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

•An LC–MS/MS method was firstly reported for determination of 7α-OH cholesterol.•This method with full validation was proven to be speedy, convenient and robust.•Key points for LC–MS method development and microsomal study were discussed in detail, with respect with the previously published.

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 × 4.6 mm i.d., 3 μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1 ml/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 100.0 ng/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.