A novel HPLC method for determination of EDTA in a cataract inhibiting ophthalmic drug.

A novel HPLC method for determination of EDTA in a cataract inhibiting ophthalmic drug product has been developed. In this method EDTA was converted to Cu(II)EDTA complex, using Cu + 2 containing mobile phase, after injection into the chromatographic system. This allowed complexation of EDTA with Cu + 2 without interference from formulation ingredients. The Cu(II)EDTA complex was separated from drug substance, impurities, degradants and other formulation excipients by a 250 x 4.1 mm anion exchange column and detected at UV wavelength 250 nm. The mobile phase consisted of 2 mM cupric nitrate, 11 mM nitric acid, and 25% (v/v) acetonitrile at pH 3.0. This stability indicating assay has been validated and shown to be specific, linear, precise, accurate and rugged for routine EDTA analysis.

Selectivity of organic solvents in micellar liquid chromatography of amino acids and peptides.

The influence of the type of organic modifiers on retention behavior in micellar liquid chromatography is studied. A group of amino acids and small peptides was used as the test mixture. It is shown that the chromatographic selectivities of 2-propanol, acetonitrile and tetrahydrofuran which belong to three different groups in Snyder's classification, are considerably similar in the presence of micelles for the test mixture. On the other hand, the selectivities of 2-propanol and butanol which belong to the same solvent selectivity group are different for these solutes in the micellar mobile phases.

Controlling solvent strength and selectivity in micellar liquid chromatography: role of organic modifiers and micelles.

Simultaneous enhancement in elution strength and selectivity which has been previously observed in micellar liquid chromatography (MLC) for a variety of compounds is further investigated. The reasons behind the occurrence of this unique phenomenon are studied, and the influence of micelles and organic solvents on elution strength and selectivity is discussed. A model is developed which explains the dependence of the solvation ability of organic solvents in MLC (represented by the solvent strength parameter, S, of solutes) and the degree of solute interactions with micelles. Whenever the difference in solvent strength parameter values of two solutes in micellar eluents, dS, is positive, maximum selectivity is observed at the weakest eluent strength. When dS less than 0, there exists an inverse relationship between retention and solvent strength parameter so that selectivity monotonically increases with volume fraction of organic solvent in micellar eluents. It is shown that usually there is no direct relationship between the solvent strength parameter in MLC and retention. As a result, selectivity enhancement due to an increase in the concentration of organic modifier (i.e. solvent strength) occurs frequently in MLC. Interestingly, for cases where selectivity decreases with an increase in organic modifier, simultaneous enhancement of selectivity and solvent strength can be observed by increasing micelle concentration. In a sense, the concentrations of organic modifier and micelles complement one another in improving selectivity at higher elution strengths. As a result of this unique phenomenon better separations in shorter analysis times can be observed. The mutual effects of micelles and organic modifier on one another would also require a simultaneous optimization of these two parameters.

Chromatographic characteristics of surfactant-mediated separations: micellar liquid chromatography vs ion pair chromatography.

The effects of concentrations of organic solvent and surfactant on elution strength and selectivity in MLC and IPC are studied. It is observed that selectivity between most pairs of solutes used in this study increases in MLC and either decreases or passes through a minimum in IPC, with the volume fraction of organic modifier. In both MLC and IPC, selectivity varies with surfactant concentration; however, the overall variation in selectivity and elution order are more pronounced in MLC. The solvent strength decreases in IPC and increases in MLC as a result of an increase in surfactant concentration. An iterative regression design is used to predict the optimum mobile-phase compositions in terms of solvent strength and selectivity. The correlation between the predicted and measured chromatograms is excellent in MLC and poor in IPC. This is due to a more regular and reproducible retention behavior in MLC which greatly facilitates the development of robust methodologies. For a mixture of amino acids and peptides, a large retention gap between the first and the last eluting solutes is observed in IPC, which makes the use of organic solvent gradient inevitable. However, a better separation for the same mixture of solutes can be achieved in MLC isocratically. Apparently, the general elution problem can be alleviated in MLC by using an optimum eluent composition. It is observed that the efficiencies of MLC and IPC are comparable. The above observations indicate that MLC can be a powerful alternative to IPC in order to achieve optimized separations in shorter analysis time.