RESUMEN
Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani. Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Fosforilcolina/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Líquida de Alta Presión/métodos , Humanos , Leishmania donovani/metabolismo , Leishmaniasis Visceral/tratamiento farmacológico , Fosfolípidos/aislamiento & purificación , Fosforilcolina/farmacologíaRESUMEN
OBJECTIVES: This study focuses on the mechanism of interaction of sitamaquine with Leishmania donovani membranes, and its accumulation within the parasites. METHODS: A biomimetic model of the outer layer of a Leishmania plasma membrane was used to examine the interactions of sitamaquine with lipids. The plasma membranes of L. donovani promastigotes were depleted of sterol using cholesterol oxidase, in order to assess the importance of sterols in drug-membrane interactions. Sterols were quantiï¬ed and sitamaquine susceptibility was assessed using the MTT test. Kinetics of sitamaquine accumulation and efflux were measured under different conditions. RESULTS: Sitamaquine interacts first with phospholipid anionic polar head groups and then with phospholipid acyl chains to insert within biological membranes and accumulates rapidly in the Leishmania cytosol according to a sterol-independent process. The rapid sitamaquine efflux observed was related to an energy-dependent mechanism since the intracellular amount of sitamaquine was enhanced three times in the absence of glucose and the efflux was inhibited in energy-depleted conditions. (1)H NMR analysis of motile lipid showed that sitamaquine did not affect lipid trafficking in Leishmania. CONCLUSIONS: We propose that sitamaquine rapidly accumulates in Leishmania by diffusion along an electrical gradient and is concentrated in the cytosol by an energy- and sterol-independent process. The affinity of sitamaquine for membranes was transitory and an energy-dependent efflux was demonstrated, suggesting the presence of an as yet uncharacterized transporter.
Asunto(s)
Aminoquinolinas/metabolismo , Antiprotozoarios/metabolismo , Membrana Celular/metabolismo , Leishmania donovani/efectos de los fármacos , Aminoquinolinas/química , Aminoquinolinas/farmacología , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Difusión , Leishmania donovani/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Esteroles/metabolismoRESUMEN
Natural cyclodextrins are cyclic oligosaccharides which can be modified to obtain more water soluble or insoluble derivatives. The main interest of cyclodextrins results from their ability to form an inclusion complex with hydrophobic molecules. Inclusion constitutes a true molecular encapsulation. This property is employed in pharmaceutical industry to facilitate the formulation of poorly water soluble and/or fragile drugs. A more recent application of cyclodextrins consists in their use in the preparation of dispersed systems such as micro- and nanoparticles or even liposomes. When incorporated in dispersed systems, cyclodextrin can enhance drug solubility, drug stability and drug loading. Interestingly, cyclodextrins themselves can also be employed to form or stabilise dispersed systems (material or emulsifying agent). For example, the interactions between cyclodextrins with components of the vegetable oils (more especially with triglycerides) allow to stabilise simple or multiple emulsions but also to form particles called "beads". Very rich in oil, this novel lipid carrier presents an important potential for the encapsulation of highly lipophilic compounds and their delivery by topical and oral routes. These two applications are more particularly developed in the present paper.
Asunto(s)
Ciclodextrinas/química , Sistemas de Liberación de Medicamentos , Triglicéridos/química , Química Farmacéutica , Industria Farmacéutica , Emulsiones , Excipientes , SolubilidadRESUMEN
The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Leishmania/química , Fosfolípidos/análisis , Dispersión de Radiación , Aerosoles/análisis , Animales , Membrana Celular/química , Cromatografía Líquida de Alta Presión/instrumentación , Luz , Reproducibilidad de los ResultadosRESUMEN
The mechanism of action of antifungal canthin-6-one series was investigated in Saccharomyces cerevisiae. After a rapid uptake, a preferential accumulation of the drug within lipid droplets was observed. The antifungal action of canthin-6-one was found as reversible. Canthin-6-one did not exhibit affinity for sterols, and membrane ergosterol was not necessary for the antifungal activity since the MICs were similar on an ergosterol-deleted and the wild-type S. cerevisiae clones. Relative amount of unsaturated alkyl chain fatty acids was significantly enhanced suggesting a stimulation of desaturase enzyme systems. No synergistic effect was observed between canthin-6-one and amphotericin B, ketoconazole and caspofungine. Canthin-6-one should now be evaluated in vivo against fungal pathogens.
