Pre-analytical stability of 7α-hydroxy-4-cholesten-3-one (7aC4): a systematic evaluation under clinically relevant storage conditions.

BACKGROUND: Pre-analytical variation is a major source of error in clinical laboratory testing. 7α-hydroxy-4-cholesten-3-one (7aC4) is an established biomarker of bile acid synthesis with increasing clinical use in the investigation of bile acid malabsorption. However, data describing its stability under clinically relevant collection, transport, and storage conditions are limited. METHODS: Venous blood from six healthy donors was analysed using a validated LC-MS/MS method. Stability of 7aC4 was assessed under a range of pre-analytical conditions, including delayed separation (up to 24h), multiple storage temperatures (4°C, 20-25°C, 37 °C, and -20°C). Samples were analysed over a multi-day time course (days 0-14). Predefined maximum permissible difference thresholds were based on assay imprecision. The study design and reporting adhered to CRESS guidelines. RESULTS: 7aC4 remained stable in clotted whole blood for up to 24h at 20-25°C, with mean percentage differences of 0.14 ± 2.33% and 4.15 ± 5.19% at 8h and 24h, respectively. Removal of serum from separator gel improved stability at 20-25°C room temperature by day 5 (-16.4 ± 3.4% vs -38.9 ± 1.6%; p = 0.028). At 37°C, substantial degradation occurred irrespective of gel status. Separated serum remained stable for up to 8 days at 4°C (-9.5 ± 2.83%) and 14 days at -20°C (2.2 ± 2.7%). CONCLUSIONS: 7aC4 demonstrates robust stability under a range of relevant conditions, including delayed separation and ambient transport. Temperature is the principal driver of analyte degradation. These findings support practical sample handling recommendations in routine practice and provide evidence-based guidance for pre-analytical management, facilitating reliable implementation of 7aC4 testing.