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Determination of 7α-OH cholesterol by LC–MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs

Author:
Changhong Yun, Taijun Yin, Katherine Shatzer, Douglas G. Burrin, Liwei Cui, Yifan Tu, Ming Hu
Source:
Journal of chromatography 2016 v.1025 pp. 76-82
ISSN:
1570-0232
Subject:
acetonitrile, cholesterol, chromatography, formic acid, liver, liver microsomes, miniature swine
Abstract:
An LC–MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were 385.1≥159.1 and 376.4≥266.3, respectively. The linear response range of 7α-OH cholesterol was covered from 1.563 to 100.0ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes.
Agid:
62878
Handle:
10113/62878