DIRECT SIMULTANEOUS DETERMINATION OF UREMIC TOXINS: CREATINE, CREATININE, URIC ACID, AND XANTHINE IN HUMAN BIOFLUIDS BY HPLC

Abstract

The determination of creatine, creatinine, uric acid, and xanthine in urine and blood serum is very important in clinical assays as they are most widely used as markers to assess renal function. A simple and direct method for the routine analysis of uremic toxins: creatine, creatinine, uric acid, and xanthine in human blood serum and urine is described. Low wavelength UV detection is achieved at 200 nm using 10 mmol/L KH2PO4, as mobile phase, at a flow rate 0.8 mL/min and a Kromasil C8, 250 × 4 mm, analytical column. Analysis was achieved within approximately 8 min. The limits of detection were 4 pg for creatine and creatinine, 20 pg for uric acid, and 6 pg for xanthine, while the limits of quantitation were 10 pg for creatine and creatinine, 60 pg for uric acid, and 20 pg for xanthine, when 20 μL were injected onto column. A rectilinear relationship was observed up to 2 ng/μL for creatine and creatinine, 12 ng/μL for uric acid, and 5 ng/μL for xanthine. The statistical evaluation of the method was examined performing day-to-day (n = 8) and within-day (n = 8) calibration, and was found to be satisfactory with high accuracy and precision results. RSD values were in the range of 0.4 to 4.3% for within-day measurements and 3.5 to 7.4% for day-to-day precision measurements. The developed method was applied to the analysis of creatinine, creatine, uric acid, and xanthine in biofluids: serum and urine, simply after dilution. The sensitivity of this method was high enough to determine the concentration of creatinine and uric acid in diluted serum (10 to 20 fold dilution) and urine (400–500 fold dilution) samples. Percentage recovery of analytes in spiked samples was in the range 91–105%. No interference was observed from endogenous compounds of human serum and urine. Correlation of analyzed samples using the developed method and conventional routine methods for creatinine and uric acid gave not significantly different results. The method appears to be a very useful tool in routine analysis of clinical samples, for simultaneous determination of creatinine, creatine, uric acid, and xanthine levels in serum and urine.