Clinical Quantitative Method Development of serum indoxyl sulfate (IS) assay using LC-MS/MS

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biomarker Analysis

Background:

• Indoxyl sulfate (IS) is a uremic toxin that accumulates with declining renal function, exacerbating kidney disease progression.
• Current IS assay reagents lack standardization due to the absence of a standard assay method.
• Establishing a reliable IS assay is crucial for monitoring renal function and disease management.

Purpose of the Study:

• To evaluate the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for establishing a standard assay method for indoxyl sulfate (IS).
• To assess the method's suitability as a reference standard for IS quantification in clinical settings.

Main Methods:

• LC-MS/MS analysis was performed using an AB SCIEX QTRAP® 4500 system.
• Method validation included linearity, reproducibility, selectivity, interferent testing, and determination of LOB, LOD, and LLOQ.
• A calibration curve was generated using the peak area ratio of IS to its deuterated internal standard (IS-d4).

Main Results:

• The LC-MS/MS method demonstrated excellent linearity (R² = 0.9995) up to 440 μmol/L.
• High precision was observed with coefficients of variation of 2.6-4.7% for repeatability and 7.9-9.2% for intermediate precision.
• The method showed high recovery (101.0-104.3%), minimal interference (<5.0%), and established LOB (0.004 μmol/L), LOD (1.248 μmol/L), and LOQ (3.23 μmol/L).
• Strong correlation (R = 0.996) was found between the LC-MS/MS method and conventional enzymatic analysis.

Conclusions:

• The validated LC-MS/MS method exhibits satisfactory basic performance and excellent precision, indicating assay stability.
• This method is suitable for establishing a standardized IS assay.
• The findings support its utility as a reliable reference method for indoxyl sulfate quantification in clinical diagnostics.