Asunto(s)
Antifúngicos/farmacología , Indoles/farmacología , Naftiridinas/farmacología , Anfotericina B/farmacología , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacocinética , Carbolinas , Sinergismo Farmacológico , Ácido Graso Desaturasas/efectos de los fármacos , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/metabolismo , Alcaloides Indólicos , Indoles/aislamiento & purificación , Cetoconazol/farmacología , Pruebas de Sensibilidad Microbiana , Naftiridinas/aislamiento & purificación , Saccharomyces cerevisiaeRESUMEN
In multiple emulsion systems, oily or aqueous transfers may occur between the dispersed droplets through the continuous phase. These transfers are controlled by both the surfactant system (micellar transport), and the partial solubility of one phase in another (molecular transport). The latter could be anticipated from the knowledge of oil polarity, if this information could easily be obtained. In this work, the relative polarity of eight oils used for various purposes has been evaluated from the comparison of their dielectric requirement for solubilization, their interfacial tension and chromatographic analysis. The results showed the complementarities of HPLC analysis and interfacial tension measurements and their superiority over the solubilization method for classifying oils as a function of their polarity.
Asunto(s)
Aceites/química , Solventes/química , Agua/química , Alcanos/química , Cromatografía Líquida de Alta Presión/métodos , Dioxanos/química , Emulsiones/química , Alcoholes Grasos/química , Aceite Mineral/química , Palmitatos/química , Transición de Fase , Reproducibilidad de los Resultados , Solubilidad , Electricidad Estática , Propiedades de Superficie , Tensión Superficial , Triglicéridos/químicaRESUMEN
Polyethylene glycol (PEG) chains covalently linked to phospholipids are often used in the preparation of lipid or even polymer colloidal particles to avoid recognition and clearance by the reticuloendothelial system and to increase their plasmatic half-life. To the best of our knowledge, no direct method allows yet to quantify these pegylated phospholipids. The aim of this work was to develop a method for the quantification of a typical pegylated phospholipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000, associated to polymeric microcapsules of perfluorooctyl bromide (PFOB). Reverse phase high-performance liquid chromatography (HPLC) was used, coupled with a corona charged aerosol detection (HPLC-CAD). Calibrations standards consisted of plain microcapsules and pegylated phospholipids (DSPE-PEG2000) in the concentration range of 2.23-21.36 microg/mL (0.22-2.14 microg injected). Calibration curve was evaluated with two different model, linear and power model. The power model describes experimental values better than the linear model, for pegylated phospholipids with the CAD detector. The correlation coefficient for the power model was 0.996, and limits of detection and quantification obtained were 33 and 100 ng, respectively. This method proved to be selective and sensitive; the accuracy of the method ranged from 90 to 115% and the relative standard deviation was Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación
, Cromatografía Líquida de Alta Presión/métodos
, Fluorocarburos/química
, Fosfolípidos/análisis
, Polímeros/química
, Calibración
, Cápsulas
, Hidrocarburos Bromados
, Estructura Molecular
, Fosfolípidos/química
, Polietilenglicoles/química
, Estándares de Referencia
, Sensibilidad y Especificidad
, Suspensiones
RESUMEN
This study compares, through micro high-temperature liquid chromatography (microHTLC), three commercial universal detectors that allow a direct detection of lipids. The detectors are: the charged aerosol detector (CAD), the evaporative light-scattering detector (ELSD) and the ion trap mass spectrometer with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) sources (APCI-MS and ESI-MS). This study shows the feasibility to use the high temperature with these detectors and hybrid behavior between concentration and mass flow rate detector in microHTLC. The detectors were compared in terms of response intensity, linearity and limit of detection for different high temperatures. The charged aerosol detector shows a linear response from 5 to 500 microg/mL and the correlation coefficients (r(2)) obtained for squalene, cholesterol and ceramide IIIB exceed 0.99.
