Electrophoretic approaches to the analysis of complex polysaccharides

Volpi, Nicola; Maccari, Francesca

doi:10.1016/j.jchromb.2006.02.049

Cited by 76 publications

(53 citation statements)

References 89 publications

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“…Entretanto, os estudos são dirigidos para a determinação das várias sequências sacarídicas que compõem a molécula e utilizam técnicas como ressonância magnética nuclear, espectrometria de massas e sistemas eletroforéticos. [8][9][10][11][12][13] Estes métodos são trabalhosos e se tornam inviáveis para a quantificação da heparina em formas farmacêuticas, pois envolvem processos de derivatização, incluindo a utilização de enzimas e a cromatografia de exclusão para a separação das diferentes frações sacarídicas. A United States Pharmacopeial Convention publicou em 2008 uma monografia atualizada para a HF, porém não descreve metodologia menos laboriosa.…”

Section: -4unclassified

Determinação de heparina fracionada em preparações farmacêuticas utilizando turbidimetria

Marotta-Oliveira¹,

Marchetti²

2011

Quím. Nova

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Recebido em 17/7/10; aceito em 5/12/10; publicado na web em 29/3/11 DETERMINATION OF FRACTIONATED HEPARIN IN PHARMACEUTICAL DOSAGE FORMS USING TURBIDIMETRY. A turbidimetric method has been used for quantification of fractionated heparin (FH) in pharmaceutical dosage. The UV detection at two wavelengths (290 and 500 nm) showed a significant increase in sensitivity of the method, specificity, and linearity to range 5.0-50.0 μg mL -1 and 50.0-200.0 μg mL, respectively (r < 0.99). At both wavelengths, the method was precise (inter-assay CV < 5.0%, and intra-assay CV < 3.0%), accurate (maximum deviation of ± 12%), and robust to the parameters evaluated. Turbidimetry proved to be easy, inexpensive and relatively fast. The results obtained attest to the reliability of the method.Keywords: fractionated heparin; quantitative analysis; turbidimetry. INTRODUÇÃOA heparina é o anticoagulante mais utilizado mundialmente há mais de 50 anos no tratamento e profilaxia da trombose venosa profunda (TVP) e de sua principal complicação, a embolia pulmonar. Constitui o segundo fármaco natural mais utilizado no mundo, superado apenas pela insulina.1 Trata-se de uma glicosaminoglicana composta de cadeias de resíduos alternados de D-glicosaminoglicana e um ácido urônico. Sua molécula é sulfatada, ácida e carregada negativamente. 2-4As heparinas fracionadas (HFs) são obtidas a partir da heparina extraída da mucosa intestinal de suínos por degradação química ou enzimática. [5][6][7] Dentre as HFs disponíveis, a enoxaparina sódica (Figura 1) é a mais estudada e tem sido veiculada em muitas formas farmacêuticas de uso parenteral. Algumas estão disponíveis no mercado como Enoxil ® (Prodotti), Dripanina ® (Ariston) e Clexane ® (Sanofi). Nos últimos anos é crescente o número de pesquisas objetivando o desenvolvimento de novos sistemas de liberação para otimização da terapia. Assim, o desenvolvimento de métodos para o doseamento da HF é bastante relevante.Diversos métodos analíticos têm sido desenvolvidos para avaliar e quantificar as glicosaminoglicanas incluindo as heparinas. Entretanto, os estudos são dirigidos para a determinação das várias sequências sacarídicas que compõem a molécula e utilizam técnicas como ressonância magnética nuclear, espectrometria de massas e sistemas eletroforéticos. [8][9][10][11][12][13] Estes métodos são trabalhosos e se tornam inviáveis para a quantificação da heparina em formas farmacêuticas, pois envolvem processos de derivatização, incluindo a utilização de enzimas e a cromatografia de exclusão para a separação das diferentes frações sacarídicas. A United States Pharmacopeial Convention publicou em 2008 uma monografia atualizada para a HF, porém não descreve metodologia menos laboriosa. Outras formas encontradas para a análise quantitativa da HF são os métodos nefelométrico, descrito por Ardry, 14 e turbidimétrico utilizado em trabalhos mais recentes. 15-17A turbidimetria é uma técnica relacionada à colorimetria, capaz de medir a turbidez de uma amostra, e possui aplicação restrita à quantificação de substân...

