| Summary: | Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid–liquid extraction with only one step—protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r<sup>2</sup> ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased.
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