Asunto(s)
Aerosoles/química , Cromatografía Liquida/instrumentación , Lípidos/análisis , Microfluídica/métodos , Espectrofotometría/instrumentación , Ceramidas/análisis , Colesterol/análisis , Cromatografía Liquida/métodos , Calor , Luz , Dispersión de Radiación , Sensibilidad y Especificidad , Solventes/química , Espectrofotometría/métodos , Escualeno/análisis , TemperaturaRESUMEN
The study of several structural variations (the length, the degree of unsaturation and hydroxylation of the alkyl chains, the number and nature of osidic residues) helped understand the behaviour of neutral glycosphingolipids (GSLs) on porous graphitic carbon stationary phase (PGC). Atmospheric pressure photoionization mass spectrometry (APPI) and tandem mass spectrometry were used to perform the detection and the identification of molecular species in positive mode where [M+H](+) and [M-H(2)O+H](+) ions provided structural information on the fatty acid and the sphingoid base. The retention of GSLs increased with the hydrocarboneous volume of their alkyl chains and with the number of osidic residues in agreement with hydrophobic properties and polar retention effect of graphite, respectively. The presence of polar groups, such as OH-group or double bond within alkyl chains, decreased their retention. The coupling of chromatography on PGC with APPI tandem mass spectrometry detection appeared a powerful technique to discriminate isobaric molecules. Isobaric solutes differing by the position of two double bonds or by the repartition of hydrocarboneous skeleton were discriminated according to their chromatographic comportment or their mass spectrum, respectively. Among isobaric molecules, only few structures differing by the nature of osidic residue were not discriminated (i.e. glucosylceramide and galactosylceramide with similar ceramide skeleton were co-eluted and no difference in mass spectra was observed).
Asunto(s)
Cromatografía Liquida/métodos , Glicoesfingolípidos/análisis , Grafito/química , Presión Atmosférica , Secuencia de Carbohidratos , Datos de Secuencia MolecularRESUMEN
The prescription of unlicensed oral medicines in paediatrics leads the hospital pharmacists to compound hard capsules, such as busulfan, an alkylating agent prescribed in preparative regimens for bone marrow transplantation. In this study, we have investigated how the general principle of process analytical technology (PAT) can be implemented at the small size of our hospital pharmacy manufacturing unit. Near infrared spectroscopy (NIRS) was calibrated for raw material identification, blend uniformity analysis and final content uniformity of busulfan hard capsules of 11 different strengths. Measurements were performed on capsules from 2 to 40 mg (n=440). After optimisation, accuracy and linearity of the NIRS quantitative method was demonstrated after comparison with a previously validated quantitative high performance thin layer chromatography (HPTLC) method. Such a comparison led to attractive NIRS precision: +/-0.7 to +/-1.0 mg for capsules from 2 to 40 mg, respectively. As NIRS is a rapid and non-destructive technique, the individual control of a whole batch of busulfan paediatric capsules intended to be administrated is possible. Actually, mastering the process of busulfan paediatric capsules with the NIRS integrated into the notion of PAT is a powerful analytical tool to assess the process quality and to perform content uniformity of at least 5mg busulfan-containing capsules.
Asunto(s)
Alquilantes/análisis , Busulfano/análisis , Espectroscopía Infrarroja Corta/métodos , Cápsulas , Niño , Cromatografía Líquida de Alta Presión , Hospitales Universitarios , Humanos , Servicio de Farmacia en Hospital , Control de CalidadRESUMEN
The advent of innovative techniques in mass spectrometry, especially in the area of imaging, prompted us to evaluate two promising techniques: secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. For this purpose, sections of cutaneous biopsies from patients affected by Fabry's disease and control patients were analyzed. In the course of this disease, two physiological glycosphingolipids [globotriasylceramide (Gb3) and the galabiosylceramide (Ga2)] accumulate in certain tissues owing to a catabolism failure. The ability of these techniques to localize sites of accumulation in body tissues and their capacity to identify the accumulated lipid structures by mass spectra were evaluated. Results demonstrated that these two techniques provide complementary information:-secondary ion mass spectrometry enabled precise localization of areas of accumulation with lateral resolution in the micrometer range;-the signal obtained with matrix-assisted laser desorption/ionization mass spectrometry was high enough to identify these structures according to their molecular weight.
