Pierre Lanteri

Determination of poly(ɛ-caprolactone) solubility parameters: Application to solvent substitution in a microencapsulation process

The evolution of regulation on chemical substances (i.e. REACH regulation) calls for the progressive substitution of toxic chemicals in formulations when suitable alternatives have been identified. In this context, the method of Hansen solubility parameters was applied to identify an alternative solvent less toxic than methylene chloride used in a microencapsulation process. During the process based on a multiple emulsion (W/O/W) with solvent evaporation/extraction method, the solvent has to dissolve a polymer, poly(epsilon-caprolactone) (PCL), which forms a polymeric matrix encapsulating or entrapping a therapeutic protein as the solvent is extracted. Therefore the three partial solubility parameters of PCL have been determined by a group contribution method, swelling experiments and turbidimetric titration. The results obtained allowed us to find a solvent, anisole, able to solubilize PCL and to form a multiple emulsion with aqueous solutions. A feasibility test was conducted under standard operating conditions and allowed the production of PCL microspheres.

Experimental designs to investigate capillary electrophoresis-electrospray ionization-mass spectrometry enantioseparation with the partial-filling technique.

An experimental design approach is described to evaluate the main electrophoretic parameters involved in the enantioseparation of pharmaceuticals by capillary electrophoresis (CE) coupled to electrospray ionization‐mass spectrometry (ESI‐MS). For all experiments, the partial‐filling technique was applied to avoid the chiral selector entering in the mass spectrometer ion source with a negative effect on the electrospray performance. To carry out enantioseparation, a volatile buffer constituted of 20 mM ammonium acetate at pH 4.0, and a polyvinyl alcohol‐coated capillary were used. Methadone was employed as the model compound and three different cyclodextrins (CDs), namely sulfobutyl ether‐β‐CD, carboxymethylated‐β‐CD and hydroxypropyl‐β‐CD, were selected in order to study the countercurrent process. Two different experimental designs were chosen: (i) a full‐factorial design to examine the effects and significance of the investigated factors, and (ii) a central composite face‐centered design to establish the mathematical model of the selected responses in function of experimental factors. The chiral selector concentration, percentage of the capillary filled with the chiral selector, and drying gas nebulization pressure were three relevant factors taken into consideration. For each CD, the methadone enantiomeric resolution, apparent selectivity, and migration time of the second enantiomer were established as responses. The latter were systematically related to experimental parameters with the help of multiple linear regression. It is noteworthy that the behaviour was different in function of the chiral selector charge. Results revealed that the nebulization pressure involved in the electrospray process and the CD concentration had a significant effect on the enantiomeric resolution, while the effect of the separation zone length was less pronounced. Finally, response surfaces were drawn from the mathematical model and experimental conditions were selected to allow a robust determination of methadone enantiomers by CE‐MS.

Development and optimisation of a single extraction procedure for the LC/MS/MS analysis of two pharmaceutical classes residues in sewage treatment plant.

A unique extraction procedure leading to the separation of 2 different pharmaceutical classes molecules has been developed and optimised by chemometric tools. From only one sampling, this analytical method allows the determination of 21 pharmaceuticals from corticosteroids and beta-blockers classes. Performing the SPE on Oasis MCX (mixed-mode cation exchange), the sequential elution of each pharmaceutical class is achievable, allowing a high purity level of extracts as well as high recovery rates. Performing a unique sample preparation results in an important save of time. The extracts were then analysed by LC/MS/MS, using a Hibar Purospher Star column for beta-blockers and an X-Bridge column for corticosteroids with formate buffer (pH 3.8)/AcN and water/AcN mobile phases, respectively. This work also includes a study of the chromatographic and mass spectrometric parameters in order to increase the analyte signal. The optimised SPE-LC/MS/MS method was then applied to environmental samples from sewage treatment plant (STP). beta-Blockers and corticosteroids were detected, respectively, in concentrations up to 318 ng L(-1) (sotalol) and 174 ng L(-1) (cortisone), in STP influents. Moreover, both pharmaceutical classes have also been detected in STP effluents. As far as we know, this is the first paper reporting the detection of corticosteroids in environmental waters. The developed analytical method can be used in further studies to investigate the environmental contamination by these drugs.

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimers disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n = 68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n = 669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.

Spray-drying Nanocapsules in Presence of Colloidal Silica as Drying Auxiliary Agent: Formulation and Process Variables Optimization Using Experimental Designs

PurposeSpray-drying process was used for the development of dried polymeric nanocapsules. The purpose of this research was to investigate the effects of formulation and process variables on the resulting powder characteristics in order to optimize them.Materials and MethodsExperimental designs were used in order to estimate the influence of formulation parameters (nanocapsules and silica concentrations) and process variables (inlet temperature, spray-flow air, feed flow rate and drying air flow rate) on spray-dried nanocapsules when using silica as drying auxiliary agent. The interactions among the formulation parameters and process variables were also studied. Responses analyzed for computing these effects and interactions were outlet temperature, moisture content, operation yield, particles size, and particulate density. Additional qualitative responses (particles morphology, powder behavior) were also considered.ResultsNanocapsules and silica concentrations were the main factors influencing the yield, particulate density and particle size. In addition, they were concerned for the only significant interactions occurring among two different variables. None of the studied variables had major effect on the moisture content while the interaction between nanocapsules and silica in the feed was of first interest and determinant for both the qualitative and quantitative responses. The particles morphology depended on the feed formulation but was unaffected by the process conditions.ConclusionThis study demonstrated that drying nanocapsules using silica as auxiliary agent by spray drying process enables the obtaining of dried micronic particle size. The optimization of the process and the formulation variables resulted in a considerable improvement of product yield while minimizing the moisture content.

