A simple surrogate approach for the quantitation of C4 (7α-hydroxy-4-cholesten-3-one) in human serum via LC-MS/MS and
Feng Yin1, Mani Deepika Vakkalanka2, Walter Wiley2
1Department of Biomarker Science and Technologies, Takeda Development Center Americas, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA.
Abstract:
We present a validated LC-MS/MS assay for the quantitation of 7α-hydroxy-4-cholesten-3-one (C4), a key intermediate in the bile acid synthesis pathway from cholesterol, in human serum. A surrogate matrix approach was employed to overcome the challenges posed by the endogenous C4 levels in the biological matrix. Human serum samples were spiked with stable isotope labeled internal standard (SIL-IS), processed using supported liquid extraction (SLE), and analyzed by LC-MS/MS. Parallelism was successfully demonstrated between human serum (authentic matrix) and 5 % bovine serum albumin in phosphate buffered saline containing 0.1 % tween 20 (5 % BSA in PBST) (surrogate matrix). The assay's linear analytical range was established from 0.200 to 200 ng/mL. This validated LC-MS/MS method exhibited excellent accuracy and precision. The overall accuracy was between 97.9 % and 101 % with %CV less than 4.0 % for C4 in human serum. C4 was found to be stable in human serum for up to 24.7 h at room temperature, up to 34 days when stored at -25 °C or - 80 °C, and after five freeze/thaw cycles. The assay has been successfully applied to human serum samples to support a clinical study.
