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.