Asunto(s)
Diagnóstico por Imagen , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/patología , Espectrometría de Masas , Piel/química , Piel/patología , Glicoesfingolípidos/química , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Interactions of solutes on porous graphitic carbon (PGC) with non-aqueous mobile phases are studied by the linear solvation energy relationship (LSER). Studies have been carried out with eight binary mixtures composed of a weak solvent (acetonitrile or methanol) and a strong solvent (tetrahydrofuran, n-butanol, CH2Cl2, 1,1,2-trichloro-2,2,1-trifluoroethane). The systematic analysis of a set of test compounds was performed for each solvent mixture in isocratic mode (50:50). The results were compared to those obtained on PGC with hydro-organic liquids and supercritical fluids. They were then correlated with the observed retention behaviour of lipid compounds, more particularly ceramides.
Asunto(s)
Ceramidas/química , Cromatografía Líquida de Alta Presión/métodos , Grafito/químicaRESUMEN
A general approach, still few exploited so far and never associated with microbore-LC, consisting of detection of various lipid classes (i.e. phospholipids, triglycerides, ceramides and glycosphingolipids) by non-covalent association with 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe is developed. This mode of detection was coupled with non-aqueous reversed-phase microbore-LC (C18) by using classical post-column fluorescence detection. The classical LC system was first adapted to microbore-chromatography (internal diameter 1 mm) without apparatus miniaturization of the solvent delivery system and the detection cell. For this purpose, the detection parameters (probe concentration, post-column flow rate, post-column reactor length and post-column system temperature) were optimized by a central composite design (CCD) using a mixture of phosphatidylcholine (PC) species as a lipid model and DPH (lambda(ex) = 350 nm, lambda(em) = 430 nm) as a fluorescence probe. The optimal conditions of detection for the various molecular species of PC were determined for a DPH concentration of 3.35 micromol/L, a post-column flow rate of 0.5 mL/min, a reactor length of 1.4 m and a temperature of 35 degrees C. The fluorescence response was linear over a wide range of PC species from 5 microg/mL to 100 microg/mL and the lower limit of detection (signal/noise = 3) was about 1 microg/mL, that is equivalent to evaporative light scattering detection (ELSD). Others molecular species of various classes of lipids, i.e. triglycerides, ceramides and glycosphingolipids were also easily detected. Thus, this study demonstrated the versatility of the proposed system of detection which was shown to be sensitive, easy to perform, non-destructive and allowed, in contrast to ELSD, for a linear response with various polarity lipid classes.
Asunto(s)
Cromatografía Liquida/métodos , Colorantes Fluorescentes/química , Lípidos/análisis , Sensibilidad y EspecificidadRESUMEN
Porphyrins are a class of photosensitizers used in photodynamic therapy (PDT). Understanding the interaction of porphyrins with membrane cells components is important in order to improve this therapy. Many analytical methods can be used for this aim. High performance liquid chromatography (HPLC) was used for the separation of porphyrins on RP and HILIC stationary phases as well as on a biomimetic membrane IAM phase. Twenty-six tetraphenyl porphyrins (TPP) were successfully separated on an IAM column, a C18 Gravity RP column, a C8 Gravity RP column, a PolarTec RP column and a HILIC column. Stationary phases were chosen as the most appropriate to cover the study of different types of interactions. Elution was performed with a 45 min linear gradient. Obtained gradient retention times were converted to gradient chromatography hydrophobicity index (CHI) and to an apparent retention factor (kapp). The partition coefficients (logP) of the 26 compounds were measured in a 2-octanol/PBS system and estimated in silico. Correlation between kapp values was studied. Moreover, a multivariate analysis was performed to explain columns relationships. Obtained results show that porphyrins are separated mainly according to hydrophobic interactions that are relative to their structure (sugar number and the disposition around the porphyrin macrocycle).
