SHA Pian, PAN Dan-dan, GU Zhu-lin, QIN Ya-wei
Objective: To develop and validate a highly sensitive analytical method that can quantitatively measure the concentration of AB-38b in normal rat brain tissue and reveal the pharmacokinetics of AB-38b in rat brain tissue. Methods: :Tolvaptan was selected as the internal standard (IS). Brain tissue samples were pretreated with methanol for protein precipitation. Liquid chromatography was performed with a Shim-pack VP-ODS C18 column (2.0 mm×150 mm,5 μm), mobile phase A of ammonium acetate, mobile phase B of methanol, a flow rate of 0.2 mL · min-1, a column temperature of 40 ℃, an auto-sampler temperature of 4 ℃, and an injection volume of 5 µL. The ionization mode was electrospray ionization (ESI), and multiple reaction monitoring (MRM) positive ion mode was used to detect AB-38b (m/z 705.2→618.3) and tolvaptan (m/z 449.3→252.2). Comprehensive methodological validation was conducted for this method, including establishment of the standard curve and the lower limit of quantification, residual effect investigation, accuracy and precision verification, matrix effect exploration, and determination of extraction recovery rates. Thirty male SD rats (240±14 g) were randomly assigned into 5 groups, with 6 rats in each group. After 12 h of fasting but free access to water, they were administrated with AB-38b at a single oral dose of 50 mg · kg-1. At the time points of 0, 15, 50, 90, and 480 min post-administration, the rats were weighed and sacrificed via the carbon dioxide method. The brain tissue was collected and weighed, and tissue homogenates were prepared with phosphate buffer. After processing according to the tissue sample treatment method, the concentrations of AB-38b in rat brain tissue samples were determined by LC-MS/MS. Results: The linear range of AB-38b was 2.5-150 ng · mL-1 (R2>0.999), and the lower limit of quantitation was 2.5 ng · mL-1. The intra-day precision and inter-day accuracy were 88.54%-104.56% and 95.24%-102.31%, respectively. The precision (RSD) was less than 9%. The extraction recovery of AB-38b and IS in rat brain homogenate samples was 81.9%-95.9%, RSD<15%. The matrix effect range of AB-38 b was 87.74%-96.21%, RSD<15%. The residual peak area at the retention time of AB-38b was less than 20% of the peak area of LLOQ, and the residual peak area at the retention time of internal standard was less than 5% of the peak area. AB-38b was stable in brain tissue homogenate at room temperature for 6 h, three freeze-thaw cycles, 24 h in the sampler, and long-term (-80 ℃ ) storage for 60 days, with RSD<12.7%. The results suggested that accuracy and precision, extraction recovery, matrix effect, residual effect, and stability all met the acceptance criteria. Conclusion: The LC-MS/MS method established in this study is rapid, sensitive, and specific, and can accurately determine the content of AB-38b in rat brain tissue.
