Analysis of Bile Acids by HPLC with ELSD using the Agilent InfinityLab Poroshell 120 EC-C18 LC Column

Importance of Topic

Bile acids are essential endogenous molecules involved in lipid digestion, cholesterol regulation, and cellular signaling. Due to their diagnostic value in liver and gastrointestinal disorders, rapid and sensitive analysis of multiple bile acids in biological matrices is critical in clinical research, pharmaceutical development, and quality control.

Study Objectives and Overview

This application note evaluates the separation of 14 bile acids plus cholesterol using HPLC with evaporative light scattering detection (ELSD) on Agilent InfinityLab Poroshell 120 EC-C18 columns. Two column formats—a 4.6 × 250 mm, 4 µm column and a 4.6 × 100 mm, 2.7 µm column—were compared to optimize analysis time, resolution, and robustness.

Materials and Methods

Standards of bile acids and cholesterol were prepared in methanol at 1 mg/mL and mixed to ~70 µg/mL. Mobile phase A was 20 mM ammonium acetate in water; mobile phase B was acetonitrile:methanol (60:40). Gradient programs differed for each column:

• 4.6 × 250 mm, 4 µm column: 0–5 min at 40 % B, 5–20 min to 60 % B, 20–25 min to 100 % B; 37 min total run time; 10 µL injection.
• 4.6 × 100 mm, 2.7 µm column: 0–2 min at 40 % B, 2–8 min to 60 % B, 8–10 min to 100 % B; 15 min total run time; 4 µL injection.

Flow rate was 1.5 mL/min and column temperature 60 °C. ELSD settings: evaporator 80 °C, nebulizer 30 °C, gas flow 1.6 SLM. Instrumentation comprised an Agilent 1290 Infinity II system with ELSD and OpenLab CDS.

Main Results and Discussion

The 4 µm superficially porous column achieved baseline separation of all 15 analytes in 32 min. Transferring the method to the 2.7 µm column halved analysis time to 13 min while maintaining resolution and reducing backpressure. Superficially porous particles delivered UHPLC-like efficiency without the high-pressure burden of sub-2 µm phases. Column temperature studies (30–60 °C) revealed that higher temperatures improved critical peak resolution and altered elution order, with 60 °C offering optimal performance.

Benefits and Practical Applications

The optimized HPLC-ELSD method provides rapid, robust profiling of conjugated and unconjugated bile acids in biological samples (bile, serum, urine). Poroshell columns enhance sample throughput, reduce solvent consumption, and resist fouling, making them well-suited for clinical and industrial laboratories engaged in metabolite analysis and QA/QC.

Future Trends and Potential Applications

Emerging directions include coupling with mass spectrometry for structural elucidation, automated high-throughput sample preparation workflows, and expansion into metabolomics and novel bile acid derivatives. Advances in column technology and detector sensitivity will further accelerate analysis times, broaden dynamic ranges, and enable real-time biomarker screening.

Conclusion

Agilent InfinityLab Poroshell 120 EC-C18 columns paired with ELSD afford efficient, high-resolution separation of bile acids and cholesterol. The shorter, 2.7 µm column format enables rapid analysis with low backpressure and robust performance, supporting diverse clinical and research applications.

Instrumental Setup

Agilent 1290 Infinity II high-speed pump, multisampler, multicolumn thermostat, ELSD, and OpenLab CDS C.01.07.

Reference

Fu R., Lei J. Analysis of Bile Acids by HPLC with ELSD using the Agilent InfinityLab Poroshell 120 EC-C18 LC Column. Agilent Technologies Application Note 5994-0807EN, 2019.