High-throughput pharmacokinetics screen of VLA-4 antagonists by LC/MS/MS coupled with automated solid-phase extraction sample preparation.
Automation of plasma sample preparation for pharmacokinetic studies on VLA-4 antagonists has been achieved by using 96-well format solid-phase extraction operated by Beckman Coulter Biomek 2000 liquid handling system. A Biomek 2000 robot is used to perform fully automated plasma sample preparation tasks that include serial dilution of standard solutions, pipetting plasma samples, addition of standard and internal standard solutions, performing solid-phase extraction (SPE) on Waters OASIS 96-well plates. This automated sample preparation process takes less than 2 h for a typical pharmacokinetic study, including 51 samples, 24 standards, 9 quality controls, and 3-6 dose checks with minimal manual intervention. Extensive validation has been made to ensure the accuracy and reliability of this method. A two-stage vacuum pressure controller has been incorporated in the program to improve SPE efficiency. This automated SPE sample preparation approach combined with liquid chromatography coupled with the high sensitivity and selectivity of tandem mass spectrometry (LC/MS)/MS has been successfully applied on both individual and cassette dosing for pharmacokinetic screening of a large number of VLA-4 antagonists with a limit of quantitation in the range of 1-5 ng/ml. Consequently, a significant throughput increase has been achieved along with an elimination of tedious labor and its consequential tendency to produce errors.
Tong
Xinchun S
XS
Basic Chemistry, Merck Research Laboratory, Merck & Co. Inc., P.O. Box 2000, RY800-B205, Rahway, NJ 07065, USA. sharon_tong@merck.com
Wang
Junying
J
Zheng
Song
S
Pivnichny
James V
JV
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D039041:Integrin alpha4beta1 / Q000037:antagonists & inhibitors* / Q000097:blood*; D008297:Male; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley
D039041:Integrin alpha4beta1