Quantitation of N-acetylneuraminic (sialic) acid in bovine glycomacropeptide (GMP)

Bovine glycomacropeptide (GMP) is the hydrophilic C-terminal peptide produced from κ-casein during cheese-making. N-Acetylneuraminic acid (NeuNAc) is one of the sugars associated with bovine GMP and the reported biological and functional properties of this dairy peptide. A sensitive RP-HPLC method has been developed to quantify NeuNAc in GMP using the fluorophore 1,2-diamine-4,5-dimethoxyl benzene dihydrochloride (DDB). The NeuNAc liberation conditions, DDB derivatisation conditions, labelling mixture and internal standard (α-keto glutaric acid) were optimised and the amount of NeuNAc in three different GMP samples determined. The optimal conditions required to liberate NeuNAc from bovine GMP were 25 mM sulphuric acid at 80 °C for 120 min. The optimal DDB derivatisation conditions were 60 °C for 150 min. Masses of the DDB derivatised NeuNAc (442 m/z) and α-KG (278 m/z) peaks were confirmed using LC–MS (ESI). The NeuNAc content of GMP samples ranged from 5.0% to 11.3% (w/w). Results were compared to the spectrophotometric thiobarbituric acid (TBA) method. TBA results for the same samples ranged from 6.4% to 13.5% (w/w). The DDB method developed had a limit of detection (LOD) of 7 pg and a limit of quantification (LOQ) of 20 pg. This sensitive method can be employed to accurately quantify the NeuNAc content of GMP.