Attestation of the primary standard sample of a new analgesic agent 1-phenyl(phenylimino)methyl)pyrrolidine-2,5-dione, development and validation of a method for quantitative determination of the substance by HPLC

Abstract 1-phenyl(phenylimino)methyl)pyrrolidine-2,5-dione (FFMP) is a derivative of a well-studied class of organic compounds, pyrrolidine-2,5-diones, which have a wide spectrum of biological activity. Investigation in vivo in laboratory mice showed that FFMP, being an almost non-toxic compound (according to K. K. Sidorov), exhibits a pronounced analgesic activity, which is 1,6 times higher than the reference drug (metamizole sodium), acting in pain caused by chemical stimuli - algogens (model of visceral pain). This paper presents the results of attestation of the primary standard sample FFMP by the “mass balance” method. The structure of the compound was confirmed by X-ray structural analysis (XRD), mass spectrometry, nuclear magnetic resonance (NMR) spectrometry and infrared spectrometry (IR). The content of related organic impurities was determined by high performance liquid chromatography (HPLC). The content of volatile substances and inorganic impurities has been determined. The “mass balance” method was used to calculate the content of the main substance in the FFMP substance. The calculated value has been proven to be equivalent to the quantification value obtained by Kjeldahl titration. Using a certified primary standard sample, a method for the quantitative determination of the main substance by HPLC was developed and validated. Chromatographic separation was achieved in the acetonitrile / 0,15% (v/v) aqueous formic acid solution (pH 2,5) in a gradient mode on a Tosoh ODS column (4,6 × 250 mm, 5 μm) with a flow rate of 1,0 mL/min in 35 min. The signal was recorded using a multiwave UV detector at a wavelength of 257 nm.