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Determination of (4-(1,3-dioxo-1,3-dihydro-2H-isoindol- 2-yl)-N'-[(4-ethoxyphenyl) methylidene] benzohydrazide, a novel anti-inflammatory agent, in biological fluids by UPLC-MS/MS: Assay development, validation and in vitro metabolic stability study.
Biomedical Chromatography : BMC 2017 October
A thalidomide analog, (4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N'-[(4-ethoxyphenyl) methylidene] benzohydrazide), has been identified as a promising broad-spectrum anti-inflammatory agent in previous study. In this study, a sensitive and selective UPLC-MS/MS assay was developed and validated for its determination in rat plasma samples. The chromatographic separation was performed on an Aquity BEH C18 column using mobile phase comprising of acetonitrile and 10 mm ammonium acetate in the ratio of 85: 15, at flow rate of 0.3 mL/min. The detection and quantification were performed in positive multiple reaction monitoring mode by parent to daughter ion transition of 414.06 ˃ 148.05 for analyte and 411.18 ˃ 191.07 for internal standard (risperidone), respectively using electrospray ionization source. The sample extraction process consisted of liquid-liquid extraction method using diethyl ether as the extracting solvent. The assay was validated by following FDA guidelines and all parameters were found to be within acceptable limits. The linearity was between 10.1 and 2500 ng/mL and the lower limit of quantification was 10.1 ng/mL. The reported results indicate that the assay could meet the requirement for analysis of this compound in amounts expected to the present in actual samples. Further, in vitro metabolic stability study was performed in rat liver microsomes by using the validated assay.
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