Development and validation of a method for the quantitative determination of rotenone in the homogenate of the cerebral cortex of rats by high-performance liquid chromatography (HPLC)

Introduction. Rotenone is a neurotoxin that causes damage of dopaminergic neurons in the substantia nigra and is used as a model for experimental parkinsonian syndrome. The development of a technique for the quantitative determination of rotenone in the brain will allow testing new strategies for the pharmacotherapy of parkinsonism by reducing the penetration of toxic substances into the brain.

The aim of the study was to develop and validate an HPLC method for the quantitative determination of rotenone in the cerebral cortex of rats.

Material and methods. Quantitative determination of rotenone was carried out using a Stayer chromatographic system (Аквилон, Russia) with a UV spectrophotometric detector UVV 104 at a wavelength of 296 nm in isocratic mode. A reverse-phase chromatographic column Luna C18 100Å (250*4.6) with a grain size of 5 μm at a temperature of 37°C was used. The composition of the mobile phase was deionized water, acetonitrile in a ratio of 70:30. Determination of rotenone concentration was carried out by the method of absolute calibration by the area of the peaks.

Sample preparation consisted in homogenization of 500 mg of crushed frontal lobe of the rat cerebral cortex in 500 μl of purified water, followed by centrifugation (1750 g), collection of the supernatant and sedimentation of the proteins by acetonitrile. The liquid layer was evaporated on a rotary vacuum. 250 µl of the mobile phase was added to the dry residue, and 100 µl was injected into the chromatograph.

Results. The method was validated for the following parameters: selectivity, linearity, accuracy, precision, limits of detection and determination, sample transfer, sample stability. The analytical range was 62.5−1000.0 ng/g brain with a correlation coefficient of more than 0.99. The limits of detection and quantification of rotenone were 25.0 and 62.5 ng/g, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for higher concentrations. The stability of the technique was demonstrated during short-term storage at room temperature, three freeze-thaw cycles at –80°C, and storage at –80°C for 60 days. There was no sample transfer.

Limitations. The chromatographic technique makes it possible to analyze the content of rotenone in the cerebral cortex of rats in the concentration range of 62.5–1000.0 ng/g.

Conclusion. A method for the quantitative determination of rotenone in the homogenate of the cerebral cortex of rats has been developed and validated.

Compliance with ethical standards. The experimental study was approved by the commission for control over the maintenance and use of laboratory animals of the Ryazan State Medical University.

Author contribution:
Gradinar M.M. — performance of chromatographic studies, sample preparation;
Shchulkin A.V. — statistical processing of the obtained results, writing an article;
Chernykh I.V. — performance of chromatographic studies, calculation of validation parameters;
Yakusheva E.N. — writing an article, general management of the work.
All co-authors — approval of the final version of the article, responsibility for the integrity of all parts of the article.

Conflict of interests. The authors declare no conflict of interest.

Funding. The study was not sponsored.

Received: January 1, 2023 / Accepted: February 2,2023 / Published: April 30, 2023