Performance evaluation of an enzymatic spectrophotometric method for milk urea nitrogen

Our objective was to determine the within and between laboratory performance of an enzymatic spectrophotometric method for milk urea nitrogen (MUN) determination. This method first uses urease to hydrolyze urea into ammonia and carbon dioxide. Next, ammonia (as ammonium ions) reacts with 2-oxoglutarate, in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and the enzyme glutamate dehydrogenase (GlDH), to form l-glutamic acid, water, and NADP⁺. The change in light absorption at 340 nm due to the conversion of NADPH to NADP+ is stoichiometrically a function of the MUN content of a milk sample. The relative within (RSD_r) and between (RSD_R) laboratory method performance values for the MUN enzymatic spectrophotometric method were 0.57% and 0.85%, respectively, when testing individual farm milks. The spectrophotometric MUN method demonstrated better within and between laboratory performance than the International Dairy Federation differential pH MUN method with a much lower RSD_r (0.57 vs. 1.40%) and RSD_R (0.85 vs. 4.64%). The spectrophotometric MUN method also had similar method performance statistics as other AOAC International official validated chemical methods for primary milk component determinations, with the average of all RSD_r and RSD_R values being <1%. An official collaborative study of the enzymatic spectrophotometric MUN method is needed to achieve International Dairy Federation, AOAC International, and International Organization for Standardization official method status.