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 ppm to 1000 ppm). Detection limits for the RCC biomarkers were 0.85ppm (quinolinic acid), 1.75ppm (gentisic acid), and 1.25ppm (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.
Keywords: biomarker detection; renal cell carcinoma; reversed phase liquid chromatography; water-rich mobile phases.