RESUMO
OBJECTIVES: Norepinephrine is usually used in emergency situations such as in intensive care units (ICUs) for the restoration of blood pressure. The objective was to study the stability of highly-concentrated solutions of norepinephrine at 0.50mg/mL and 1.16mg/mL, diluted in glucose 5% (G5%) in polypropylene syringes, protected or not from light, up to 48h. MATERIALS AND METHODS: Chemical stability was analysed by high-performance liquid chromatography coupled to photodiode array detection at each time of the analysis. The method was validated according to the International Conference on Harmonisation Q2(R1). Physical stability was evaluated by visual and subvisual inspection. Three syringes for each condition were prepared. At each time of the analysis, three samples were analysed for each syringe. pH values were evaluated at each moment of the analysis. RESULTS: Solutions of norepinephrine at 0.50 and 1.16mg/mL, diluted in G5%, with or without protection from light, retained more than 95.0% of the initial concentration after a 48-hour storage at 20-25°C. No visual and subvisual modification occured during the stability study. No degradation product appearing during the stressed degradation was observed during the study but an additional peak with a relative retention at 0.66 was observed and constant. This peak was identified as 5-hydroxymethylfurfural, a degradation product of glucose. CONCLUSION: Norepinephrine diluted in G5% at 0.50mg/mL and 1.16mg/mL was physically and chemically stable over a period of 48hours at room temperature. These stability data of highly concentrated solutions provide additional knowledge to assist intensive care services in daily practice.
Assuntos
Unidades de Terapia Intensiva/estatística & dados numéricos , Norepinefrina/análise , Vasoconstritores/análise , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Furaldeído/análogos & derivados , Furaldeído/análise , Concentração de Íons de Hidrogênio , Luz , Norepinefrina/administração & dosagem , Polipropilenos , Reprodutibilidade dos Testes , Seringas , Temperatura , Vasoconstritores/administração & dosagemRESUMO
A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid-phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C18 column (150 × 4.6 mm, 3.6 µm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/sangue , Peptídeos/farmacocinética , Animais , Fluorescência , Injeções Intraperitoneais , Limite de Detecção , Masculino , Peptídeos/administração & dosagem , Ratos Wistar , Sensibilidade e Especificidade , Extração em Fase Sólida/métodos , Espectrometria de Fluorescência/métodos , Espectrometria de Massas em TandemRESUMO
Cyclodextrins (CyDs) currently displays even today the image of a natural macrocyclic compound largely dominant in the formation of inclusion complexes with small hydrophobic molecules. During the past 10years, advances in this field allowed to achieve more and more sophisticated CyDs derivatives opening a simple access in scale-up quantities to original and better CyD-based gene delivery systems. In addition, possibility to combine covalent and supramolecular approaches offers new venues for the design of tailor-made CyD-based nanovehicles to improve their transfection ability and gene transfer in cells. In this account, we describe our recent progress in the construction of a novel CyD-based G0 (generation number) core dendrimer, scalable to CyD oligomers by a strategy using protonable guanidine tethers and whose concept can be generalized for the assembly of CyD pre-coated dendrimers. The synthetic strategy based on an original Staudinger-Aza-Wittig tandem coupling reaction. We present an outline of the different analytical strategies to characterize CyD-ODN (cyclodextrin-oligodeoxynucleotide) complexes. Among them, Capillary electrophoresis (CE) was used to perfectly characterize our CyD-siRNA and CyD-DNA complexes and shown to be a very attractive method with advantages of low sample consumption, rapid analysis speed, and high efficiency that make this technology a major tool for association constant measurement. Finally, we present the different biological methods that can be used, in vitro, to study gene delivery, and more precisely ones we have performed to evaluate the capability of our original model bis-guanidinium-tetrakis-ß-cyclodextrin dendrimeric tetrapod, to deliver efficiently DNA or siRNA in eukaryotic cells.
