References of "Fillet, Marianne"
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See detailCapillary electrophoresis-mass spectrometry of derivatized amino acids for targeted neurometabolomics – pH mediated reversal of diastereomer migration order
Moldovan, Radu-Cristian ULiege; Bodoki, Ede; Servais, Anne-Catherine ULiege et al

in Journal of Chromatography. A (in press)

A targeted CE-MS approach was developed for the chiral analysis of biologically relevant amino acids in artificial cerebrospinal fluid (aCSF). In order to achieve chiral resolution, the five amino acids ... [more ▼]

A targeted CE-MS approach was developed for the chiral analysis of biologically relevant amino acids in artificial cerebrospinal fluid (aCSF). In order to achieve chiral resolution, the five amino acids (Ser, Asn, Asp, Gln and Glu) were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate ((+)-FLEC). The diastereoselectivity was found to be highly dependent on pH for all analytes and the optimized background electrolyte (BGE) consisted of 150 mM acetic acid, adjusted to pH 3.7 with NH4OH. Furthermore, a reversal of the migration order of Asp derivatives was observed. This phenomenon seems to be caused by intra-molecular interactions affecting the pKa of the second ionizable group (the side chain carboxyl). The applicability of this method was evaluated using aCSF. A solid phase extraction (SPE) protocol was developed for the selective extraction of the FLEC derivatives. A full evaluation of the matrix effect and extraction yield was performed concluding that the matrix effect is marginal and the recoveries are between 46 and 92%. The method offers adequate sensitivity (limits of detection below 1 μM). [less ▲]

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See detailCapillary electrophoretic mobility shift competition assay for the assessment of weak drug-protein interactions
Farcas, Elena ULiege; Hanson, Julien ULiege; Pochet, Lionel et al

in Analytica Chimica Acta (in press)

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic ... [more ▼]

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used in FBDD for primary screening of weak biomolecular interactions between fragments and target protein. The accuracy and reliability of the present method was demonstrated by measuring the interaction between two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat. The measured IC50 were found to be in good accordance with the previously reported ones. Furthermore, we built a small chemical library to evaluate the performance and the advantage of our newly developed screening-bioassay compared to the direct affinity capillary electrophoresis-binding assay. The results demonstrate the high discriminatory power of the method and above all its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, greatly expanding the scope of the assay. Finally, we proved that this approach is able to discriminate between competitive binders and irreversible binders. Altogether, this work demonstrates that capillary electrophoresis could constitute an important added value in the arsenal used to screen fragments in drug discovery. [less ▲]

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See detailCESI-MS Workflow for Protein Quantification
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; CAVALIER, Etienne ULiege et al

Poster (2018, October)

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as ... [more ▼]

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as migration and injection conditions. Nevertheless, these parameters influence each other and require methodical optimization to get the most of each instrument. In the present work we share our experience with sheathless CE-MS and neutral coating to analyze peptide and protein samples. Methods: Experiments were conducted on a CESI 8000 capillary electrophoresis holding a neutral coated OptiMS cartridge and coupled to a QT 6500 mass spectrometer. Separation buffer and voltage, curtain gas and source temperature were conserved through experiments. Separation pressure and source voltage were optimized while applying voltage and pressure on separation buffer spiked with the peptide used (pI 9.5 marker from the Advance cIEF starter kit by Sciex). A daily reference run was used to compare modifications to the injection despite variable capillary performance. Finally, shifts in spray voltage due to injection parameters were determined using sequences of runs with different spray voltages. Preliminary results: Decreasing separation pressure from 5 to 1.5 psi increased peptide intensity; electrokinetic injection (EKI) increased peak intensity compared to hydrodynamic injection (HDI); the HDI of a water plug before the EKI increased peak intensity further, as well as a high percentage of acetonitrile in the sample medium. Finally, we compared our initial and our final conditions. In both cases, a positive Q1 scan of 1000 Da/s for m/z 300 to 1000 was acquired, and the electropherograms display the extracted ion current for a 1 m/z interval centered on the m/z of the doubly charged peptide. In the initial method, the peptide was diluted in BGE and was introduced by HDI (1 % of total length); 5 psi pressure were applied to both inlet and outlet; source voltage was 1800 V. When analyzing a 1:160 (v/v) dilution of the peptide, the intensity recorded for [M+2H]2+ was 9.3e7 counts. In the final method, the peptide was diluted in 75:25 acetonitrile:water (v/v) and was introduced by EKI (+ 10 kV 100s). Before the EKI, a HDI of water (0.5 % of total length) was performed, and after the EKI, separation buffer was introduced by HDI (0.5 % of total length). The separation pressure was changed to 1.5 psi and the spray voltage adjusted to 1600 V. When analyzing a 1:160000 (v/v) dilution of the peptide, the recorded intensity of [M+2H]2+ was 9e8 counts. Therefore, following these guidelines, we were able to increase intensity by a 10000 factor. Novel aspect: Frequent monitoring of spray voltage and peak intensity in similar conditions allows good inter-run comparison and troubleshooting. [less ▲]

