Second-derivative spectrometric determination of urea in milk using the diacetyl monoxime reagent

The application of derivative spectrometry to resolve overlapping spectra and improve the sensitivity and selectivity of the colorimetric determination of urea in milk using diacetyl monoxime is presented. With first-derivative (1D) spectrometry, the lambda(max) of the colored complex was established to be 525 nm. The absorption band at lambda(max) = 525 nm in normal absorption (0D) spectrometry was resolved into three clearly distinct spectral bands with minima at 497, 530, and 566 nm with second-derivative (2D) spectrometry. With the second-derivative (2D530) technique, the depth of the trough of the strongest signal at 530 nm was used to determine urea in milk samples. Linear calibration curves for urea were obtained over a concentration range of 0.2-1.4 mg/L, with correlation coefficients greater than 0.999. The LOD and LOQ of the method were 0.01 and 0.03 mg/L, respectively. Recoveries of 99-100% of spiked urea in milk with RSD values below 2.0% were obtained. The second-derivative method is simple, affordable, sensitive, and selective for the determination of urea in milk.