resistance results occurred in 21% of episodes (8/38). In conclusion, T2Bacteria/T2Resistance results can influence early treatment decisions in critically ill patients with sepsis or septic shock in real-life practice. Large, controlled studies remain necessary to confirm a favorable impact on patients' outcomes and antimicrobial stewardship interventions.

Subcutaneous catabolism of peptide therapeutics: bioanalytical approaches and ADME considerations.

Many peptide drugs such as insulin and glucagon-like peptide (GLP-1) analogues are successfully administered subcutaneously (SC). Following SC injection, peptides may undergo catabolism in the SC compartment before entering systemic circulation, which could compromise their bioavailability and in turn affect their efficacy.This review will discuss how both technology and strategy have evolved over the past years to further elucidate peptide SC catabolism.Modern bioanalytical technologies (particularly liquid chromatography-high-resolution mass spectrometry) and bioinformatics platforms for data mining has prompted the development of in silico, in vitro and in vivo tools for characterising peptide SC catabolism to rapidly address proteolytic liabilities and, ultimately, guide the design of peptides with improved SC bioavailability.More predictive models able to recapitulate the interplay between SC catabolism and other factors driving SC absorption are highly desirable to improve in vitro/in vivo correlations.We envision the routine incorporation of in vitro and in vivo SC catabolism studies in ADME screening funnels to develop more effective peptide drugs for SC delivery.

Oligomerization, albumin binding and catabolism of therapeutic peptides in the subcutaneous compartment: An investigation on lipidated GLP-1 analogs.

Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Doping control analysis of small peptides: A decade of progress.

Small peptides are handled in the field of sports drug testing analysis as a separate group doping substances. It is a diverse group, which includes but is not limited to growth hormone releasing-factors and gonadotropin-releasing hormone analogues. Significant progress has been achieved during the past decade in the doping control analysis of these peptides. In this article, achievements in the application of liquid chromatography-mass spectrometry-based methodologies are reviewed. To meet the augmenting demands for analyzing an increasing number of samples for the presence of an increasing number of prohibited small peptides, testing methods have been drastically simplified, whilst their performance level remained constant. High-resolution mass spectrometers have been installed in routine laboratories and became the preferred detection technique. The discovery and implementation of metabolites/catabolites in testing methods led to extended detection windows of some peptides, thus, contributed to more efficient testing in the anti-doping community.

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

The Role of Pharmacogenetics in Antithrombotic Therapy Management: New Achievements and Barriers Yet to Overcome.

BACKGROUND: Pharmacogenetics investigates the response to pharmacological treatments based on individual genetic background. Actually, numerous pharmacogenetic tests help to predict the response to drugs used in different medical areas, contributing to the so-called personalized medicine. OBJECTIVE: This review aims to update the available data on the genotype-guided treatment with both the anticoagulant and antiplatelet agents. Moreover, it sheds light on the pitfalls in the implementation of cardiovascular pharmacogenetics. METHODS: A review of the literature on the studies investigating the effects of the genotype- guided anticoagulant and antiplatelet treatment was performed. RESULTS: Considering the extensive use of antithrombotic drugs, pharmacogenetics has particular importance in this field. Several polymorphisms influence the response to both anticoagulant and antiplatelet agents, and tests, based on their identification, are now available. CONCLUSION: Recent randomized clinical trials demonstrated that pharmacogenetics might successfully contribute to optimizing the antiplatelet therapy also in patients particularly complicated to treat. However, despite accumulating evidence on the utility and feasibility of some pharmacogenetics tests, several barriers still exist in their implementation in clinical practice.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Comparison of different protein precipitation and solid-phase extraction protocols for the study of the catabolism of peptide drugs by LC-HRMS.

LC-HRMS-based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent-related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid-phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP-2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (HR ) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed-mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP-2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the HR and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvß3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)⁻cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvß3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.

A liquid chromatography high-resolution mass spectrometry in vitro assay to assess metabolism at the injection site of subcutaneously administered therapeutic peptides.

Subcutaneous (SC) injection is the most common administration route for peptide therapeutics. Catabolism at the injection site can be a specific and major degradation pathway for many SC administered peptides. In some cases, it can significantly affect pharmacokinetics, particularly bioavailability, and have detrimental effects on the efficacy of the drug. This work describes a liquid chromatography-high resolution mass spectrometry based in vitro assay to assess peptide metabolism in the SC tissue (SCiMetPep assay). The SCiMetPep assay was developed using human, Sprague-Dawley rat and Göttingen minipig SC tissue homogenate supernatant, and allows for both determination of degradation rate (half-life) of the parent peptide and identification of metabolites generated from enzymatic proteolysis. The assay was developed and validated using known peptides including human insulin and four GLP-1 analogues (lixisenatide, exenatide, liraglutide and semaglutide). Different experimental parameters were evaluated in order to optimize the homogenization process of the SC tissue and the peptide incubation conditions. In vitro metabolism of these peptides was in good agreement with in vivo data reported in the literature. Finally, when SCiMetPep assay was applied on a series of structurally related peptides, a fairly good correlation was found between SC metabolic stability and bioavailability, suggesting that catabolism at the injection site can have a major role in the absorption, distribution, metabolism, and excretion (ADME) of peptide therapeutics. The SCiMetPep showed the ability to identify analogs with improved SC metabolic stability and hence higher bioavailability. The assay can be used in the early phases of drug discovery to identify peptide metabolic soft spots at the injection site and guide the peptide drug discovery process.

