The Combined Quantification of the 15 Human Bile Acids and Their Precursor 7α‐Hydroxy‐4‐Cholesten‐3‐One in Human Serum: LC‐MS/MS Method Development and Validation

Bile acids (BAs) have been considered potential biomarkers for various metabolic diseases, which prompted the need for accurate and sensitive quantification methods. This study aimed to develop a sensitive, validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying primary, secondary, free, and conjugated BAs, along with their precursor, 7α-hydroxy-4-cholesten-3-one (C4), in human serum. The analytical approach involved a rapid protein precipitation-based extraction technique in the presence of deuterated internal standards. Mass spectrometry detection employed electrospray ionization in negative and positive ion modes for BAs and C4, respectively. BA levels obtained by LC-MS/MS were validated by comparing them to total BAs measured by a sensitive enzymatic assay. The chromatographic cycle time for the method was 21 min per injection, employing multiple reaction monitoring. All BAs were separated with negligible carryover, matrix effect, and good recovery. Good linearity was observed in the concentration range of (0.0-10.0) μM for BAs and (0.0-0.4) μM for C4. The detection limit for BAs was between 0.0002 and 0.0138 μmol/L, and the sensitivity was 0.00156-0.0125 μmol/L. Acceptable precision results were obtained on intraday and interday analyses for BAs and C4. The LC-MS/MS quantification of total BAs closely matched levels from the colorimetric assay. This study presents a straightforward LC-MS/MS method for simultaneously quantifying BAs and their precursor, C4, in human serum. The low sample volume, rapid extraction, validated results, and detailed protocol make the established method reproducible and suitable for use.