MAREKERA FREEDOM STENYG12M5762Pharmaceutical Chemistry Assignment II: Analysis of Streptomycin1.)Adapted from: Goodman & Gilmans The Pharmacological Basis of Therapeutics, 12e.p 808The streptomycin molecule contains a number of functional groups which may serve as bases for chemical assay. Furthermore, the molecule has a free or potentially free carbonyl group and marked reducing properties. Any of these groups can be used as a basis for assay purposes on highly purified material, but these reactions are not sufficiently specific for samples of lower potency or biological fluids. 1 Streptomycin and its impurities, like most aminoglycosides and carbohydrates, lack a good chromophore and therefore require high concentrations to be detected by UV absorbance. Many ingredients of manufacturing process intermediates and final pharmaceutical formulations are chromophoric and can interfere with the direct detection of streptomycin A and its impurities by absorbance. Refractive index detection has similar limitations hence this will not form a good method for an assay.2 Aminoglycosides, due to their extremely polar, hydrophilic character shown by abundance of hydroxyl and amine groups on the molecule, are analyzed mostly by chromatography on silica gel, but C-18 plates can also be used. Polar organic solvents (methanol, acetone, chloroform) mixed with 25% aqueous ammonia are the most popular mobile phases. Since the majority of aminoglycosides lack UV absorption, they must be derivatized by spraying or dipping after development with fluorescamine, vanillin, or ninhydrin solutions. They can be also detected by charring, treating with iodine vapor, or derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or with a mixture of diphenylboronicanhydride and salicylaldehyde.3. Thus high-performance liquid chromatography (HPLC) analysis is suitable for the analysis of streptomycin.2.) High Performance Liquid ChromatographyIn HPLC, a liquid sample, or a solid sample dissolved in a suitable solvent, is carried through a chromatographic column by a liquid mobile phase. Separation is determined by solute/stationary-phase interactions, including liquidsolid adsorption, liquidliquid partitioning, ion exchange and size exclusion, and by solute/mobile-phase interactions. 4 For HPLC a high surface area of the interface between stationary and mobile phase is essential where different components in the mixture are discriminated to provide resolution of components. Amines, thiols, and hydroxyl groups oxidize readily at electrodes such as the carbon electrodes used for electrochemical detection. The multiple amino and hydroxyl groups in the aminoglycoside structures are apparently the source of their electrochemical activity. The advantage of electrochemical detection in aminoglycoside analysis is that it doesnot require derivatization. However, the structure of the detected compound cannot be identified, and confirmation of a specific chromatographic peak is not possible . The inability to do these is a particular disadvantage when retention times and elution orders change. 5Liquid ChromatographyAs water-soluble, polar, and relatively large compounds, aminoglycosides typically would be analyzed by liquid chromatography Most high-performance liquid chromatographic (HPLC) methods concern gentamicin analysis, but because of the chemical similarity within the group, these methods are often applicable to other aminoglycosides. The most significant characteristics of aminoglycosides relevant to HPLC analysis are the lack of a chromophore and the high hydrophilicity. To improve detectability and separation, the aminoglycosides are usually derivatized prior to or after chromatographic separation.Reversed-Phase HPLCBecause of their polar and ionic nature, aminoglycosides usually are not partitioned on reversed-phase columns, Partition to C18 columns can be improved by using acetate buffer in the mobile phase. The acetate ion acts as a counter ion, forming ion pairs with aminoglycosides. Usually aminoglycosides are derivatized with a nonpolar group prior to reversed-phase analysis to improve the partition to column and the separation characteristics. The most commonly used derivatization reagents are o-phthalaldehyde (OPA) and 1-fluoro-2, 4-dinitrobenzene (FDNB). The separation columns usually contain C8 or C18 materials, and mobile phases consist of acidic buffers with methanol and/or acetonitrile. 4Ion-Exchange ChromatographyIon-exchange chromatography (IE) should be a popular method for analysis of polar aminoglycosides that are ionized in solutions. However, IE requires careful regulation of pH, temperature, and ionic strength, and it has not become widely used in aminoglycoside analysis.Normal-Phase HPLCNormal-phase chromatography (adsorption chromatography) is usually used for simple compounds that are not ionized and that dissolve readily in organic solvents. The hydrophilicity and poor solubility in