Assuntos
Ciclodextrinas/química , Portadores de Fármacos/química , Técnicas de Transferência de Genes , Sequência de Carboidratos , Corantes , Ciclodextrinas/análise , DNA/administração & dosagem , Portadores de Fármacos/análise , Sistemas de Liberação de Medicamentos , Modelos Moleculares , Dados de Sequência Molecular , Sais de Tetrazólio , TiazóisRESUMO
The development of new packaging able to preserve sensitive biomolecules against oxidative stress is an important field. Several studies refer to antioxidant properties carried out by colloidal gold nanoparticles (AuNP). Herein, the purpose was to check whether this property is preserved when AuNP are immobilized on a glass support. After nanostructured film preparation, the physicochemical characterization proved that AuNP were well-individualized in the films with a high density of immobilization. Two radicals: ABTSâ¢+ and DPPH⢠were used to investigate their antioxidant capacity. The results showed that immobilized AuNP had a preserved antioxidant capacity characterized by a different kinetic: more controlled and more prolonged but with the same efficiency (vs the same quantity of colloidal AuNP). The AuNP films demonstrated a capacity to prevent from degradation a molecule containing a thiol function. A 10-fold increase of N-acetylcysteine half-life was measured using the immobilized AuNP, highlighting the interest of the developed and adaptable support.
Assuntos
Antioxidantes/farmacologia , Embalagem de Medicamentos , Nanopartículas Metálicas/química , Acetilcisteína , Coloides/química , Nanopartículas Metálicas/ultraestrutura , Compostos de Sulfidrila/químicaRESUMO
The facile synthesis of a novel bis-(guanidinium)-tetrakis-(beta-cyclodextrin) tetrapod, the first example of a new host family, was described, and the ability of the cyclodextrin CyD tetrapod to form molecular association with siRNA and DNA guest molecules was demonstrated. Affinity capillary electrophoresis was used to determine the binding constant with the evaluation of the shift in the electrophoretic mobility mu of injected siRNA when various CyD tetrapod concentrations were added to the run buffer. A significant association constant (K(a) =16,000 M(-1)) was obtained with borate buffer when double-stranded siRNA was primarily opened with the help of temperature. An efficient cellular transfection of siRNA into human embryonic lung fibroblasts was observed by fluorescence microscopy.
Assuntos
DNA/genética , Dendrímeros/síntese química , Dendrímeros/metabolismo , Desenho de Fármacos , Guanidinas/síntese química , Guanidinas/metabolismo , RNA Interferente Pequeno/genética , Transfecção/métodos , beta-Ciclodextrinas/síntese química , beta-Ciclodextrinas/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Dendrímeros/toxicidade , Guanidinas/toxicidade , RNA Interferente Pequeno/metabolismo , beta-Ciclodextrinas/toxicidadeRESUMO
A rapid and simple method for the determination of main components and related substances of both neomycin sulfate and framycetin sulfate by HPLC and evaporative light scattering detection (ELSD) is described. The method was also used to determine the neomycin B and the sample sulfate content. Detection and quantitation of aminoglycoside antibiotics are problematic because of the lack of UV absorbing chromophore. The use of a universal detector avoids the need for sample derivatization or use of specific detector based on pulsed amperometry described to be difficult in routine assays. Separation was performed with a Polaris C18 150 mm x 4.6 mm i.d., 3 microm reversed-phase column with a solution of 170mM trifluoroacetic acid (TFA) mobile phase at a flow rate of 0.2 mL/min. The chromatographic parameters were optimized with the help of experimental design software. Mass spectrometry (MS) was employed to confirm the ELSD profile. The final method was validated using methodology described by the International Conference of Harmonization in the field of Active Pharmaceutical Ingredients. Commercial samples of different sources were analyzed and results were in good agreement with specifications of the European Pharmacopoeia.
Assuntos
Antibacterianos/análise , Cromatografia Líquida de Alta Pressão/métodos , Framicetina/análise , Neomicina/análise , Espalhamento de Radiação , Luz , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A rapid and simple method for the separation and quantitation of gentamicin sulfate by HPLC coupled with evaporative light scattering detection (ELSD) has been developed. Detection of the different components of gentamicin is problematic because of the lack of UV absorbing chromophore. The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry. Separation was performed on a highpurity C18 125 mm x 4 mm i.d., 3 microm, reversed phase column with 48.5 mM trifluoroacetic acid-methanol (97:3, v/v), as mobile phase at a flow rate of 0.7 ml/min. The influence of the gas nature, gas pressure and temperature of the drift tube of the detector on the detection response was investigated. Optimization was performed with the help of a specific experimental design software. This method allows the determination of the composition in components C1, C1a, C2, C2a and C2b of gentamicin sulfate samples. Mass spectrometry was employed to confirm the ELSD chromatographic profile. The method was validated using methodology described by the International Conference of Harmonization in the field of Medicinal Substances. Commercial samples of different sources were analyzed and results were in good agreement with specifications of both European and United States Pharmacopoeia.