Asunto(s)
Porfirinas/química , Algoritmos , Biomimética , Calibración , Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Lineales , Membranas Artificiales , Análisis Multivariante , Octanoles/química , Fosfolípidos/química , Fotoquimioterapia/métodos , Porfirinas/análisis , Análisis de Componente Principal , Programas InformáticosRESUMEN
Potentialities of polymerized vinyl alcohol on silica gel were assessed for class separation of simple lipids, sphingolipids, glyceroglycolipids and phospholipids by high-performance liquid chromatography. A screening of pure solvents in binary gradient elution and a chemometric approach was used to define a rugged two segment linear gradient formed from four solvents for total lipid class separation. Triethylamine and formic acid were added in all mobile phase components for acidic phospholipid separation and evaporative light scattering response enhancement. Simple analytical procedures are described for the analysis of complex lipid materials.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lípidos/aislamiento & purificación , Alcohol Polivinílico/química , Luz , Dispersión de RadiaciónRESUMEN
A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.
Asunto(s)
Ceramidas/análisis , Ceramidas/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Estructura MolecularRESUMEN
This study was devoted to the development of an analytical method for ceramide analysis in packed subcritical fluid chromatography (pSubFC). Monofunctional grafted silica support was found to be more suitable for ceramide analysis. Five Kromasil columns were coupled and the parameters, temperature, pressure and percentage of organic modifier in CO2 were optimised, considering selectivity and analysis time. The final conditions were 31 degrees C, 6% of methanol (MeOH) and 13 MPa. In these conditions the selectivity for structural differences (methylene group, unsaturation or two different bases) were studied. As classically observed, the methylene selectivity decreased with the increase of the eluotropic strength. Moreover, unlike in non-aqueous reversed-phase liquid chromatography (NARP-LC), adding a further unsaturation and two further methylene groups on ceramide results to an increase of retention in pSubFC. Moreover, this last technique allowed to separate ceramides with the same total number of carbons containing unsaturated fatty acids, when the distribution of carbon number of the two chain is very different. These results had enabled to plot retention chart in order to predict ceramide structure in view to identify additional ceramide. This retention chart was finally compared with the one already obtained in NARP-LC.
Asunto(s)
Ceramidas/análisis , Cromatografía Liquida/métodosRESUMEN
Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.
Asunto(s)
Ceramidas/análisis , Cromatografía Líquida de Alta Presión/métodos , Luz , Dispersión de Radiación , Espectrometría de FluorescenciaRESUMEN
A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min-1, using a 10% min-1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb3) in the urine of patients affected with Fabry disease. A liquid-liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Enfermedad de Fabry/orina , Glicoesfingolípidos/orina , Trihexosilceramidas/orina , Humanos , Sensibilidad y EspecificidadRESUMEN
When dealing with solid tumors in vivo, pegylated long-circulating carrier systems show, after intravenous administration, an attractive extravasation profile with an enhanced localization in the tumoral interstitium. These systems could be of help for the delivery of cancer fighting drugs, such as Tamoxifen, a well known antiestrogen used in breast cancer therapy that possesses an extended biodistribution in vivo. This work aimed at encapsulating Tamoxifen in long-circulating poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) 1:4 nanospheres. Tamoxifen-loaded poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) nanospheres were successfully synthesized and characterized in terms of hydrophilicity/hydrophobicity by a model made up from near infrared spectra using principal component analysis. Zeta potential, drug loading, encapsulation efficiency, as well as biological effect, in vitro release and nanospheres integrity were also investigated. Even though near infrared spectroscopy could not detect Tamoxifen, it revealed that Pluronic F68 was associated with the pegylated nanospheres. HPLC measurements demonstrated that Tamoxifen was encapsulated in the pegylated nanospheres following a partition equilibrium between the polymeric and the aqueous phases. The Tamoxifen encapsulated in the nanospheres still showed a transcription inhibitory activity in ex vivo experiments. However, zeta potential and in vitro release suggested that Tamoxifen was essentially localized at the nanoparticles surface, resulting in an important and immediate drug release.