Analysis of gentisic acid and related renal cell carcinoma biomarkers using reversed-phase liquid chromatography with water-rich mobile phases

Abstract

The problem of longer retention times using water-rich mobile phases in reversed-phase liquid chromatography (RPLC) has been addressed using hydrophobic alcohols such as butanol in very low quantities (approximately 0.1%) as the organic modifier. Advantages of water-rich mobile phases in RPLC for the separation of water-soluble and weakly retained compounds are improved separation of congeners and better tuning of RPLC separations. This is demonstrated in the separation of gentisic acid and related renal cell carcinoma (RCC) biomarkers in urine with a Zorbax C₁₈ column and a mobile phase of 0.1% (volume/volume) butanol in water with 0.6% (volume/volume) acetic acid. Calibration curves for the RCC biomarkers were linear over the concentration range investigated (5–1000 ppm). Detection limits for the RCC biomarkers were 0.85 ppm (quinolinic acid), 1.75 ppm (gentisic acid), and 1.25 ppm (4-hydroxybenzoic acid). Recovery tests using synthetic urine samples containing 20 ppm, 100 ppm, and 700 pm of each RCC biomarker were successful for all compounds.

Graphical Abstract

Keywords:

• Biomarker detection
• renal cell carcinoma
• reversed-phase liquid chromatography
• water-rich mobile phases

Additional information

Funding

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number P20GM103447. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.