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See detailPartial filling capillary electrophoretic mobility shift competition assay: a versatile and reliable tool for the assessment of weak biomolecular interactions
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Hanson, Julien ULiege et al

Conference (2018, September 10)

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both ... [more ▼]

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both academia and pharmaceutical industry. FBDD detects low molecular-weight (MW) ligands (fragments) that bind to biologically important targets, then a structure-guided fragment growing or merging approach is performed giving rise to potent molecules with drug-like properties. However, the analytical arsenal able to point out weak interactions is rather expensive, time consuming or unable to reflect the physiological environment. In this framework, we developed a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used for primary screening of weak biomolecular interactions between fragments and the target of interest. The affinity capillary electrophoresis (ACE) competitive approach is based on the monitoring of the competition of fragments with a known target inhibitor (PL) for the same active site. The consequence of the competition is a modification of PL electrophoretic mobility, modification that can be measured and used for ligand screening and/or IC50 determination. To achieve our goal, particular attention has been paid to the optimization of the binding environment parameters: an optimal buffer was used for the binding measurements, a partial filling approach was considered to gain sensitivity and to reduce protein consumption and a neutral dynamic coating was performed to reduce protein adsorption to the capillary wall. Moreover, the binding partners concentrations and the electrophoretic conditions were carefully optimized. It is noteworthy that the interactions occur in solution, using the protein in its native form, thus mimicking the physiological environment.The accuracy and reliability of the proposed method was demonstrated by monitoring the competition of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat with a known thrombin inhibitor, pefabloc (PEFA). The measured IC50 were found to be in good accordance with the previously reported ones. Additionally, a small chemical library was built to evaluate the performance of the newly developed screening-bioassay. The optimized method proved to be remarkably reproducible (migration time RSDs < 1.2%) and selective. The results prove the high discriminatory potency of the method and its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, significantly expanding the applicability of the assay compared to a direct approach [1]. Finally, the ability of this approach to discriminate between competitive and irreversible thrombin binders was also demonstrated.References [1] E. Farcas, C. Bouckaert, A.-C. Servais, J. Hanson, L. Pochet, M. Fillet, Analytica Chimica Acta, 2017, 984, 211-222 [less ▲]

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See detailEccentric training for tendon healing after acute lesion: a rat model
Kaux, Jean-François ULiege; Libertiaux, Vincent; Leprince, Pierre ULiege et al

in Towards healthy tendon ageing... (2018, September)

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See detailTransverse diffusion of laminar flow profiles as a generic capillary electrophoresis method for in-line nanoreactor mixing: application to the investigation of antithrombotic activity
Farcas, Elena ULiege; Pochet, Lionel; Fillet, Marianne ULiege

in Talanta (2018), 188

Capillary electrophoresis (CE)instrumentwas used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened ... [more ▼]

Capillary electrophoresis (CE)instrumentwas used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened molecules was monitored in a quick and fully automated fashion using only few nanoliters of reactants. To achieve this goal, the targeted enzyme (thrombin) and the chromogenic substrate with or without the screened inhibitor were injected as separate plugs. The mixing of the reactants was then realized using electrophoretically mediated microanalysis (EMMA) or fast transverse diffusion of laminar flow profiles (TDLFP) procedure. The latest provided better mixing performance and was chosen to investigate the inhibitory potency of a fragment library. This very straightforward and fast CE activity assay showed results in good accordance with a previously developed CE affinity assay that confirms the potential of CE at the early stages of drug discovery, providing not only an efficient nanoscale bioreactor but also a selective and integrated separation device. [less ▲]

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See detailVolumetric absorptive microsampling for targeted metabolomics of whole blood
Kok, Miranda ULiege; Nys, Gwenaël ULiege; Fillet, Marianne ULiege

Conference (2018, May 23)

Volumetric absorptive microsampling (VAMS) enables the collection of small and accurate quantities of biological fluids. Therefore, this sampling technique is of great interest for volume-limited samples ... [more ▼]

