B-190 Development of an LC-MS/MS Method for Analysis of 7α-hydroxy-4-cholesten-3-one and Investigation Into its Utility as a Biomarker for Bile Acid Diarrhoea in the United Kingdom

Abstract

Abstract Background Bile Acid Diarrhoea (BAD) is a common gastrointestinal condition estimated to affect 1% of the population. It is, however, significantly under-diagnosed. This is partly due to lack of diagnostic methods. In the United Kingdom (UK) the 75-SeHCAT retention scan is considered the gold-standard diagnostic method and is the only routinely available diagnostic test for BAD. SeHCAT retention of <15% is considered consistent with BAD. The SeHCAT test is, however, expensive, time consuming, and exposes the patient to a dose of radiation. 7-alpha-hydroxy-4-cholesten-3-one (7αC4) is a precursor in the bile acid synthesis pathway and has demonstrated utility as a marker of BAD. There is limited data comparing 7αC4, which is measured in the United States, to the SeHCAT test, as SeHCAT is not licensed in the United States. 7αC4 can be measured by LC-MS/MS, thus can provide a more convenient, low cost and high throughput option for diagnosis of BAD. Aims 1) To develop and validate an LC-MS/MS method for analysis of 7αC4. 2) To analyse serum samples taken from patients referred for SeHCAT scans. 3) To determine the utility of 7αC4 compared to SeHCAT for diagnosis of BAD. Methods The method was adapted from Donato et al. 750 µL of ice-cold acetonitrile containing 50 nmol/L D7-7αC4 as internal standard was added to 250 µL serum. Samples were vortexed for 30 s, centrifuged at 3000g for 10 min, and supernatant transferred into a vial for LC-MS/MS analysis. The LC-MS/MS system utilised a Gemini 5um NX-C18 column coupled to a Sciex 6500 TripleQuad mass spectrometer. Ethical approval was obtained to collect serum on patients undergoing SeHCAT retention scans for investigation of BAD. 40 patients who attended for SeHCAT scans had serum collected and stored at −80 degrees Celsius until analysis for 7αC4. Sensitivity and specificity of 7αC4 was calculated and ROC curve analysis performed. Results The 7αC4 method demonstrated an LoQ of 1.5 nmol/L, linearity up to 1095 nmol/L, and intra-assay %CV of 3.0%–7.7%. Using SeHCAT retention of <15% as positive for BAD, ROC curve analysis demonstrated an AUC of 0.765. 7aC4 at a concentration of 175 nmol/L provided a sensitivity of 30% and a specificity of 92%. 7αC4 at a concentration of 50 nmol/L provided a sensitivity of 93% and a specificity of 46%. Conclusion The 7αC4 method demonstrated good analytical performance and was suitable for use in a routine laboratory. 7αC4 results of <50 nmol/L suggest BAD is unlikely, and results of >175 nmol/L make a diagnosis of BAD likely. Results in between these two values may be considered indeterminate and referral for SeHCAT scanning may be considered appropriate. The 7αC4 assay provides an opportunity to stratify patients for SeHCAT testing in the UK, improving diagnosis and management of BAD in patients with chronic diarrhoea and streamlining the patient pathway.