Abstract Background Ammonia is produced by catabolism of protein and other compounds which contain nitrogen. Elevated ammonia levels are toxic for the central nervous system. High plasma ammonia is a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in urea cycle metabolism. The reliable and accurate quantification of ammonia is influenced by assay, reagent, and sample management. Current commercially available GLDH enzyme-based ammonia assay reagents experience high bias between matched Lithium Heparin and K2-EDTA samples, poor accuracy, imprecision at the low end, large HIL (hemolysis, icterus and lipemia) interferences and shorter shelf life. To improve upon the listed deficiencies, we developed an improved GLDH based ammonia assay in a two-part format with improved reagent composition. Methods Ammonia reagent in 2-part format R1 and R2 were assessed for accuracy, precision, interference by HIL reportable range, sensitivity (LoB, LoD, LoQ), shelf life, matched matrix comparison (Lithium Heparin vs K2-EDTA) and method comparison. All testing were performed using Li Heparin plasma samples in Beckman DxC700AU analyzer. Accuracy was tested using Verichem ammonia standards which are ACS ammonia standard material. Twenty-day precision studies were performed using three levels of ammonia concentrations using Bio-Rad Liquichek Ammonia controls and plasma samples. Interference was tested in two ammonia concentrations (40-50 µmol/L) and (200-250 µmol/L) using commercially available spiking material for clinical chemistry assays such as conjugated and unconjugated bilirubin, Intralipid and in-house prepared hemolysate. Infinity Ammonia Reagent on Beckman Coulter AU Chemistry Analyzers was used for method comparison studies Results Next generation ammonia showed good accuracy with Verichem ammonia standards and acceptable within-run and total precision. Studies showed significant improvement in sensitivity and longer shelf life (>18 months). Next generation ammonia is less affected by interferences from icterus (100 mg/dL), lipemia (1000 mg/dL intralipid) and hemolysis (100 mg/dL). Comparison studies show no significant bias between match Lithium Heparin and K2-EDTA matched samples. Conclusions Next Generation Ammonia reagent showed very good overall performance required for reliable and accurate quantification of ammonia in human plasma sample.
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