HPLC with enzymatic detection as a candidate reference method for serum creatinine.
We present a candidate Reference Method for determining the concentration of serum creatinine. The method is based on HPLC combined with enzymatic determination. Creatinine plus 14[C]creatinine is extracted by cation-exchange chromatography, subjected to reversed-phase HPLC, and finally quantified enzymatically. Enzymatic measurement ensures no interference from co-eluting compounds, which has been a problem for some reported HPLC assays relying on ultraviolet detection. The average corrected recovery was 100.1% (SEM = 1.1%; n = 15). The accuracy was verified by assaying five sera with target values determined by isotope dilution mass spectrometry. The total interassay CV was less than or equal to 2.5%. We applied the method to study the specificity of HPLC-ultraviolet detection, using 72 plasma samples from hospitalized patients; no interference was noted. Thus, HPLC-ultraviolet detection appears to be specific, provided that sample cleanup is based on cation-exchange chromatography. Our diode-array detector studies of peak homogeneity supported this conclusion. Still, combined HPLC-enzymatic determination ensures even greater accuracy, ranking close to that by isotope dilution mass spectrometry.
Department of Clinical Chemistry, Rigshospitalet, University Hospital, Copenhagen, Denmark.
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D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003404:Creatinine / Q000097:blood*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007124:Immunoenzyme Techniques; D007201:Indicator Dilution Techniques; D012015:Reference Standards
D003404:Creatinine