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.

Keywords: LC-MS/MS; chronic kidney diseases; indoxyl sulfate; renal tubular function; uremic toxins.

FIGURE 1

Product spectra of (a) indoxyl sulfate, m/z 212.0 @ –24 V and (b) internal standard (isotope labeled; 13C6), 218.0 @ –27 V

FIGURE 2

Representative chromatograms of (a) indoxyl sulfate and (b) internal standard for a blank and LLOQ (0.1 μg/mL) spiked surrogate matrix (BSA). Panel C represents the chromatograms for a mouse plasma sample

FIGURE 3

Bland–Altman plot represents a reanalysis of 42 samples of mouse plasma

FIGURE 4

Baseline levels of plasma indoxyl sulfate (IS) in various healthy species. Each dot represents an individual animal

FIGURE 5

Indoxyl sulfate (IS) concentrations in control and diabetic nephropathy mice (STZ). At all time points, (a) elevated plasma IS concentrations and (b) decreased urinary IS concentrations were found in diabetic mice. Data are presented as mean ± SD of individual values obtained by using the current method. The non‐parametric Mann–Whitney U test was performed for statistical analysis