A robust, accurate, sensitive LC–MS/MS method to measure indoxyl sulfate, validated for plasma and kidney cells

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.

Traditional Chinese medicine improved diabetic kidney disease through targeting gut microbiota

“…In DKD mice exhibiting renal tubular injury, a significant increase in plasma IS levels was observed (Ahmed et al. 2022 ). Meprin-β was found to improve diabetic kidney injury, promoting the production of uremic toxins (IS, N-methyl-pyridone-carboxamide) and altering the metabolite balance in the kidney (Gooding et al.…”

Determination of indoxyl sulfate by spectrofluorimetric method in human plasma through extraction with deep eutectic solvent

“…Numerous approaches have been developed to assess the IS level in biological fluids including chromatographic methods like chromatography (GC) and liquid chromatography (LC) and non-chromatographic methods such as electrochemical [ 49 , 50 ], enzymatic [ 51 ], and spectroscopic [ 52 ] techniques. LC methods with mass (MS) or MS/MS [ 53 – 64 ] detectors are common for the analysis of IS in biological fluids. However, they are time-consuming, laborious, and need specialized and expensive equipment.…”

Asperulosidic Acid Ameliorates Renal Interstitial Fibrosis via Removing Indoxyl Sulfate by Up-Regulating Organic Anion Transporters in a Unilateral Ureteral Obstruction Mice Model

“…The plasma sample preparation method was modified according to a previous report [47]. An aliquot of 50 µL mouse plasma was transferred to a 1.5 mL tube, then 10 µL internal standard solution and 600 µL acetonitrile were mixed.…”