title: Measurement Uncertainty of Chromogenic LAL Assays: Reaction Time and Proportion of Endotoxin and LAL Reagent Affect Release of p-Nitroaniline.
abstract: Limulus Amebocyte Lysate (LAL) assays are widely used for detection and quantification of bacterial endotoxins in pharmaceuticals and medical devices. However, there are only a few studies on the measurement uncertainty of LAL assays. The aim of this work was to identify and quantify the main sources of measurement uncertainty for end point and kinetic-chromogenic LAL assays. Response surface methodology was used to study how the release of p-nitroaniline (pNA) is affected by reaction time and proportion of endotoxin and LAL reagent in end point and kinetic-chromogenic LAL assays, respectively. Increased release of pNA was observed when reaction time was increased. In addition, if different volumes of sample (or endotoxin standard) and LAL reagent are used, the pNA release rate will be affected. These results may be due to the increased interaction between the bacterial endotoxin and LAL-activated enzyme. Final measurement uncertainties (95% confidence interval) were 90-120% and 90-127% of bacterial endotoxin content for end point and kinetic-chromogenic assays, respectively. These values are reasonable for the scope of the method and allow the application of these measurement uncertainties in routine analysis of pharmaceuticals and medical devices.
authors: Universidade de São Paulo, Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Av. Prof. Lineu Prestes, 580 Cidade Universitária, São Paulo SP, Brazil CEP 05508-000.
authors: Celina Silva
authors: CS
authors: Ostronoff
authors: Felipe Rebello
authors: FR
authors: Lourenço
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001681:Biological Assay / Q000379:methods*; D002863:Chromogenic Compounds; D004340:Drug Contamination; D004731:Endotoxins / Q000737:chemistry*; D007700:Kinetics; D008033:Limulus Test / Q000379:methods*; D013997:Time Factors; D035501:Uncertainty
chemical_list: D000814:Aniline Compounds; D000900:Anti-Bacterial Agents; D002863:Chromogenic Compounds; D004731:Endotoxins; C019498:4-nitroaniline