The all-D-enantiomeric-peptide RD2 was developed for the treatment of Alzheimer’s disease. This study aimed to develop a specific and highly sensitive liquid chromatographymass-spectrometric (UHPLC-ESI-QTOF) method for quantifying RD2 in the mouse brainand to validate it according to the ICH M10 guideline to investigate the pharmacokinetic profile of RD2 in its target organ. Sample preparation, chromatographic separation and quantification were very challenging due to RD2’s highly hydrophilic properties, the complex matrix and the required lower limit of quantification (LLOQ). Chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size) within 5 min at 50 ◦C with a flow rate of 0.5 mL·min−1. Mobile phasesconsisted of water and acetonitrile with 0.2% formic acid and 0.015% heptafluorobutyric acid. Ions were generated by electrospray ionization in the positive mode, and RD2 was quantified by QTOF-MS. The developed extraction method revealed complete recovery.The linearity of the calibration curve was in the range of 2 ng·mL−1 to 500 ng·mL−1 (R2 > 0.99) with a LLOQ of 5 ng·mL−1. The intraday and interday accuracy and precision ranged from 0.4% to 12.2% and from 1.0% to 12.0%. RD2 remained stable in thefreshly homogenized brain even after several freeze–thaw cycles, but stability decreased over time during long-term storage at −80 ◦C. Using this validated method, RD2-spiked brain homogenate samples and samples of a pharmacokinetic study with RD2 in micewere analyzed.
Jonas et al. (Thu,) studied this question.