Volumetric absorptive microsampling (VAMS) enables the collection of small and accurate quantities of biological fluids. Therefore, this sampling technique is of great interest for volume-limited samples or serial collection of samples. Here, we present and discuss the potential of VAMS for targeted mass spectrometry (MS)-based metabolomics. In total, 24 amino acids and 12 organic acids were selected as target metabolites. Two ultra-high performance liquid chromatography (UHPLC) methods coupled to tandem MS have been developed and optimized for the separation and quantitation of these metabolites. A reversed-phase UHPLC-MS/MS method was used to analyze organic acids, whereas hydrophilic interaction chromatography (HILIC)-MS/MS was selected for the determination of amino acids. VAMS devices were used to collect 10 µL of whole blood via a simple finger prick. After collection, the samples were dried for two hours before sample preparation. A design of experiments was conducted to find an extraction solvent providing the maximum recovery of the target analytes from the dried VAMS samples. Overall, the optimum extraction solvent was acetonitrile-water in a proportion of 60:40 (v/v), resulting in the detection of all target metabolites in whole blood with good repeatability. Furthermore, the stability of the analytes in dried whole blood supported on VAMS devices was investigated. We showed that the amino and organic acids were stable for at least 4 days when stored at room temperature. This is in contrast to the instability of these compounds in blood, thereby showing great possibilities of VAMS in metabolomics studies. [less ▲]

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See detailDevelopment of injectable liposomes and drug-in-cyclodextrin-in-liposome formulations encapsulating estetrol to prevent cerebral ischemia of premature babies.
Palazzo, Claudio; Laloy, Julie; Delvigne, Anne Sophie et al

in European Journal of Pharmaceutical Sciences (2018), 9(127), 52-59

Neonatal Hypoxic-Ischemic Encephalopathy (HIE), a brain disease due to brain hypoxia along with ischemia and reduced cerebral blood flow, is one of the primary reasons of severe injury among babies ... [more ▼]

Neonatal Hypoxic-Ischemic Encephalopathy (HIE), a brain disease due to brain hypoxia along with ischemia and reduced cerebral blood flow, is one of the primary reasons of severe injury among babies prematurely born. No efficacy treatment is available to the present day. Estetrol (E4), a major estradiol metabolite, has an important role in the brain development and protection. The aim of this study is to develop new injectable liposome and drug-in-cyclodextrin-in-liposome (DCL) formulations, encapsulating E4 in order to enhance its crossing through the blood-brain barrier (BBB). Liposome and DCL formulations were prepared and were physiochemically characterized. Stability in foetal bovine serum (FBS) was evaluated. LDH and MTS tests on endothelial, neuronal and BBB model cells, as well as hemocompatibility of the nanovectors were performed in vitro. In vitro BBB passage was evaluated using human BBB cell line (hCMEC/D3). All the formulations had average particle size below 150 nm, polydispersity index below 0.10 and ζ potential around + 30 mV. The encapsulation efficacy for liposomes was between 3% and 10% while those of DCL are between 15% and 35%. The effect of liposome and DCL formulations on cell viability and integrity was evaluated. The results showed no toxic effects on all the tested cell lines. Hemocompatibility tests showed no hemolysis, platelet aggregation or effects on coagulation, confirming the possibility of the formulations to be intravenously administrated. BBB passage tests highlighted the capability of the formulations to pass the BBB and reach the brain. Therefore, the formulations are promising drug delivery system to target estrogens to the brain, due to their physiochemical characteristics. [less ▲]

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See detailVolumetric absorptive microsampling: Current advances and applications.
Kok, Miranda ULiege; Fillet, Marianne ULiege

in Journal of Pharmaceutical and Biomedical Analysis (2018), 147(5), 288-296

Recently, volumetric absorptive microsampling (VAMS) has been introduced for the sampling of biological fluids, and more particularly whole blood, on a porous hydrophilic tip. VAMS enables the collection ... [more ▼]

Recently, volumetric absorptive microsampling (VAMS) has been introduced for the sampling of biological fluids, and more particularly whole blood, on a porous hydrophilic tip. VAMS enables the collection of small, accurate and precise blood volumes (10 or 20muL) regardless of the hematocrit. After drying, the samples can be stored or directly analyzed. The stability of various compounds in dried samples supported on VAMS tips varies from one day to a few months at room temperature, and increases at lower temperatures. The complete tip is used during a simple and straightforward sample preparation. Compounds can be extracted with a variety of solvents, and thereafter directly analyzed. A design of experiments is recommended to determine the optimal extraction conditions for a reproducible recovery. The recovery of compounds might be influenced by the hematocrit. In the last two years, various pharmacokinetic and therapeutic drug monitoring studies have been conducted with VAMS. This review covers the general aspects related with the use of VAMS and its applicability is demonstrated through examples. [less ▲]

