Development and Validation of UHPLC-MS/MS Method for Quantifying of Agarotriose: An Application for Pharmacokinetic, Tissue Distribution, and Excretion Studies in Rats

A sensitive, rapid, and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for the first time to quantify agarotriose (A3) in rat plasma, tissues, urine, and feces. A3 and stachyose (internal standard) were separated by a BEH amide column at 65 °C under the mobile phase of 10 mmol L−1 ammonium acetate-acetonitrile (42:58, v/v) with 350 µL min−1. The acquisition of transitions was carried out in multiple reaction monitoring (MRM) pattern operating with positive ionization at m/z 509.16 ≥ 329.15 for A3 and m/z 689.15 ≥ 527.11 for stachyose. The linearity ranges of A3 were 10 to 5000 nmolL−1 for plasma, 20 to 10000 nmolL−1 for tissues, and 40 to 20000 nmolL−1 for urine and feces. The accuracy and precision ranged from 90.9% to 111.6% and 0.7% to 10.1%, respectively. The stability was between 86.1% and 102.5%. The extraction recovery was consistent and reproducible. The matrix effect ranged from 1.5% to 11.4%. The pharmacokinetic, tissue distribution, and excretion studies were successfully conducted with the validated method. Results showed that A3 could be absorbed by rats, and the absolute bioavailability was 6.7%. Furthermore, it was rapidly distributed in rat tissues and mainly eliminated via feces excretion (67.0%) after oral administration. For intravenous bolus, 85.5% was recovered, and renal excretion was the primary pathway (77.6%) for cumulative recovery.