A sensitive HPLC-MS-MS assay for quantitative determination of midazolam in dog plasma.
The clinical pharmacokinetics of midazolam have been extensively studied, due to its high clearance by CYP3A4 and sensitivity to drug-drug interactions. In order to investigate the potential to model drug-drug interactions with midazolam in the dog, a selective and sensitive high performance liquid chromatography-tandem mass spectroscopy (HPLC-MS-MS) method has been developed, with sufficient sensitivity to allow analysis of dog plasma samples generated following administration of a clinically relevant dose. The method involves extraction of midazolam and internal standard (flunitrazepam) from dog plasma, using 96-well Oasis MCX solid phase extraction plates. The assay has been validated over a concentration range of 0.1-10 ng/ml and its specificity, accuracy and precision demonstrated. The relative bias of the assay was within +/-15% for all standards with intra- and inter-assay precision (coefficient of variation-%CV) of less than 15%. The assay was applied to the analysis of plasma samples (0.2 ml), generated following intravenous or oral administration of midazolam to male beagle dogs, at a dose level of 0.05 mg/kg, and pharmacokinetic parameters were derived from the resulting data.
Harris
S R
SR
Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK. stephan_harris@sandwich.pfizer.com
Gedge
J I
JI
Nedderman
A N R
AN
Roffey
S J
SJ
Savage
M
M
D000284:Administration, Oral; D000818:Animals; D014151:Anti-Anxiety Agents / Q000097:blood*; D019540:Area Under Curve; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D005445:Flunitrazepam / Q000097:blood; D007275:Injections, Intravenous; D008297:Male; D013058:Mass Spectrometry; D008874:Midazolam / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D014151:Anti-Anxiety Agents; D005445:Flunitrazepam; D008874:Midazolam