Comparison of the effect of Sodium Chloride concentration on protein determination: Bradford and Biuret methods.

Protein quantification methods using spectrophotometry are widely used in laboratory routines for different purposes. Samples generally contain non-protein components that can interfere with the quality of the analysis. A simple and quick test with different concentrations of sodium chloride demonstrated that the Bradford method is significantly affected by the presence of salt, while Biuret remains stable. Therefore, the choice of method is an important factor in reducing errors and ensuring more reliable results.

Negative interference of icteric serum on a bichromatic biuret total protein assay.

BACKGROUND: Bilirubin is stated to be a negative interferent in some biuret assays and thus could contribute to pseudohypoproteinemia in icteric samples. OBJECTIVE: The purpose of the study was to evaluate the magnitude of and reason for a falsely low total protein concentration in icteric serum when the protein concentration is measured with a bichromatic spectrophotometric biuret assay. METHODS: Commercially available bilirubin was dissolved in 0.1 M NaOH and mixed with sera from 2 dogs to achieve various bilirubin concentrations of up to 40 mg/dL (first set of samples) and 35 mg/dL (second set of samples, for confirmation of first set of results and to explore the interference). Biuret total protein and bilirubin concentrations were determined with a chemistry analyzer (Cobas 6000 with c501 module). Line graphs were drawn to illustrate the effects of increasing bilirubin concentrations on the total protein concentrations. Specific spectrophotometric absorbance readings were examined to identify the reason for the negative interference. RESULTS: High bilirubin concentrations created a negative interference in the Cobas biuret assay. The detectable interference occurred with a spiked bilirubin concentration of 10.7 mg/dL in one set of samples, 20.8 mg/dL in a second set. The interference was due to a greater secondary-absorbance reading at the second measuring point in the samples spiked with bilirubin, which possibly had converted to biliverdin. CONCLUSION: Marked hyperbilirubinemia is associated with a falsely low serum total protein concentration when measured with a bichromatic spectrophotometric biuret assay. This can result in pseudohypoproteinemia and pseudohypoglobulinemia in icteric serum.

Validation for the determination of biuret in water-soluble, urea-based commercial inorganic fertilizer materials, urea solutions, and sulfur-coated urea products by reversed-phase liquid chromatography: single-laboratory validation of an extension of AOAC official methods 2003.14.

A single-laboratory validation (SLV) study for the LC determination of biuret in dry and liquid urea-based commercial fertilizers was conducted. A total of 23 samples were used: 11 commercial dry urea products, two urea ammonium nitrate products, eight liquid urea-based commercial fertilizers, and four sulfur-coated urea samples from different sources. In addition, one biuret standard from Aldrich and one sample from the Magruder check sample program were used as validation samples. The proposed method is an extension of AOAC Official Method 2003.14 and is based on dissolution of the test portion in the LC mobile phase and determination by HPLC. The system is linear over a concentration range of 1.00-4.50 mg/L biuret, with a correlation coefficient > or = 0.9999. The biuret was well- separated from urea in the commercial urea samples, and from other constituents in the commercial liquid fertilizer with no observed interferences. Recoveries were determined by spiking four of the validation materials with a known amount of biuret standard and measuring the biuret level according to the method. The averaged recovery was 97%. Method precision was determined by quadruplicate analyses of four of the liquid and six of the commercially available dry urea validation materials using three and four replicate analyses. For the liquid fertilizer analyses, the RSD ranged from 7.04 to 13.31%. For the dry urea analyses, the RSDs ranged from 5.68 to 14.34%. Instrument precision was evaluated at the test initiation by using seven injections of five biuret standard solutions. SD varied from 0.27 to 1.02%, with RSDs averaging 1.14%. The LOD was determined to be 0.009% biuret in material, while the LOQ was determined to be 0.031% biuret in material. In addition to the intralaboratory study, interlaboratory studies were performed by two other outside laboratories using this method. Over a concentration range of 0.2 to 0.9% biuret, the average SD was 0.11%, the average RSD was 21.16%, and the average HorRat value was 4.73%. Furthermore, comparative studies for biuret using AOAC Official Methods 960.04 and 976.01 with the proposed LC method were performed. The three methods produced very close results; however, the two AOAC methods generate hazardous wastes and are more tedious. On the basis of accuracy and precision of the results for this SLV study, it is recommended that this method be collaboratively studied for the determination of biuret in dry and liquid urea-based commercial fertilizer materials.

Detection and quantification of additives (urea, biuret and poultry litter) in alfalfas by NIR spectroscopy with fibre-optic probe.

The additives (urea, biuret and poultry litter) present in alfalfa, which contribute non-proteic nitrogen, were analysed using near infrared spectroscopy (NIRS) technology together with a remote reflectance fibre-optic probe. We used 75 samples of known alfalfa without additives and 75 samples with each of the additives, urea (0.01-10%), biuret (0.01-10%) and poultry litter (1-25%). Using the discriminant partial least squares (DPLS) algorithm, the presence or absence of the additives urea, biuret and poultry litter is classified and predicted with a high prediction rate of 96.9%, 100% and 100%, obtaining the equations of discrimination for each additive. The regression method employed for the quantification was modified partial least squares (MPLS). The equations were developed using the fibre-optic probe to determine the content of urea, biuret and poultry litter with multiple correlation coefficients (RSQ) and prediction corrected standard errors (SEP (C)) of 0.990, 0.28% for urea, 0.991, 0.29% for biuret and 0.925, 2.08% for poultry litter. The work permits the instantaneous and simultaneous prediction and determination of urea, biuret and poultry litter in alfalfas, applying the fibre-optic directly on the ground samples of alfalfa.

Identification of N1-glycolylbiuret in the gamma radiolysis of aerated aqueous solution of cytosine. Influence of the pH.

N1 glycolylbiuret has been identified as a radiation product of cytosine in aerated aqueous solution at pH 4.5. When varying the pH of the solution before irradiation from acidic values towards neutral ones, G value of N1 glycolylbiuret reached a maximum at pH 4.5.