Development, validation and application of first derivative spectroscopy ratio method for estimation of Bradford assay.

Several, novel and different spectrophotometric methods based on UV and first-order derivative spectrophotometry, were developed to increase accuracy and improve sensitivity of the Bradford assay for protein quantification in hair samples. The linear calibration function was established in concentration range 2-90 µg/mL at pH 0.6. Significant improvement over the classical method was observed using three different methods based on first-order derivative spectra: signals at 550 nm (1D550), numerically integrated area under curve from 545 to 565 nm (AUC1D545-565) and the ratio of peaks at 550 and 420 nm (1D550/1D420). The correlation coefficients were 0.9924, 0.9932 and 0.9991, respectively. Accordingly, the best correlation was observed by the 1D550/1D420 ratio method. The LOD of 15.67 µg/mL (1D550), 14.80 µg/mL (AUC1D545-565), 5.45 µg/mL (1D550/1D420) and LOQ of 47.48 µg/mL (1D550), 44.85 µg/mL (AUC1D545-565), 16.51 µg/mL (1D550/1D420), were calculated. The sample recoveries for 1D550/1D420 ratio method have proven that its accuracy (95.05%) is within the acceptable interval of recoveries. The obtained 1.55% RSD for precision fell well within the criteria accepted in bioanalytical method validation. This method was applied for determination of total proteins in hair samples. Also, the influence of different chemical treatments on total protein amount in hair sample was investigated.