Comparison of electrochemical, electrophoretic and spectrophotometric methods for creatinine determination in biological fluids

Abstract

Quantitation of creatinine in biological fluids was achieved using both a biosensor based electrochemical flow cell and a micellar electrokinetic chromatography (MEKC) separation method. The results obtained from each method were compared with the widely recognised clinical method based on the Jaffé reaction. This is a standard spectrophotometric assay based on the Jaffé reaction of creatinine with alkaline picrate. The biosensor for creatinine was constructed by co-immobilising the enzymes creatininase, creatinase and sarcosine oxidase (SO) using a bovine serum albumin (BSA)–glutaraldehyde (GA) procedure at a platinum electrode. Detection of the hydrogen peroxide product of the third enzyme-catalysed reaction (SO) allowed the measurement of creatinine at +0.7 V versus Ag/AgCl. The biosensor was incorporated into a flow cell arrangement which allowed rapid determination of creatinine concentrations in urine samples with a limit of detection of 4.5 μM, a response time of 1 min, a linear range of 0–500 μM and a precision of 1.14% ( n=10). The results for five urine samples were compared with the Jaffé reaction method and based on this number of samples results from the spectrophotometric method were 36.7% higher than those determined using the flow cell method. A MEKC method for creatinine determination in serum samples was also developed and employed for serum analysis, with only a simple filtration step as sample preparation. The method showed a limit of detection of 11.5 μM, a linear range of 0–4.4 mM and a peak area precision of 2.7% ( n=10). The method was applied to creatinine determination in serum samples ( n=13) from patients undergoing renal dialysis and the results compared to the Jaffé reaction procedure. The Jaffé method generated values on average 26.7% greater than the corresponding MEKC results.