Gwenaël Nys

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry

&NA; Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20 &mgr;l of whole blood from tested subjects. In this study, we present the development and validation of ultra‐high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof‐of‐concept pharmacokinetic studies of E4 after VAMS collection in a murine model. Graphical abstract Figure. No caption available. HighlightsA UHPLC–MS/MS method is proposed for the analysis of estetrol without derivatization.NH4F as LC additive is used to reach needed sensitivity in negative mode.An innovative microsampling technique is used to collect 10 &mgr;l of blood from mice.The method was successfully validated for the quantitation of estetrol.A pharmacokinetic study is performed as proof‐of‐concept.

Sampling only ten microliters of whole blood for the quantification of poorly soluble drugs: Itraconazole as case study

Nowadays in animal studies, it is important to comply with the so-called Three Rs rule by replacing or reducing the number of tested animals. Volumetric absorptive microsampling (VAMS) can be used to collect small quantities (10 or 20μL) of whole blood, thereby limiting the amount of animals needed. In this study, a quantitative method was developed and subsequently validated for the poorly soluble drug itraconazole (ITZ) using VAMS and ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS). A proof of concept study showed that the optimized method is applicable to test the bioavailability of drug formulations containing ITZ. Using VAMS, smaller blood volumes can be taken per sampling point (10-20μL instead of the conventional 0.2-0.5mL) avoiding the sacrifice of animals. Moreover, the same rats can be used to compare different drug formulations which strengthens the validity of the results. In long-term bioavailability studies, it is necessary to guarantee the stability of the tested drugs supported on VAMS devices. In this study, we show that ITZ was only stable for 24h after collection with VAMS, but for at least two weeks by the storage of extracted samples at -80°C.

Impact of injection solvent composition on protein identification in column-switching chip-liquid chromatography/mass spectrometry

In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupled to mass spectrometry. Many researches have been carried out to study the effects on identification performances of chromatographic parameters such as the stationary phase and column dimensions, mobile phase composition and flow rate, as well as the gradient slope and length. However, little attention is usually paid to the injection solvent composition. In this study, we investigated the effect of the injection solvent on protein identification parameters (number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Tryptic peptides from six different proteins, covering a wide range of physicochemical properties, were employed as training set. Design of experiments was employed as a tool to highlight the factors related to the composition of the injection solvent that significantly influenced the obtained results. Optimal results for the training set were applied to analysis of more complex samples. The experiments pointed out optimising the composition of the injection solvent had a strong beneficial effect on all the considered responses. On the basis of these results, an approach to determine optimal conditions was proposed to maximise the protein identification performances and detection sensitivity.

Validation of a new method by nano-liquid chromatography on chip tandem mass spectrometry for combined quantitation of C3f and the V65 vitronectin fragment as biomarkers of diagnosis and severity of osteoarthritis.

Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and the V65 vitronectin fragment in serum. The method was first carefully optimized and then validated in serum biological matrix. Stable isotopes for the two biomarkers of interest were used as stable isotope labeled peptide standards. A weighted 1/x2 quadratic regression for C3f and a weighted 1/x quadratic regression for the V65 vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the concentration range of 2.5-200ng/mL for C3f and 2.5-100ng/mL for the V65 vitronectin fragment. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method. Our data confirm that C3f and the V65 vitronectin fragment are biomarkers of OA severity, but also that C3f fragment is further related to OA severity whereas the V65 vitronectin fragment is more related to early OA detection.

Comparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling

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 50pg/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.

Development of injectable liposomes and drug-in-cyclodextrin-in-liposome formulations encapsulating estetrol to prevent cerebral ischemia of premature babies

Abstract 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 &zgr; 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. Graphical abstract Figure. No Caption available.

Microfluidics contribution to pharmaceutical sciences: From drug discovery to post marketing product management

Graphical abstract Figure. No caption available. HighlightsMicrofluidics use at early phases of target discovery and validation are reviewed.Synthesis and formulation are improved when realized in microfluidic devices.Rejection rate in clinical trials can be reduced by organs‐on‐a‐chip drug testing.Less animals are employed by using miniaturized sampling and analytical instruments. ABSTRACT Drug discovery and development is a long‐lasting process in which many challenges have to be addressed at every stage, from the discovery of the target biomolecule to the commercialization of the discovered drugs and its quality control. From thousands of hits identified during early drug discovery only one drug is eventually efficient and safe enough to be commercialized. This high rejection rate, especially during preclinical and clinical studies have led to an exponential increase of costs to develop new medicines thereby strongly impacting healthcare systems. In this context, miniaturized devices have the potency to significantly reduce the cost and the time needed to develop new therapeutics by streamlining drug development and rejecting drug candidates earlier in the process prior to costly animal and human trials. In this review, we present recent advances involving miniaturized technologies in the field of drug discovery such as target discovery, drug screening, drug synthesis and formulation, in‐vitro and in‐vivo testing and quality control. We discuss opportunities enabled by miniaturized devices but also their limitations and challenges that need to be resolved in order to spread their use in the pharmaceutical industries.