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See detailInflammation-Generated Extracellular Matrix Fragments Drive Lung Metastasis
BEKAERT, Sandrine ULiege; Fillet, Marianne ULiege; Detry, Benoit et al

in Cancer Growth and Metastasis (2017), 10

Mechanisms explaining the propensity of a primary tumor to metastasize to a specific site still need to be unveiled, and clinical studies support a link between chronic inflammation and cancer ... [more ▼]

Mechanisms explaining the propensity of a primary tumor to metastasize to a specific site still need to be unveiled, and clinical studies support a link between chronic inflammation and cancer dissemination to specific tissues. Using different mouse models, we demonstrate the role of inflammation-generated extracellular matrix fragments ac-PGP (N-acetyl-proline-glycine-proline) on tumor cells dissemination to lung parenchyma. In mice exposed to cigarette smoke or lipopolysaccharide, lung neutrophilic inflammation produces increased levels of MMP-9 (matrix metalloproteinase 9) that contributes to collagen breakdown and allows the release of ac-PGP tripeptides. By silencing CXCR2 gene expression in tumor cells, we show that these generated ac-PGP tripeptides exert a chemotactic activity on tumor cells in vivo by binding CXCR2. [less ▲]

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See detailQuantitative profiling of endogenous polar metabolites from low volumes of blood samples
Kok, Miranda ULiege; Fillet, Marianne ULiege

Conference (2017, November 08)

The etiology of many diseases is not yet completely understood. The involved biological processes might be resolved using a metabolomics approach, because metabolomics provides unique challenging ... [more ▼]

The etiology of many diseases is not yet completely understood. The involved biological processes might be resolved using a metabolomics approach, because metabolomics provides unique challenging opportunities to correlate the metabolome with a physiological or pathophysiological status and provides a vision on the relationships between genes, gene expression, environment and lifestyle. Here, we present the development of two ultra-high performance liquid chromatography (UHPLC) methods coupled to mass spectrometry (MS) for the separation and quantitation of polar metabolites in blood samples. A reversed-phase UHPLC-MS/MS method has been developed to quantify anionic energetic metabolites, whereas hydrophilic interaction chromatography (HILIC)-MS/MS has been used to determine amino acids. Two sample pretreatment procedures have been developed for an optimal recovery of the respective metabolites from whole blood samples. One method involved the precipitation of proteins with organic solvents and acids. In addition, volumetric absorptive microsampling has been used for the sample preparation. Small and accurate quantities of biological fluids (10 or 20 µL) can be collected with this sampling technique, which is of great interest for volume-limited samples or serial collection of samples. The developed methods have been validated and will be applied to determine differences in metabolite concentrations between blood samples from patients and controls. This can lead to a breakthrough in the understanding of diseases and can open new therapeutic perspectives. [less ▲]

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See detailComparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling
Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege; Kok, Miranda ULiege et al

in Journal of Chromatography. A (2017), 1524

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the ... [more ▼]

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC-MS/MS methods In this study, we present the development of a nanofluidic-LC-MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC-MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC-MS/MS method. A limit of quantitation of 50. pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC-MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples. © 2017 Elsevier B.V. [less ▲]

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See detailQuantitative profiling of endogenous polar metabolites from low volumes of blood samples
Kok, Miranda ULiege; Fillet, Marianne ULiege

Poster (2017, October 05)

The etiology of many diseases is not yet completely understood. The involved biological processes might be resolved using a metabolomics approach, because metabolomics provides unique challenging ... [more ▼]

The etiology of many diseases is not yet completely understood. The involved biological processes might be resolved using a metabolomics approach, because metabolomics provides unique challenging opportunities to correlate the metabolome with a physiological or pathophysiological status and provides a vision on the relationships between genes, gene expression, environment and lifestyle. Here, we present the development of two ultra-high performance liquid chromatography (UHPLC) methods coupled to mass spectrometry (MS) for the separation and quantitation of polar metabolites in blood samples. A reversed-phase UHPLC-MS/MS method has been developed to quantify anionic energetic metabolites, whereas hydrophilic interaction chromatography (HILIC)-MS/MS has been used to determine amino acids. Two sample pretreatment procedures have been developed for an optimal recovery of the respective metabolites from whole blood samples. One method involved the precipitation of proteins with acetonitrile and acids. In addition, volumetric absorptive microsampling has been used for the sample preparation. Small and accurate quantities of biological fluids (10 or 20 µL) can be collected with this sampling technique, which is of great interest for volume-limited samples or serial collection of samples. The developed methods have been validated and will be applied to determine differences in metabolite concentrations between plasma samples from patients and controls. This can lead to a breakthrough in the understanding of diseases and can open new therapeutic perspectives. [less ▲]

