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Motolimod (VTX-2337) is an agonist of toll-like receptor 8. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantifying motolimod in rat plasma and subsequently used in a pharmacokinetic study. Proteins were precipitated from plasma samples using acetonitrile prior to the analysis. GS-9620 was used as an internal standard. High-performance liquid chromatography was performed using a Spursil C18-EP column (3 μm, 50 × 2.1 mm). Aqueous ammonium formate and acetonitrile were used as the mobile phase. Motolimod was detected using an electrospray ionization interface under multiple reaction monitoring conditions in the positive ion mode. The developed method produced a linear correlation over a concentration range of 1-1000 ng/mL (r = 0.9944). Intra- and inter-day precision values ranged from 4.8%-10.7% (the lower limit of quantification precision value was 16.3 %), whereas intra- and inter-day accuracy values ranged from 0.3%-9.1 %. The mean recovery of motolimod from rat plasma was 109.4 %. Additionally, motolimod was found to be stable under various conditions (three freeze-thaw cycles, 6-h storage at room temperature, short- and long-term stability tests, and processing). The developed method was successfully used in a pharmacokinetic study in rats. Motolimod showed non-linear pharmacokinetics following its intravenous administration to rats at 0.6-6 mg/kg. Additionally, very low exposure (<1 %) was obtained following oral administration of the drug to rats. The results also showed that motolimod has a low metabolic stability in the liver microsomes and exhibits extensive binding to the plasma proteins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C 18 column (2.1 × 50 mm, 3.5 μm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 μg/mL with a lower limit of quantification of 5 μg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice., (Copyright © 2018 Elsevier B.V. All rights reserved.)