Optimization of liquid chromatographic method for the separation of folic acid and its two impurities
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In this article, the multiobjective optimization of reversed-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of folic acid and its two impurities, p-aminobenzoic acid and N-(4-aminobenzoyl)-L-glutamic acid, is presented. During the preliminary study, the independent variables whose impact should be further examined in the optimization were defined (acetonitrile content in the mobile phase, molarity of sodium 1-heptanesulfonate in the aqueous phase, and pH of the aqueous phase). The face-centered central composite design was chosen for the method optimization. As the dependent variables, the separation between the impurities and the retention factor of folic acid were followed. The analysis of variance (ANOVA) test was done, and good correlation among results was confirmed. For the more accurate and reliable optimization, Derringer's desirability function was applied. On the basis of restrictions adopted for the selected targets, the resulting optimal comp...osition of the mobile phase was acetonitrile -3 mM sodium 1-heptanesulfonate (5.3:94.7 V/V), pH of the aqueous phase adjusted to 2.1 with the concentrated ortho-phosphoric acid. Furthermore, the method was subjected to method validation. It proved to be reliable and suitable for the routine analysis of investigated substances in active pharmaceutical ingredients, as well as in pharmaceutical preparations.
Keywords:p-aminobenzoic acid / N-(4-aminobenzoyl)-L-glutamic acid / central composite design / desirability-based optimization / folic acid
Source:Instrumentation Science & Technology, 2012, 40, 2-3, 138-149
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