Methods for quantification of carbohydrates
Inventors
Burki, Rajendar • Matur, Ramesh Venkat • Mantena, Narender Dev • Datla, Mahima
Assignees
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Publication Number
US-12276645-B2
Publication Date
2025-04-15
Expiration Date
Abstract
The present invention provides a colorimetric based method for quantifying carbohydrates in a given aqueous sample. The method provided by the invention uses 2-Phenoxyethanol as a novel reagent for quantifying carbohydrates in a given sample. The present invention is a rapid, sensitive, simple and direct method for carbohydrate quantification.
Core Innovation
The invention relates to a method for quantification of carbohydrates in an aqueous sample. The method comprises admixing sulphuric acid and 2-Phenoxyethanol with an aqueous sample containing carbohydrates to obtain a reaction mixture, incubating the reaction mixture to form a coloured complex, and measuring the absorbance of the coloured complex to quantify an amount of carbohydrates present in the aqueous sample.
The assay uses the formation of the coloured complex to quantify carbohydrates by measuring absorbance associated with the reaction mixture after incubation. The method is used for carbohydrates including monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, and their derivatives and combinations.
The described context includes application to carbohydrate quantification in complex matrices, including low-concentration pharmaceutical/biopharmaceutical/vaccine matrices, with stated performance metrics such as limit of detection and limit of quantification.
Claims Coverage
The document includes two independent claims: one directed to a method for carbohydrate quantification using sulphuric acid and 2-Phenoxyethanol to form a coloured complex, and one directed to a kit containing sulphuric acid and 2-Phenoxyethanol for the same purpose. Across the method claim, the claim set specifies multiple inventive features, including the reagent pairing, incubation to form the coloured complex, absorbance measurement, and constraints on measurement wavelength and assay proportionality and reagent composition/concentrations.
Sulphuric acid and 2-Phenoxyethanol reaction mixture for coloured complex formation
A method for quantification of carbohydrates in an aqueous sample comprising admixing sulphuric acid and 2-Phenoxyethanol with an aqueous sample containing carbohydrates to obtain a reaction mixture and incubating the reaction mixture to form a coloured complex.
Absorbance measurement of the coloured complex to quantify carbohydrates
Measuring the absorbance of the coloured complex to quantify an amount of carbohydrates present in the aqueous sample.
Absorbance wavelength range for quantification
Measuring the absorbance of the coloured complex at a wavelength in the range of 490 nm to 510 nm.
Proportional relationship between carbohydrate quantity and absorbance
Determining that the quantity of carbohydrate in an aqueous sample is proportional to the measured absorbance of a coloured complex.
Sulphuric acid to sample volume ratio
Using sulphuric acid at a volume that is 1 to 3 times the volume of the aqueous sample.
2-Phenoxyethanol concentration constraint in the reaction mixture
Using a reaction mixture containing 2-Phenoxyethanol at a concentration ranging from 0.1% v/v to 2.5% v/v.
Carbohydrate selection from defined carbohydrate classes
Selecting carbohydrates from monosaccharides, disaccharides, polysaccharides, uronic acids, hexosamines, their derivatives, and combinations thereof.
Carbohydrate quantification kit containing sulphuric acid and 2-Phenoxyethanol
A kit for quantifying carbohydrates in an aqueous sample comprising sulphuric acid, 2-Phenoxyethanol and a combination thereof.
Overall, the claim coverage centers on carbohydrate quantification by forming a coloured complex from an aqueous carbohydrate sample using sulphuric acid and 2-Phenoxyethanol, followed by absorbance measurement, with additional constraints specifying an absorbance wavelength range, proportionality of carbohydrate quantity to absorbance, constrained sulphuric acid-to-sample volume ratio, constrained 2-Phenoxyethanol concentration, and defined carbohydrate categories. A complementary kit claim covers sulphuric acid and 2-Phenoxyethanol as kit components.
Stated Advantages
Provides rapid, sensitive, simple quantification for carbohydrates.
Stated performance metrics include a limit of detection (LOD) of less than 4 μg/mL and a limit of quantification (LOQ) of 5–8 μg/mL.
Stated specificity against impurities/excipients.
Stated improved sensitivity compared with anthrone (625 nm) and phenol-sulphuric acid (490 nm) assays.
Stated non-impairment by uronic-acid-containing polysaccharides.
Stated suitability for pneumococcal conjugate vaccine matrices, including monoconjugate and multivalent forms, with stated recovery.
Documented Applications
Quantification of carbohydrates in pneumococcal conjugate vaccines, including monoconjugate and multivalent pneumococcal conjugate vaccine matrices.
Carbohydrate quantification in low-concentration pharmaceutical/biopharmaceutical/vaccine matrices.
Use in an assay context comparing performance to anthrone and phenol-sulphuric acid assays.
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