Toxicokinetic evaluation of atrasentan in mice utilizing serial microsampling: validation and sample analysis in GLP...

Area of Science:

• Pharmacokinetics and Drug Metabolism
• Analytical Chemistry
• Biomedical Science

Background:

• Development of a semi-automated 96-well plate method for atrasentan determination in mouse whole blood.
• Utilization of whole blood matrix to overcome limitations of small sample volumes and avoid plasma preparation.
• Application of a stable-labeled internal standard (d18-atrasentan) for accurate quantification.

Purpose of the Study:

• To develop and validate a sensitive HPLC-MS/MS method for atrasentan quantification in mouse whole blood.
• To enable robust toxicokinetic evaluations under Good Laboratory Practice (GLP) conditions.
• To obtain reliable drug exposure data from serially sampled mice.

Main Methods:

• Semi-automated protein precipitation from 10-µl whole blood aliquots.
• HPLC separation on a Zorbax SB-C18 column with a gradient mobile phase.
• MS/MS detection using positive ion mode and multiple-reaction monitoring (MRM).

Main Results:

• Validated method achieved a dynamic range of 5-1400 ng/ml.
• Successfully addressed challenges including anticoagulant interference and phospholipid management.
• Demonstrated excellent assay reproducibility with 100% of incurred samples meeting reanalysis criteria.

Conclusions:

• The validated HPLC-MS/MS method provides high-quality exposure data for atrasentan in mice.
• The method is suitable for GLP toxicokinetic studies requiring precise quantification.
• Enables reliable assessment of drug pharmacokinetics in preclinical research.