Experience in development of bioanalytical methods by HPLC with UV detection

Introduction. The HPLC method with UV-detection is one of most often used modern method for the quantitative analysis of drugs in biosamples obtained during pharmacokinetics, bioequivalence or drug monitoring studies. This method may be characterized by flexibility, allowing to create many assays with different variants of chromatographic separation conditions, detection, sample handling procedures, etc., oriented to specific target analytes, taking into account their properties and peculiarities. However, as experience is gained and data are compiled, patterns are formed, and the most common, convenient variations, techniques, or combinations may be identified. Material and methods. An overview of experimental experience in the development of bioanalytical methods using HPLC-UV method for quantitative analysis of natural and synthetic drugs in biosamples obtained after pharmacokinetics and bioequivalence studies is presented. Results and discussion. Analysis was performed in reverse phase HPLC on analytical C18 type columns in almost cases. Isocratic elution regimes were selected in most cases (68%), retention times of the target analytes were the range of 4–8 minutes. In 63% of cases mixtures of aqueous solution of trifluoroacetic acid and acetonitrile in different ratios were selected as eluent. Methods developed for pharmacokinetic studies with laboratory animals (rats, rabbits) and for bioequivalence studies done with the participation of healthy volunteers are combined in the presented materials. Three regions of the UV spectrum were selected as most commonly used for detection – 205–220 nm (25%), 220–254 nm (31%), 270–295 nm (35%), and 9% of other wavelengths. These results characterizes general features of absorption spectra of organic compounds. Extraction (in 36% of cases) and precipitation (in 64% of cases) were used for sample handling prior to dosing in HPLC. Precipitation of plasma proteins with acetonitrile (64%) proved to be most common; methanol (15.5%) and acidic or other precipitating agents (20.5%) were optimal in about the same number of cases. The lower quantification limit founded from validation tests for most techniques was in the range of about 0.01–0.1 μg/ml, consistent with literature (0.05–0.1 μg/ml).