Method for the Analysis of Glycosaminoglycans, and Their Derivatives and Salts by Nuclear Magnetic Resonance
AssigneeLaboratorios Farmaceuticos Rovi, S.A.
InventorsGuillermo FRANCO RODRIGUEZ, Ibon GUTIERRO ADURIZ
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Abstract
A nuclear magnetic resonance method of analysis for glycosaminoglycans in general, and of heparins and low molecular weight heparins and their derivatives in particular, is provided. The method allows for their identification and for relative quantification of respective characteristic signals by 1H-NMR and/or 1H-13C HSQC.
Claims47
1) A nuclear magnetic resonance (NMR) method for identifying and/or quantifying the content of one or more individual saccharides in one or more glycosaminoglycans in a composition, the method comprising at least the step of:
- providing a composition comprising:
- i) at least one glycosaminoglycan (GAG) comprising plural saccharides comprising respective anomeric or target hydrogen atoms exhibiting respective anomeric or target 41 chemical shift signals when analyzed by NMR;
- and ii) at least one reference compound comprising a hydrogen atom having a NMR signal Ti longitudinal relaxation time of 1 s or less, wherein the at least one reference compound exhibits a reference 1I-INMR chemical shift signal separated from said anomeric or target 1H chemical shift signals, and said reference 41 chemical shift signal exhibits a concentration dependent intensity;
- conducting at least one NMR analysis, on said composition, selected from the group consisting of one-dimensional (1D) 1H-NMR analysis, two-dimensional (2D) 1H-13C-NMR analysis, or a combination of said analyses, to provide at least one GAG NMR spectrum;
- thereby providing the content of said one or more individual saccharides and/or providing the identity of said one or more individual saccharides.
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11) A nuclear magnetic resonance (NMR) method for identifying and/or quantifying the content of one or more individual saccharides in one or more glycosaminoglycans in a composition, the method comprising at least the step of:
- providing a composition comprising:
- i) at least one glycosaminoglycan (GAG) comprising plural saccharides comprising respective anomeric or target hydrogen atoms exhibiting respective anomeric or target 1H chemical shift signals when analyzed by NMR and further comprising respective anomeric or target carbon atoms covalently bound to said anomeric or target hydrogens atoms and exhibiting respective anomeric or target 13C chemical shift signals when analyzed by NMR;
- and ii) at least one reference compound comprising a hydrogen atom having a NMR signal tl longitudinal relaxation time of 1 s or less and a respective carbon atom covalently bound to said hydrogen atom, wherein said at least one reference compound exhibits a reference 1H NMR chemical shift signal separated from said anomeric or target 1H chemical shift signals and further exhibits a reference 13C NMR chemical shift signal separated from said anomeric or target 13C chemical shift signals, and said reference 1H chemical shift signal and said reference 13C chemical shift signal each exhibits a concentration dependent intensity;
- conducting at least one NMR analysis, on said composition, selected from the group consisting of one-dimensional (1D) 13C-NMR analysis, two-dimensional (2D) 1H-13C-NMR analysis, or a combination of said analyses, to provide at least one GAG NMR spectrum;
- thereby providing the content of said one or more individual saccharides and/or providing the identity of said one or more individual saccharides.
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41) A method for identifying and/or quantifying the content of one or more individual saccharides in one or more glycosaminoglycans in a composition, the method comprising at least:
- providing said composition comprising at least a) one or more glycosaminoglycan comprising one or more individual saccharides comprising anomeric or target hydrogen exhibiting respective anomeric or target 41 chemical shift signal in the range of about 3.2 ppm to about 6 ppm when analyzed by NMR;
- b) dimethylmalonic acid as reference compound comprising a hydrogen atom having a NMR signal T1 longitudinal relaxation time of 1 s or less, wherein said reference compound exhibits a reference 1H NMR chemical shift signal separated from said anomeric or target 1H chemical shift signals, and said reference 1H chemical shift signal exhibits a concentration dependent intensity;
- and c) chemical shift reference standard defining 0 ppm;
- conducting at least one NMR analysis, on said composition, selected from the group consisting of one-dimensional (1D) 1H-NMR analysis, two-dimensional (2D) 1H-13C-NMR analysis, or a combination of said analyses, to provide at least one GAG NMR spectrum;
- and
- comparing the intensities of said anomeric or target 1H chemical shift signals to the intensity of the reference 1H chemical shift signal;
- thereby providing the content of and/or providing the identity of said one or more individual saccharides in said one or more glycosaminoglycans.
Dependencies are inferred from the claim text.