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Simplified HPLC Method for Urinary and Circulating Creatinine

2004; American Association for Clinical Chemistry; Volume: 50; Issue: 1 Linguagem: Inglês

10.1373/clinchem.2003.024141

1530-8561

Dimitrios Tsikas, Alexander Wolf, Jürgen C. Frölich,

Electrochemical sensors and biosensors

HPLC with various detection methods, including ultraviolet absorbance, is frequently used to separate and measure creatinine and creatine in serum, plasma, and urine. Current HPLC and other analytical methods for the measurement of creatinine, including capillary electrophoresis and gas chromatography–mass spectrometry, have been reviewed recently (1). In 1990, Paroni et al.(2) reported a cation-pairing HPLC method with ultraviolet detection at 236 nm for the measurement of creatinine in serum and urine. This method involves use of cimetidine as an internal standard, treatment of the sample (100 μL of serum or 30-fold dilution of urine) with acetone (400 μL) to precipitate proteins, complete drying of the supernatant (300 μL) after sample centrifugation, reconstitution with the mobile phase, and injection into the HPLC apparatus. Paroni et al. (2) reported a good correlation between their procedure and other methods, including the Jaffe method. We have modified several steps of the HPLC method originally described by Paroni et al. (2). The modifications outlined in detail below have simplified the previous method and made it more easily adapted to automated analysis. HPLC analyses were performed with a Pharmacia LKB Model 2248 pump and an analytical column [125 × 3 mm (i.d.)] packed with Nucleosil 120-3 C18 from Macherey-Nagel. The mobile phase consisted of water–acetonitrile (95:5 by volume) containing 10 mmol/L of the sodium salt of 1-octanesulfonic acid (Sigma-Aldrich), which served as the cation-pairing agent. The pH of the mobile phase was adjusted to 3.2 with orthophosphoric acid. The flow rate was 1 mL/min. [In the method of Paroni et al. (2), the more lipophilic cation-pairing agent 1-decanesulfonate (at 10 mmol/L) in water–methanol (50:50 by volume) was used.] The Model Spectroflow 783 variable ultraviolet/visible detector (Kratos Analytical) was set at 236 nm for creatinine or 215 nm for creatine and creatinine. Analyses were performed …