High throughput bioanalysis of serum 7a-hydroxy-4-cholesten-3-one (C4) using LC-MS/MS: Devising an end-to-end single vial solution for a sample limited application.

Crohn's disease (CD) is characterized by chronic ileal/ileocolonic inflammation, and in some cases, can result in bile acid malabsorption (BAM) and subsequent bile acid diarrhea (BAD). Although BAD is common in CD, diagnosis is difficult. In patients with CD who had ileal resection (IR), elevated serum 7a-hydroxy-4-cholesten-3-one (C4), a cholesterol-derived stable intermediate in bile acid synthesis, is associated with diarrhea attributable to BAM and therefore, may have diagnostic utility. The previously existing validated methodology to measure C4 in human serum required 100 μL with an analytical range of 0.5-100 ng/mL, making it incompatible with the clinical trial sample set we had available for analysis due to limited serum volume and an extended assay range requirement (up-to 1 μg/mL). We present here a simplified, end-to-end single vial approach performed in a 96-well format for clinical sample C4 analysis for application in sample limited settings.