HPLC with enzymatic detection as a candidate reference method for serum creatinine

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Accurate serum creatinine measurement is crucial for diagnosing kidney disease.
• Existing High-Performance Liquid Chromatography (HPLC) assays can suffer from interference from co-eluting compounds, particularly when using ultraviolet detection.
• A need exists for a highly specific and accurate reference method for serum creatinine determination.

Purpose of the Study:

• To develop and validate a candidate Reference Method for serum creatinine concentration.
• To enhance accuracy and specificity in creatinine measurement compared to existing HPLC methods.
• To compare the performance of the new method against isotope dilution mass spectrometry (IDMS).

Main Methods:

• Serum creatinine was quantified using a combination of High-Performance Liquid Chromatography (HPLC) and enzymatic determination.
• Creatinine was extracted via cation-exchange chromatography, followed by reversed-phase HPLC separation.
• Quantification was achieved through enzymatic measurement, ensuring specificity against interfering substances.

Main Results:

• The developed method demonstrated an average corrected recovery of 100.1% (SEM = 1.1%).
• Interassay coefficient of variation (CV) was consistently below or equal to 2.5%.
• Assays using the method on five sera with IDMS-determined target values showed high accuracy.

Conclusions:

• The combined HPLC-enzymatic method provides high accuracy for serum creatinine determination, comparable to IDMS.
• HPLC with cation-exchange chromatography cleanup followed by UV detection is specific, but the enzymatic endpoint offers superior accuracy.
• This candidate Reference Method offers a reliable and precise approach for serum creatinine analysis.