Optical calibration and performance of the adaptive secondary mirror at the Magellan telescope.

In this paper we describe the procedure for the optical calibration of large size deformable mirrors, acting as wavefront correctors for adaptive optics systems. Adaptive optics compensate the disturbance due to the atmospheric turbulence to restore the telescope resolution. We will showcase in particular the activities performed for the Adaptive Secondary Mirror (ASM) of the Magellan Adaptive Optics system (MagAO), which is an instrument for the 6.5 m Magellan Clay Telescope, located at Las Campanas Observatory, in Chile. The MagAO ASM calibration is part of the MagAO-2K project, a major MagAO upgrade that started in 2016 with the goal of boosting adaptive optics (AO) correction at visible wavelengths to image exoplanets. For the first time, the optical quality of MagAO mirror is reported. We describe the procedures developed to achieve high SNR interferometric measurements of the ASM modes under the presence of dome convection noise and telescope vibrations. These measurements were required to produce an improved control matrix with up to 500 modes to close the AO loop on sky with enhanced performances. An updated slaving algorithm was developed to improve the control of actuators vignetted by the central obscuration. The calibrations yielded also a new ASM flattening command, updating the one in use since the MagAO commissioning in 2013. With the new flattening command, a 22 nm RMS surface error was achieved. Finally, we present on-sky results showing the MagAO performance achieved with the new calibrations.

An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides.

In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compounds, including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metabolism, and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclinical in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quantitative determination of metabolic stability of peptides and qualitative identification/structural elucidation of their metabolites in preclinical plasma metabolic stability studies using liquid chromatography-high-resolution Orbitrap™ mass spectrometry (LC-HRMS). Sample preparation was based on protein precipitation: experimental conditions were optimized after evaluating and comparing different organic solvents in order to obtain an adequate extraction of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liquid chromatography: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelectric points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in positive full scan + data-dependent MS/MS acquisition mode, and then processed to calculate plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder™. Finally, as an example of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial peptides is described. This approach was developed for the evaluation of in vitro plasma metabolic stability studies of peptides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes). Graphical Abstract Left: trend plot for omiganan and major metabolites. Right: stability plot for five antimicrobial peptidesafter incubation with mouse plasma.

Liposomes as potential masking agents in sport doping. Part 1: analysis of phospholipids and sphingomyelins in drugs and biological fluids by aqueous normal-phase liquid chromatography-tandem mass spectrometry.

In the present work, aqueous normal-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H]+ were detected in positive precursor ion scan of m/z 184 for the classes of phosphatidylcholines, lyso-phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso-phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M-H]- were instead acquired in negative precursor ion scan of m/z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m/z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO]- ions for phosphatidylcholines and [M + H]+ ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid-based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd.

Liposomes as potential masking agents in sport doping. Part 2: Detection of liposome-entrapped haemoglobin by flow cytofluorimetry.

This work presents an analytical procedure for the identification and characterization of liposome-entrapped haemoglobins, based on flow cytofluorimetry. Flow cytofluorimetric detection is carried out following labelling by two distinct fluorescent reagents, an anti-haemoglobin antibody, fluorescein isothiocyanate conjugated, and an anti-poly(ethylene glycol) antibody, streptavidin-phycoerythrin conjugated. This experimental strategy allows the detection of liposome-entrapped haemoglobins in aqueous media, including plasma; the efficacy of the proposed approach has been verified on whole blood samples added with the liposomal formulation (ex-vivo). Additionally, the proposed technique allows the characterization of several key parameters in the study of liposomal haemoglobins, including, for instance (1) the determination of the degree of haemoglobin entrapment by liposomes; (2) the poly(ethylene glycol) insertion efficiency; and (3) the evaluation of liposome-entrapped haemoglobins stability following storage at 4 °C, allowing to follow both the process of haemoglobin loss from liposomes and the liposome degradation. The procedure is proposed for the detection and characterization of liposome-entrapped haemoglobin formulations to control their misuse in sport, but is also suggested for further applications in biological and clinical laboratory investigations. Copyright © 2016 John Wiley & Sons, Ltd.

Use of 'dilute-and-shoot' liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma.

This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.