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Section: -4unclassified

Determinação de heparina fracionada em preparações farmacêuticas utilizando turbidimetria

Marotta-Oliveira¹,

Marchetti²

2011

Quím. Nova

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“…Although, at present, only single examples of polysaccharides have been recognized as being involved in biomineralization, recently developed glycomics research methods may now provide rapid of access to the analysis of these materials. Analytical electrophoretic separation techniques, including agarose-gel, high-performance capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), have been introduced into the areas of structural analysis and quantification of complex polysaccharides, and notably of the acidic bacterial polysaccharides [37]. In this respect, considering that PS3 represents only 3% of the total acidic polysaccharides (PS1, 2 and 3), many new polysaccharides involved in the biomineralization remain to be discovered.…”

Section: Acidic Polysaccharides Mediating Formation Of Complex Calcitmentioning

confidence: 99%

Growth and Form: What is the Aim of Biomineralization?

Bäuerlein¹

2007

Handbook of Biomineralization

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Abstract12-Amino-acid peptides with binding-selected sequences and random functional groups synthesize the same inorganic material. Bacteria use structured polymers as filaments of cytoskeletal protein and of polysaccharides in templating biominerals. Nucleation appears to be involved not only in crystal formation, but also in the formation of porous or amorphous inorganic material. A seed of one iron ion for an iron-oxide cluster could be obtained in the low-iron-state of an archeal ferritin crystal. Porous inorganic materials were described to grow out in huge channeled complexes of organic compounds or crystallized seawater. The evolutionary progress of biomineralization runs apparently in the development of complex, inorganic-organic hybrid materials, composites, which are often structured hierarchically. The mechanical properties of a mineral undergo a change by presence and partition of the organic material, the most prominent example being the human skeleton. In order to describe such a complex system, a mathematical procedure -the finite element analysis -was introduced with great success. This analysis is governed amazingly by only one physical quantity, force, together with the modulus of elasticity. The overwhelming extent of research into the mechanical properties of skeletons has resulted in the conclusion that the aim of biomineralization appears to be stability.

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“…GAGs can be quantified and analyzed by NMR [14], MS [15], LC-MS [16], enzymatic post column fluorescence HPLC [17], electrophoretic methods [18], and by more general methods for monosaccharide composition analysis such as high-performance anionexchange chromatography (HPAEC) with pulsed amperometric detection (PAD) [19]. However, a simple, direct, sensitive, and robust quantification method is needed for routine GAG quantification.…”

Section: Introductionmentioning

confidence: 99%

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

Frazier

Roodhouse²,

Hourcade³

et al. 2008

TOGLYJ

111

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Glycosaminoglycans (GAGs) are linear polysaccharides that are found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. There are five classes of animal GAGs: heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA). Many biological functions can be monitored directly by their impact on GAG quantity. Thus, simple, sensitive, and robust GAG quantification methods are needed for the development of biomarkers. We have systematically compared three available GAG quantification assays including an HPLC-based assay, a simplified Alcian Blue assay, and a miniaturized carbazole assay. The carbazole and Alcian Blue assays were reproducible and simple to perform in general lab settings, but had important limitations: The carbazole assay could not detect KS and it overestimated GAGs that were contaminated with salts or dissolved in PBS. The Alcian Blue assay detected only those GAGs that were sulfated. In contrast, while the HPLC method was time-consuming, it was a robust and sensitive assay that not only detected all GAGs but also quantified glucosamine-GAGs and galactosamine-GAGs simultaneously. The HPLC assay was not affected by salt or level of GAG sulfation and it yielded reproducible values for all types of GAGs tested. These results suggest that an automated HPLC assay would be generally useful for the routine measurement of a panel of GAG-based biomarkers while the carbazole assay and the Alcian Blue assays could prove valuable for more specific purposes.

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Electrophoretic approaches to the analysis of complex polysaccharides

Cited by 76 publications

References 89 publications

Determinação de heparina fracionada em preparações farmacêuticas utilizando turbidimetria

Determinação de heparina fracionada em preparações farmacêuticas utilizando turbidimetria

Growth and Form: What is the Aim of Biomineralization?

The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods

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