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


RESULTS
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.


CONCLUSIONS
Good quality exposure data were obtained from every serial sampled mouse in the study.
Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA. xia.wan@abbott.com
Katty X
KX
Wan
Michael T
MT
Reimer
Maria P
MP
Metchkarova
Amy J
AJ
Richter
Auratip
A
Paramadilok
Olga
O
Kavetskaia
Tawakol
T
El-Shourbagy
D000818:Animals; D000077868:Atrasentan; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D008297:Male; D051379:Mice; D010599:Pharmacokinetics; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification; D011759:Pyrrolidines / Q000097:blood* / Q000592:standards; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards
D010743:Phospholipids; D011759:Pyrrolidines; D000077868:Atrasentan