title: Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography.
abstract: We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 μg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 μM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.
authors.affiliation: Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
authors.forename: Hsiaoling
authors.initials: H
authors.lastname: Wang
authors.affiliation: Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
authors.forename: Nagarani
authors.initials: N
authors.lastname: Pampati
authors.affiliation: Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
authors.forename: William M
authors.initials: WM
authors.lastname: McCormick
authors.affiliation: Division of Biological Standard and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993. Electronic address: lokesh.bhattacharyya@fda.hhs.gov.
authors.forename: Lokesh
authors.initials: L
authors.lastname: Bhattacharyya
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines / Q000032:analysis*; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D009584:Nitrogen / Q000032:analysis*; D010047:Ovalbumin / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis*; D000075462:Serum Albumin, Human / Q000032:analysis*
chemical_list: D022122:Anthrax Vaccines; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D009584:Nitrogen; D000075462:Serum Albumin, Human
keywords: Kjeldahl digestion; analytical biochemistry; biotechnology; ion chromatography; oxalate complexation with copper and aluminum; protein nitrogen determination; proteins; suppressed conductivity detection; vaccines; validation