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A robust,accurate, sensitive LC–MS/MS method to measure indoxyl sulfate,validated for plasma and kidney cells
Authors:Sabbir Ahmed  Rolf W Sparidans  Jingyi Lu  Silvia M Mihaila  Karin G F Gerritsen  Rosalinde Masereeuw
Institution:1. Division of Pharmacology, Department of Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands;2. Division of Pharmacology, Department of Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands;3. Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands

Abstract:Proximal tubular damage is an important prognostic determinant in various chronic kidney diseases (CKDs). Currently available diagnostic methods do not allow for early disease detection and are neither efficient. Indoxyl sulfate (IS) is an endogenous metabolite and protein-bound uremic toxin that is eliminated via renal secretion, but accumulates in plasma during tubular dysfunction. Therefore, it may be suitable as a tubular function marker. To evaluate this, a fast bioanalytical method was developed and validated for IS in various species and a kidney cell line using LC–MS/MS. An isotope-labeled IS potassium salt as an internal standard and acetonitrile (ACN) as a protein precipitant were used for sample pretreatment. The analyte was separated on a Polaris 3 C18-A column by gradient elution using 0.1% formic acid in water and ACN, and detected by negative electrospray ionization in selected reaction monitoring mode. The within-day (≤ 4.0%) and between-day (≤ 4.3%) precisions and accuracies (97.7 to 107.3%) were within the acceptable range. The analyte showed sufficient stability at all conditions investigated. Finally, applying this assay, significantly higher plasma and lower urine concentrations of IS were observed in mice with diabetic nephropathy with tubular damage, which encourages validation toward its use as a biomarker.
Keywords:chronic kidney diseases  indoxyl sulfate  LC–MS/MS  renal tubular function  uremic toxins
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