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Bioanalysis. 2012 Jun;4(11):1351-61. doi: 10.4155/bio.12.91.

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

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  • 1Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. xia.wan@abbott.com



A semi-automated 96-well protein precipitation followed by HPLC-MS/MS method for the determination of atrasentan (2R-[4-methoxyphenyl]-4S-[1,3-benzodioxol-5-yl]-1-[N,N-di-(N-butyl)-aminocarbonyl-methyl]-pyrrolidine-3R-carboxylic acid) in mouse whole blood was developed, validated and utilized in GLP toxicokinetic evaluations. Six 40-µl whole blood samples were collected from a single mouse over the course of a 12 h blood collection window. To avoid sample volume losses, whole blood was selected as the matrix in place of the more typically used plasma. A 10-µl assay volume was used to ensure sufficient volumes are available for dilutions, repeats and incurred sample reanalysis. The samples (10-µl aliquot) were fortified with stable-labeled internal standard (d18-atrasentan) and lysed thoroughly prior to protein precipitation. The chromatographic separation was performed on a Zorbax(®) SB-C18 (50 x 2.1 mm; 5 µm) HPLC column with a mobile phase consisting of 25 mM ammonium acetate and 0.25% (v/v) acetic acid in 50/50 (v/v) acetonitrile/water. The MS measurement was conducted under positive ion mode using multiple-reaction monitoring of m/z 511→354 for analyte and 529→354 for stable-labeled internal standard. The peak area ratio (analyte:stable-labeled internal standard) was used to quantitate atrasentan.


A dynamic range of 5-1400 ng/ml was established after validation. The challenges associated with a small-volume whole-blood assay involved anticoagulant overloading with commercial blood collection tubes, managing phospholipids to ensure a robust assay and automation. In-depth discussions are provided in this article. The validated method was then used for GLP toxicokinetic evaluations. To demonstrate the method reproducibility, approximately 10% of the incurred samples from the study were repeated in singlet. Excellent assay reproducibility was demonstrated where 100% of samples met incurred sample reanalysis acceptance criteria.


Good quality exposure data were obtained from every serial sampled mouse in the study.

[PubMed - indexed for MEDLINE]
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