Evaluation of nitrate as internal standard for quantitative determination of urea in urine by Raman spectroscopy

Abstract

Nitrate was tested as internal standard in a new method for urea determination in urine by Raman spectroscopy. The proposed method furnished better precision in comparison with conventional methods. Besides, it is simple, robust and provides faster results, minimum sample preparation and lower reagent consumption and minimum waste generated than the reference spectrophotometric method. Nitrate was evaluated as internal standard (IS) for urea determination in urine by Raman spectroscopy. The influence of main operating parameters (laser power and integration time) on the Raman scattering of urea (N-C-N stretching at 1003 cm-1) and nitrate (N-O stretching at 1045 cm-1) was evaluated and the results showed good correlation between analyte and IS signals. The method was then developed and applied in five urine samples which presented urea concentrations in the 7.3 - 18.3 g L-1 range. For comparative purposes, samples were also analyzed by the reference method based on the enzymatic hydrolysis of urea followed by the spectrophotometric determination of ammonium ions. Results obtained by the proposed IS method were in agreement with the reference method at the 95% confidence level (paired t-test). Relative standard deviations (n = 12) of a sample analyzed by the reference method, proposed IS method and conventional external calibration method were in the ranges of 3.8 - 6.3%, 1.4 -3.9% (IS) and 1.8 - 7.5%, respectively. Recoveries improved from 85 - 108% to 97% - 103% intervals when samples were analyzed by the conventional external calibration and IS proposed method, respectively.