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
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.