Application Brief
This is an Application Brief and does not contain a detailed Experimental section.
HPLC Separation of Bile Acids Using a CORTECS™ Premier Phenyl Column
HPLC Separation of Bile Acids Using a CORTECS™ Premier Phenyl Column
Kenneth D Berthelette, Maureen DeLoffi, Keil Brinster, Heather Throckmorton, Tom Walter
Waters Corporation, United States
Published on September 01, 2025
Abstract
The determination of bile acids is important in understanding the metabolism and absorption of fats. Additionally, some bile acids have been shown to be associated with colon cancer risk. Six of these compounds were analyzed by LC-MS using a MaxPeak™ Premier CORTECS Phenyl Column. Good separation was achieved for two sets of isobaric compounds present in the panel.
Benefits
- Baseline resolution of isobaric bile acids: taurodeoxycholic acid/taurochenodeoxycholic acid and glycodeoxycholic acid/glycochenodeoxycholic acid
Introduction
Bile acids are endogenous compounds which aid in the digestion, transport, and absorption of fats in the body. The concentrations of these compounds in blood plasma have been shown to be associated with an increased risk of developing colon cancer.1 Consequently, the determination of these compounds is important for health monitoring. As such, achieving good chromatographic separation so that they can be accurately measured and monitored is a necessity.
A 2.1 x 50 mm, 2.7 µm CORTECS Premier Phenyl Column was used to separate six bile acids using high-performance liquid chromatography - mass spectrometry (HPLC-MS). MS detection was employed because these analytes do not contain a chromophore. Since some of these compounds are isobaric (have the same molecular weight), the isobars must be resolved chromatographically so that they may be accurately identified and quantified. The CORTECS Premier Phenyl Column employs solid-core silica particles shown to improve column efficiency compared to fully-porous particles of the same size.2 Additionally, by using MaxPeak Premier Technology, interactions between the analytes and the column hardware are greatly reduced. These interactions can lead to peak tailing, reduced peak area, iron adduct formation, and in rare occasions, metal-catalyzed reactions of the analyte.3-7
Results and Discussion
Six bile acids were combined to create a mixture with each compound at a concentration of 50 µg/mL in 90:10 water:acetonitrile, Figure 1. A standard screening gradient was used with formic acid modified mobile phases. A constant concentration (0.1%) of formic acid was maintained throughout the gradient. The starting condition of 5% acetonitrile was maintained for 1.00 minute, followed by a linear gradient to 95% acetonitrile in 6.86 minutes. The 95% acetonitrile concentration was maintained for 1.14 minutes before returning to the starting condition and re-equilibrating for 2.28 minutes. A flow rate of 0.5 mL/min was used, and the column was maintained at 40 °C. Negative ion electrospray ionization MS detection was carried out using a Waters™ ACQUITY™ QDa™ Detector. Shown in Figure 2 is a stacked plot of single ion recordings (SIR) of the four mass-to-charge ratios for the six bile acids. Peaks were identified based on retention times in comparison to individual standards.
The isobaric pairs taurodeoxycholic acid/taurochenodeoxycholic acid and glycodeoxycholic acid/glycochenodeoxycholic acid are both baseline resolved with good peak shapes. Achieving good results for taurocholic acid and its deoxy forms is made easier by using a MaxPeak Premier Column. When ionized, the sulfonate group of these compounds is known to interact with metal surfaces in the column hardware causing peak tailing and decreased peak area.8 However, these results are quite good with no discernible peak area loss or peak tailing.
Conclusion
Bile acids are critically important in the human body as they regulate how fats are metabolized and adsorbed. They may also be associated with the risk of colon cancer. Six bile acids were analyzed using a CORTECS Premier Phenyl Column. Baseline separation of two pairs of isobaric compounds was successfully achieved, allowing their accurate identification based on retention times. MaxPeak Premier Column hardware resulted in minimized interactions between the analytes and the column hardware, while the CORTECS solid-core particles produced high column efficiency. These two features are a match made in analytical heaven for critical separations of challenging analytes.
References
- Kuhn T, et al. Prediagnostic Plasma Bile Acid Levels and Colon Cancer Risk: A Prospective Study. J. Natl. Cancer Inst. (2020) 112(5):516–524.
- Walter TH, Shiner S, Izzo G, Savaria M, Iraneta PC, Berthelette K, Danaceau JP, Chambers EE and Fountain KJ. High Efficiency Narrow-Bore Columns Packed with 1.6 and 2.7 µm Solid Core Particles, Chromatography Today, 8 (2015), 22.
- Chul Jung M, Lauber M. Demonstrating Improved Sensitivity and Dynamic Range with MaxPeak High Performance Surfaces (HPS) Technology: A Case Study on the Detection of Nucleotides. Waters Application Note. 720007053.
- Layton C, Rainville P. Advantages of Using MaxPeak HPS Technology for the Analysis of Targeted Cancer Growth Inhibitor Therapies. Waters Application Note. 720007565.
- Boissel C, Walter TH, Shiner S. ACQUITY Premier Solution Improves the UPLC-MS Analysis of Deferoxamine – an Iron Chelating Drug. Waters Application Note. 720007239.
- Berthelette K, Aiello M, Collins C, Kalwood J, Walter TH. Analysis of Radioligand Therapy Components Using Reversed-Phase and HILIC Columns. Waters Application Note. 720008710.
- Myers DP, Hetrick EM, Liang Z, Hadden CE, Bandy S, Kemp CA, Harris TM, Baertschi SW. On-Column nitrosation of amines observed in liquid chromatography impurity separations employing ammonium hydroxide and acetonitrile as mobile phase. J Chrom A. 2013; 1319: 57–64.
- Berthelette K, DeLoffi M, Collins C, Kalwood J, Walter TH. Correlation Between the Adsorption of Acidic Analytes on Stainless Steel Columns and Their Ionic Charge. Waters Application Note. 720008792.
720008967, September 2025
