Box-Behnken Design is a useful tool for the optimization of the chromatographic analysis. The goal of this study was to select the most significant factors that influenced the following parameters of the chromatographic separation: retention time, relative retention time, symmetry of the peaks, tailing factor, a number of theoretical plates, Foley – Dorsey parameter, resolution factor, peak width at half height. The results underwent the ANOVA test to find the statistically significant variables and interactions between them. The level of significance was for p < 0.05. The polynomial equations described quantitatively the statistically significant parameters and the interactions between them. The statistical analysis indicated both the best conditions for the separation of the compounds and the variables that were most influential for peaks’ parameters. The four-factor analysis performed for LEVO and MOXI indicated that ACN, TEA and pH are the most significant factors that influence the separation. The analysis for the pair CIPRO and LEVO required six factors. The statistical analysis proved that the most significant factors are ACN, MeOH and TEA. In the separation of these two compounds on the HPLC column, the interaction ACN × MeOH was also significant.
Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.
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