The study was carried out to develop procedure for determining concentration of formaldehyde to be used for crosslinking of gelatin in the presence of drugs having amino groups. Gentamicin sulfate was used as a drug candidate due to its high content of amino acids. Gelatin crosslinking is accelerated by aldehyde-containing compounds and inhibited by amino group-containing compounds. The major modifications from already existing procedures are that the trinitrobenzenesulphonic acid (TNBS) reaction is used to detect ε-amino groups of Type A gelatin in the presence of formaldehyde and further it is supported with colorimetric analysis of free formaldehyde content using a chromotropic acid regent. Since formaldehyde crosslinks amino groups, the TNBS assay can be effectively utilized for determination of complete crosslinking of gelatin with analysis of free amino acid content in crosslinked formulation. The effect of the presence of amino groups on gelatin crosslinking was estimated in the presence of gentamicin sulfate. The ε-amino content of uncrosslinked Type A gelatin was found to be 28.6 mol/gelatin molecule of 1000 residues and in case of crosslinked gelatin it varies with varying concentration of formaldehyde. The procedure stated here should be applicable to a broad range of drugs containing amino groups which are used along with gelatin or other proteinaceous materials which are applicable after crosslinking with formaldehyde.
Key words: Chromotropic acid reagent, colorimetric assay, gentamicin sulfate, trinitrobenzene sulfonic acid, ε-amino groups.
