Development, validation and testing of the HPLC-UV bioanalytical method for quantification of etmaben in blood plasma and organs of rats

Introduction. Currently, the search and development of innovative cardiotropic drugs is an urgent task for pharmaceutical science. One such drug is a derivative of malonic acid, etmaben, which was synthesized at the St. Petersburg State Chemical and Pharmaceutical University (SPCPU). To conduct preclinical and clinical studies, it is necessary to develop and validate a bioanalytical method that allows for the quantitative determination of etmaben in complex biological matrices with minimal time and resource requirements.

Aim. The aim of the study is to develop and validate a method for the determination of etmaben in the blood plasma and organs of laboratory animals using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) followed by testing and obtaining a pool of data on the distribution of etmaben in the organs and tissues of experimental animals.

Materials and methods. The development and validation of the bioanalytical method, as well as the quantitative determination of etmaben, were performed on a Flexar liquid chromatograph (PerkinElmer, USA) equipped with a UV detector and a thermostatted autosampler, using a Kromasil 100, 150 × 2.1, C8, 3.5 μm column (AkzoNobel, Netherlands). Detection was performed at a wavelength of 270 nm. A pharmacological experiment to test the method was conducted on 20 outbred male rats (Kurchatov Institute – "Rappolovo", Russia), divided into a control group (intact animals, n = 10) and a group with experimental chronic heart failure (CHF) (n = 10), a model of which was induced by ligation of the left coronary artery. All animals received etmaben intragastrically at a dose of 60 mg/kg for 30 days. Whole blood samples were collected for analysis on day 30, and organs (heart and kidneys) were isolated. Etmaben was quantified in plasma and tissue homogenates using HPLC-UV after sample preparation, including protein precipitation with acetonitrile (HPLC Grade, cat. no. A/0627/17, Thermo Fisher Scientific, USA).

Results and discussion. Optimal conditions for the chromatographic determination of etmaben in biological matrices (blood plasma and tissue homogenates) using HPLC-UV were developed. The developed method was validated using the following validation parameters: selectivity, calibration curve, accuracy, precision, lower limit of quantification (LLOQ), carryover effect, no effect of sample dilution and stability. The degree of etmaben recovery from the matrix was also assessed. The LLOQ for etmaben was 5.0 ng/mL. The developed method was successfully applied to assess etmaben distribution in the organs and tissues of laboratory animals at specific stages of preclinical testing. In CHF, plasma concentrations of etmaben doubled, and in pathologically altered myocardium, they increased by 47 % compared to control animals.

Conclusion. A bioanalytical method for determining etmaben using HPLC-UV was developed and validated. It was tested during preclinical trials to study the drug's distribution in organs and tissues of experimental animals. The observed selective accumulation of etmaben in damaged myocardium confirms its therapeutic relevance for the treatment of CHF.

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