Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography-mass spectrometry.
A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography-mass spectrometry (LC-MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15-1788) was linear in the concentration range of 1-150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to <or=0.23%. Extractions were performed in a concentration interval of 30-3000 nM without any sign of column overload. The method was successfully used for determining the microsomal metabolic stability of flumazenil. As a result, the described analysis system is currently used for metabolic screening of PET tracer candidates in our laboratory.
Lavén
M
M
Department of Analytical Chemistry, Institute of Chemistry, Uppsala University, Box 599, 751 24 Uppsala, Sweden.
Markides
K
K
Långström
B
B
D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D005442:Flumazenil / Q000032:analysis; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005442:Flumazenil