Validation of a modified spectrophotometric method for the determination of nitrate in dry milk using 2-sec-butylphenol.

A rapid, simple and direct visible spectrophotometric method, based on the quantitative nitration of 2-sec-butylphenol in concentrated sulfuric acid, is described for the determination of nitrate in milk. The nitration product was extracted into toluene and the yellow nitrophenoxide compound formed in alkaline aqueous solution was determined at 418 nm. Carrez reagents were used for protein precipitation. Interference from lactose, chloride and nitrite ions and the effect of reagent concentration was controlled. The calibration graph was linear over the range 0.5-5 micrograms ml-1 NO3-; y = -0.0019 + 0.0681 x; r = 0.9997. The limits of detection and quantification were found to be 0.18 and 0.5 microgram ml-1 NO3-, respectively. The accuracy of the method did not depend on nitrate content in spiked milk. The mean recovery of the spiked milk was found to be 99.81% with the confidence limits of 0.67%, a relative standard deviation (RSDr) of 1.79% for repeatability and an RSDR value of 1.90% for reproducibility. Adequate agreement was found between results obtained by the nitration method and those of the French official reduction spectrophotometric reference method (AFNor). For the Indonesian dry (powdered) milk sample studied the nitrate levels were in the range 10.7-29.5 mg kg-1 NO3-.