Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1'-hydroxylation kinetics in human liver microsomes.
The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1'-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10-5000 ng/mL was obtained in rat plasma with analysis times of 1 min per sample. Kinetic constants for midazolam 1'-hydroxylation in human liver microsomes yielded an apparent Km of 10.0 microM and Vmax of 6.4 nmol/min/mg. Studies investigating the inhibition of 1'-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03 microM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations.
Steenwyk
Rick C
RC
Pfizer Inc., Pharmacokinetics, Dynamics and Metabolism, Kalamazoo, MI, USA. rick.c.steenwyk@pfizer.com
Hutzler
J Matthew
JM
Sams
Jim
J
Shen
Zhouxin
Z
Siuzdak
Gary
G
D000818:Animals; D001274:Atmospheric Pressure; D002478:Cells, Cultured; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000378:metabolism*; D008874:Midazolam / Q000032:analysis* / Q000097:blood / Q000378:metabolism*; D051381:Rats; D012825:Silicon / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D008874:Midazolam; D012825:Silicon