Quantification of creatinine in biological samples based on the pseudoenzyme activity of copper–creatinine complex

Glomerular filtration rate (GFR), the marker of chronic kidney disease can be analyzed by the concentration of cystatin C or creatinine and its clearance in human urine and serum samples. The determination of cystatin C alone as an indicator of GFR does not provide high accuracy, and is more expensive, thus measurement of creatinine has an important role in estimating GFR. We have made an attempt to quantify creatinine based on its pseudoenzyme activity of creatinine in the presence of copper. Creatinine in the presence of copper oxidizes paraphenylenediamine dihydrochloride (PPDD) which couples with dimethylamino benzoicacid (DMAB) giving green colored chromogenic product with maximum absorbance at 710 nm. Kinetic parameters relating this reaction were evaluated. Analytical curves of creatinine by fixed time and rate methods were linear at 8.8–530 μmol L−1 and 0.221–2.65 mmol L−1, respectively. Recovery of creatinine varied from 97.8 to 107.8%. Limit of detection and limit of quantification were 2.55 and 8.52 μmol L−1 respectively whereas Sandell's sensitivity and molar absorption coefficient values were 0.0407 μg cm−2 and 0.1427 × 104 L mol−1 cm−1 respectively. Precision studies showed that within day imprecision was 0.745–1.26% and day-to-day imprecision was 1.55–3.65%. The proposed method was applied to human urine and serum samples and results were validated in accordance with modified Jaffe's procedure. Wide linearity ranges with good recovery, less tolerance from excipients and application of the method to serum and urine samples are the claims which ascertain much advantage to this method.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► We have developed a pseudoenzymatic reaction for the quantification of creatinine. ► The pseudoenzymatic activity is quantified by a chromogenic probe involving paraphenylenediamine dihydrochloride and dimethylamino benzoicacid. ► The proposed method is compared with Jaffe's method and its applicability is tested in human urine and serum samples.