Simplified HPLC method for urinary and circulating creatinine.

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 L of serum or 30-fold dilution of urine) with acetone (400 L) to precipitate proteins, complete drying of the supernatant (300 L) 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 MachereyNagel. 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 at ambient temperature (22–26 °C). Creatinine and creatine (98% purity) were purchased from Sigma-Aldrich. Stock solutions (20 mmol/L) of creatinine and creatine were prepared in distilled water and diluted appropriately with the mobile phase. The major modifications were in the sample treatment procedures. In the present study, for quantitative measurements of urinary creatinine, we diluted 10 L of centrifuged (800g for 5 min) native urine with 990 L of the mobile phase (final dilution, 1:100 by volume) and injected a 200L aliquot. For quantitative measurements of circulating creatinine and creatine, we mixed 100 L of plasma (anticoagulated with EDTA, citrate, or lithium heparin) or serum with 100 L of acetonitrile, incubated the mixture for 3 min, centrifuged it at 2400g for 10 min at 5 °C, diluted 50 L of the supernatant with 950 L of the mobile phase (final dilution, 1:40 by volume), and injected a 200L aliquot into the HPLC instrument. Precipitation of proteins from plasma or serum by acetonitrile was complete, whereas we found that treatment of urine samples (by this procedure or by acetone precipitation) was unnecessary (data not shown). These modifications simplify the method, considerably shorten the time for sample preparation because no drying of samples is required, and make the use of an internal standard such as cimetidine unnecessary if the HPLC method has adequate precision. Mixtures containing equimolar concentrations of synthetic creatine and creatinine in the range 0–200 mol/L were used to prepare calibration curves in urine, plasma, or serum. The mean (SD) retention time of creatinine was 7.26 (0.04) min (CV 0.6%; n 12) and did not depend on the amount of creatinine injected. By contrast, the retention time of creatine decreased with increasing amounts injected, and the variation in the retention time was relatively large [mean (SD) retention time, 3.36 (0.16) min; CV 4.8%; n 12]. The linear regression equations for peak area (y; in arbitrary units) and concentration ( mol/L) of the injected calibrator (x) were: y 9312x 9220 (r 0.998) for creatine at 215 nm; y 41 494x 40 668 (r 0.999) for creatinine at 215 nm; and y 10 215x 14 885 (r 0.998) for creatinine at 236 nm. Thus, the creatinine assay at 215 nm was approximately four times more sensitive than that at 236 nm. Nevertheless, detection at 236 nm allows for more specific analysis of creatinine and is recommended for use. To estimate the limit of detection of the method for creatinine, we injected 200L aliquots of a 500 nmol/L solution in the mobile phase, which was equivalent to 100 pmol of creatinine, and measured the absorbance at 236 nm; the experiment was performed in quadruplicate. The HPLC peak observed at the retention time of creatinine was measured with a signal-to-noise (S/N) ratio of 21:1 and an imprecision (CV) of 5.6%. A urine sample with a basal creatinine concentration of 24.1 mmol/L was serially diluted with the mobile phase in 1:10 (by volume) steps and analyzed by HPLC. Injection of a 200L aliquot of a 1:10 dilution of the sample, which corresponded to 48.2 pmol of creatinine, yielded a creatinine peak with a S/N ratio of 8:1. A serum sample (100 L) with a basal creatinine concentration of 82 mol/L was treated with acetonitrile (100 L) and diluted with the mobile phase to a final dilution of 1:200. Injection of a 200L aliquot of this dilution, corresponding to 82 pmol of creatinine, yielded a creatinine peak with a S/N ratio of 6:1. These concentrations were comparable to those obtained from analysis of aqueous solutions of synthetic creatinine. Paroni et al. (2 ) reported a limit of detection (S/N ratio of 3:1) of 0.5 mg/L for plasma creatinine, which corresponds to an injected amount of 22 pmol. Thus, the detection limit of the present method appears to be similar to that of the method of Paroni et al. (2 ). Representative chromatograms from the analysis of synthetic, urinary, and circulating creatinine are shown in Fig. 1A. A 20 mmol/L solution of creatinine in distilled Technical Briefs