Assuntos
Antibacterianos/análise , Gentamicinas/análise , Cromatografia Líquida de Alta Pressão , Luz , Espectrometria de Massas , Reprodutibilidade dos Testes , Espalhamento de Radiação , SoftwareRESUMO
The stoichiometric coefficients and apparent formation constants (Kf) of alpha-terpineol, thymol, geraniol and linalool complexes with beta-cyclodextrin (beta-CD) were determined using HPLC with a porous graphitic carbon (PGC) chromatographic support. Measurements were performed with four different methanol-water mobile phases. All the terpene derivatives under study form 1:1 guest-CD complexes. Graphs of Kf as a function of the mobile phase composition appeared different from those classically described for RP-C18 and suggest that the PGC stationary phase could play an active role in the complexation process. Solute-CD inclusion and solute-stationary phase interactions may be involved in this specific behavior.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ciclodextrinas/química , Terpenos/química , beta-Ciclodextrinas , Cromatografia Líquida de Alta Pressão/instrumentação , Grafite/químicaRESUMO
Specific programming of automated HPLC systems allows total on-line qualification, validation and stability monitoring using the concept of deferred standards. Setting up such a process for routine analyses in an automated HPLC system requires specific autosampler programming as well as specific monitoring software. With an autosampler, a double injection procedure is programmed, the first introducing the sample, and the second, a few minutes deferred, the deferred control standard. Two additional compounds are therefore added to the sample before and during the chromatographic process: the intemal standard for sample quantification and the deferred standard for system control. Specific methodologies are described of how to obtain classical quantitative analysis information as well as system qualification validation stability information. Experiments were performed to develop specified methodologies to monitor the quality of quantitative analysis during the life of the column by using the deferred standard concept to probe the effects of column ageing on separation characteristics.
Assuntos
Cromatografia Líquida de Alta Pressão/normas , Automação , Cromatografia Líquida de Alta Pressão/métodos , Compostos Policíclicos/análise , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.
Assuntos
Eritrócitos , Separação Celular , Fracionamento Químico/métodos , Humanos , Concentração OsmolarRESUMO
Near InfraRed Spectroscopy (NIRS) is a potentially powerful tool for assessing the homogeneity of industrial powder blends. In the particular context of hospital manufacturing, we considered the introduction of the technique at a small pharmaceutical process scale, with the objective of following blend homogeneity in mixtures of seven components. This article investigates the performance of various NIRS-based methodologies to assess powder blending. The formulation studied is prescribed in haematology unit, as part of the treatment for digestive decontamination in children receiving stem-cell transplantation. It is composed of the active pharmaceutical ingredients (APIs) colimycin and tobramycin and five excipients. We evaluated 39 different blends composing 14 different formulations, with uncorrelated proportions of constituents between these 14 formulations. The reference methods used to establish the NIRS models were gravimetry and a High Performance Liquid Chromatography method coupled to an Evaporative Light Scattering Detection. Unsupervised and supervised qualitative and quantitative chemometric methods were performed to assess powder blend homogeneity using a bench top instrument equipped with an optical fibre. For qualitative evaluations, unsupervised Moving Block Standard Deviation, autocorrelation functions and Partial Least Square Discriminant Analysis (PLS-DA) were used. For quantitative evaluations, Partial Least Square Cross-Validated models were chosen. Results are expressed as API, and major excipient percentages of theoretical values as a function of blending time. The 14 different formulations were only satisfactorily discriminated by supervised algorithms, such as an optimised PLS-DA model. The homogeneity state was demonstrated after 16 min of blending, quantifying three components with a precision between 1.2% and 1.4% w/w. This study demonstrates, for the first time, the effective implementation of NIRS for blend homogeneity evaluation, as early as the preformulation step in a small hospital manufacturing unit. It shows how NIRS involving sampling with an optic fibre can be useful to characterise, optimise and control a small-scale mixing processes on the basis of the distribution of APIs and excipients during blending.