Experimental design in supercritical fluid extraction of cocaine from coca leaves

An optimisation procedure for the supercritical fluid extraction (SFE) of cocaine from the leaves of Erythroxylum coca var. coca was investigated by means of experimental design. After preliminary experiments where the SFE rate-controlling mechanism was determined, a central composite design was applied to evaluate interactions between selected SFE factors such as pressure, temperature, nature and percentage of the polar modifier, as well as to optimise these factors. Predicted and experimental contents of cocaine were compared and robustness of the extraction method estimated by drawing response surfaces. The analysis of cocaine in crude extracts was carried out by capillary GC equipped with a flame ionisation detector (GC-FID), as well as by capillary GC coupled with a mass spectrometer (GC-MS) for peak identification.

Improvement of an encapsulation process for the preparation of pro- and prebiotics-loaded bioadhesive microparticles by using experimental design

The purpose of this study was to design a new vaginal bioadhesive delivery system based on pectinate-hyaluronic acid microparticles for probiotics and prebiotics encapsulation. Probiotic strains and prebiotic were selected for their abilities to restore vaginal ecosystem. Microparticles were produced by emulsification/gelation method using calcium as cross-linking agent. In the first step, preliminary experiments were conducted to study the influence of the main formulation and process parameters on the size distribution of unloaded microparticles. Rheological measurements were also performed to investigate the bioadhesive properties of the gels used to obtain the final microparticles. Afterwards an experimental design was performed to determine the operating conditions suitable to obtain bioadhesive microparticles containing probiotics and prebiotics. Experimental design allowed us to define two important parameters during the microencapsulation process: the stirring rate during the emulsification step and the pectin concentration. The final microparticles had a mean diameter of 137μm and allowed a complete release of probiotic strains after 16h in a simulated vaginal fluid at +37°C.

Optimisation of pressurised liquid extraction by experimental design for quantification of pesticides and alkyl phenols in sludge, suspended materials and atmospheric fallout by liquid chromatography–tandem mass spectrometry

The study proposes an analytical method to quantify 10 alkylphenols and 12 pesticides at ultra-trace levels by liquid chromatography in reverse mode coupled to positive electrospray ionisation-tandem mass spectrometry. The extraction procedure from environmental solids was optimised by pressurised liquid extraction using acetonitrile:isopropanol (1:1, v/v). The influence of several extraction experimental factors, temperature, pressure, duration and number of cycles, related to the PLE was investigated by an original and efficient chemometric approach. The optimised extraction method (80 degrees C, 40 bar, 10 min, 1 cycle) exhibited recoveries between 67 and 127% with RSD mostly under 13%. The whole method was applied to real samples: sludge, suspended materials, atmospheric fallouts and roof deposit. Pollutant levels were between 1 microg kg(-1) and 5.9 mg kg(-1).

Optimization of fast CE analyses of ecstasy derivatives by use of experimental designs

SummaryThe separation by capillary electrophoresis of five ecstasy derivatives has been evaluated by means of experimental designs. A full-factorial design and a central composite design were employed to optimize the experimental conditions for a fast separation. With a conventional capillary (62.5 cm length) analytes were separated in less than 8 min. When the capillary length was reduced the analysis time decreased substantially. Comparisons were made between different procedures (e.g. working at a constant field or at constant voltage) and finally, a fast separation of five Ecstasy derivatives was achieved in 1 min.

Surface characterisation of amine-containing latexes by charge titration and contact angle measurements

Abstract Surface properties of cationic amino-containing latexes, as obtained by emulsifier-free emulsion copolymerisation of styrene with vinyl benzyl amine hydrochloride using 2,2′-azobis (2-amidinopropane) dihydrochloride (V50) as initiator, were investigated. Surface charge density was first examined using 1 H NMR, conductometry and two spectroscopic methods (UV, fluorescence). Whereas the first two methods only provide semi-quantitative information on the overall surface density, the latter two were more accurate and suitable for discriminating amidine (issued from the initiator) and amine (brought by the cationic monomer) surface groups. In addition, it was possible to show differences in the nature of surface groups according to the mode of polymerisation process (batch versus shot-growth). Latex surface polarity was examined through contact angle measurements which allowed one to determine surface tension using the mean harmonic approximation method. Such amino latexes were found to exhibit a much lower surface polarity than sulphate-charged ones.