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See detailIdentification and quantitation of intact virus-like particles of human papillomavirus (HPV-VLP) using capillary electrophoresis
Bettonville, Virginie ULiege; Nicol, Jérôme; Furst, Tania et al

Conference (2017, September 19)

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See detailChallenges in the determination of amyloid oligomeric species by two electrophoretic techniques
Napp, Aurore ULiege; Houbart, Virginie ULiege; Demelenne, Alice ULiege et al

Poster (2017, September 18)

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already ... [more ▼]

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already occurred. Knowing that, it is of high interest to have one (or more) reliable biomarker(s) at our disposal to diagnose Parkinson before the first symptoms appear. Alpha-synuclein (aSyn) is a protein physiologically expressed at high level by neuronal cells, under a monomeric form. This protein would play a critical role in the development of the disease because under certain conditions, aSyn is capable of self-assembly to form fibrils like those found in Lewy bodies. Other intermediate soluble forms like dimers and oligomers are also formed. As these forms seems to be the toxic species, they are the center of many attentions. The quantification of each form would be a great help, but for the moment only the total forms (of monomeric or oligomeric) can be quantified. In this study, aSyn oligomers were generated after optimization of incubation conditions (pH, temperature, agitation, …). Then, different approaches were investigated to detect and follow the different species formed during the aggregation. We analyzed the oligomers by capillary gel electrophoresis (CGE) and SDS-PAGE. We found that capillary gel electrophoresis is a promising automated technique to analyze aSyn oligomers, due to the fact that it separates the aggregates according to their size, like the SDS-PAGE, but with more advantages. To gain sensitivity and selectivity by CGE, we used a laser-induced fluorescence detector. As aSyn do not have a native fluorescence, we derivatized it. After careful screening and optimization of various derivatization reagents, we could quantify with high sensitivity aSyn oligomers by CGE-LIF. We realized different calibration curves, and we had promising results that will allow us to quantify the different aSyn oligomeric forms in biological fluids. [less ▲]

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See detailSampling only 10 μL of whole blood to study the bioavailability of itraconazole formulations in rats
Kok, Miranda ULiege; Thiry, Justine ULiege; Evrard, Brigitte ULiege et al

Conference (2017, September 12)

Background Volumetric absorptive microsampling (VAMS) offers a unique opportunity to collect small and accurate quantities of biological fluids. This sampling technique is of great interest for volume ... [more ▼]

Background Volumetric absorptive microsampling (VAMS) offers a unique opportunity to collect small and accurate quantities of biological fluids. This sampling technique is of great interest for volume-limited samples and for the collection of multiple samples from the same animal. This serial sample collection may reduce the number of required study animals, thereby fulfilling the three Rs rule (refine, reduce, replace). Here, we demonstrate the applicability of VAMS to study the bioavailability of drug formulations in rats. Methods Four itraconazole-containing formulations were successively administered to rats with a wash-out period of one week. VAMS was used to collect 14 whole blood samples of only 10 μL each within a time frame of 48 hours after administration of the different drug formulations. Particular attention was paid to sample preparation and stability. The extraction of itraconazole and its main metabolite hydroxy-itraconazole was optimized to provide a high recovery and minimal matrix effects. A developed and validated LC–MS/MS method was used for the quantification of the two compounds. Pharmacokinetic profiles for the different formulations were constructed and compared. Results The stability of itraconazole and hydroxy-itraconazole in dried VAMS samples of whole rat blood could not be guaranteed for more than a day when the samples were stored at room temperature. However, samples were stable for at least two weeks when stored at -80°C after sample preparation. Differences in pharmacokinetic profiles were observed for the tested drug formulations. Whole blood concentrations of itraconazole and its main metabolite were significantly higher after administration of three in-house produced formulations compared to concentrations obtained with a commercially available product. Moreover, these in vivo results could be partly related to in vitro dissolution rates of the various formulations. Conclusions VAMS is an attractive approach for bioavailability studies. Due to the low blood volumes per sampling point, the same rats can be used to compare various drug formulations. Therefore, the number of required animals can be drastically reduced. Moreover, this helps to suppress the inter-individual variability and strengthens the validity of results. [less ▲]

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