Assuntos
Composição de Medicamentos/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Calibragem , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão , Colistina/química , Desenho de Equipamento , Luz , Tamanho da Partícula , Preparações Farmacêuticas/química , Pós/química , Espalhamento de Radiação , Software , Tecnologia Farmacêutica/métodos , Tobramicina/químicaRESUMO
A rapid and simple method for the simultaneous determination of tobramycin and colistin sulphate in a pharmaceutical formulation by reversed phase HPLC and evaporative light scattering detection is described. Chromatographic separation was carried out in gradient mode using a Zorbax SB C18 column (150mmx4mm, 3.5microm) with mobile phases of acetonitrile and water containing trifluoroacetic at 1ml/min. The method was validated using methodology described by the International Conference of Harmonization. The method was shown to be specific, precise, accurate and linear. Real samples were analyzed to demonstrate the applicability of the chromatographic method in a routine use.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Colistina/análise , Tobramicina/análise , Luz , Padrões de Referência , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e EspecificidadeRESUMO
Thanks to the recent technological advancements, evaporative light-scattering detection (ELSD) is regarded as a valuable alternative to UV detection for liquid chromatographic analysis of substances that do not contain a chromophore. In the field of substances for pharmaceutical use, LC-ELSD appears to be suitable for aminoglycosides, most of which (for ex. gentamicin) are presently controlled in the Ph. Eur. by pulsed amperometric detection. Other substances (ex sugars, triglycerides) presently employing refractometrric detection, could be conveniently analysed by LC-ELSD. ELS detection is regarded as robust and relatively simple, although not particularly sensitive. A key feature of ELSD is that - unlike refractometry - it can operate in gradient mode, thus allowing application of more selective liquid chromatographic methods. ELSD can also be used to set up MS-compatible methods, as the mobile phase constraints are essentially the same. Due to all the above, ELSD is becoming increasingly used in pharmacopoeial methods.
Assuntos
Cromatografia Líquida/métodos , Preparações Farmacêuticas/análise , Espalhamento de Radiação , Aminoglicosídeos/análise , Carboidratos/análise , Contaminação de Medicamentos , Luz , Farmacopeias como Assunto , Controle de Qualidade , Reprodutibilidade dos Testes , Triglicerídeos/análiseRESUMO
Sedimentation field-flow fractionation (SdFFF) instrumentation is now mature. Methodological procedure and particle separation development rules are well established even in the case of biological species. However, in some biological applications, retention properties of samples not predicted by any field-flow fractionation (FFF) elution models are observed. It is demonstrated that the trapping of cellular material in the separation system is not related to geometrical instrumentation features but to channel wall characteristics. The physicochemical particle-wall attractive interactions are different depending on the flow-rate and field intensity applied. Separation power in SdFFF for biological species is therefore limited by the intensity of these interactions. In terms of separation, a balance is to be found between external field and flow intensity to limit particle-wall interactions.
Assuntos
Separação Celular/métodos , Eritrócitos , Cimento de Policarboxilato , Fracionamento Químico , Fenômenos Químicos , Físico-Química , Eritrócitos/química , Humanos , Cintilografia , TecnécioRESUMO
The retention behavior of natural alpha-, beta-, and gamma-cyclodextrins on a porous graphitic carbon (PGC) stationary phase is investigated. Unusual retention properties for reversed-phase chromatographic conditions are observed with acetonitrile-methanol and water-methanol mixtures as mobile phases. It is assumed that the retention process is governed not only by the standard solvophobic effect but also by specific interactions described as "CD-PGC" effect. The retention factor versus the volumetric methanol fraction in the mobile phase show second-order curves expressing this double mechanism hypothesis. van't Hoff plots demonstrate the contribution of these two retention processes. The retention factor of each natural cyclodextrin is shown to depend on the mobile phase property to act as a proton acceptor, according to the solvent selectivity classification described by Snyder. The "CD-PGC" effect is interpreted as an equilibrium between different interactions: cyclodextrin-PGC stationary phase, London dispersion forces, and cyclodextrin-mobile phase hydrogen bonding. The balance of these interactions may monitor the orientation of the cyclodextrin molecule facing the carbon surface, which is therefore suspected to be the major parameter of this retention mechanism.