Determination of plasma and brain levels of isotretinoin in mice following single oral dose by high-performance liquid chromatography.
An isocratic reversed-phase high-performance liquid chromatographic method was established and validated according to FDA's Guidance for Industry, "Bioanalytical Method Validation", for the determination of isotretinoin in plasma and brain tissue from mice following single and multiple oral doses of Accutane. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized and extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by high-performance liquid chromatography (HPLC). Benz[alpha]anthrancene-7,12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and was 30 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30-600 ng/mL, and 15-300 ng/0.1g for brain. Maximum concentrations of isotretinoin in both plasma and brain were observed at 1h after single oral dosing (25 mg/kg). The maximum concentrations in plasma and brain were 2.36 microg/mL and 0.34 microg/g, respectively. The mean area under curve (AUC) in plasma was 6.13 microg h/mL. The mean eliminate half-life in plasma was estimated as 46 min.
Yang
Yongsheng
Y
Food and Drug Administration, Center for Drug Evaluation and Research/Division of Product Quality Research, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Faustino
Patrick J
PJ
Pine
P Scott
PS
Davis
Hirsch
H
Grunberg
Neil
N
Phillips
Jennifer
J
Lyon
Robbe C
RC
Yu
Lawrence X
LX
Ciavarella
Anthony B
AB
Del Grosso
Alfred V
AV
Hanig
Joseph P
JP
D000284:Administration, Oral; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D001923:Brain Chemistry / Q000187:drug effects / Q000502:physiology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015474:Isotretinoin / Q000008:administration & dosage* / Q000097:blood* / Q000737:chemistry; D008297:Male; D051379:Mice
D015474:Isotretinoin