Study On The High Accuracy Purity Assessment Methods For The Essential Pure Amino Acids

Abstract:Amino acids are the building blocks of proteins. They are the basis in the metabolic process. Almost of all life activities are related to amino acids. They are also the important nutrition compositions in organisms. Lack of any of the essential amino acids can result in physiological abnormalities, influence the normal metabolism of antibody and finally lead to diseases of human body. Amino acids play an important role in the fields of proteomics, biochemistry and clinical medicine. Certified reference materials of pure amino acids are the basis of the accurate quantification and traceability of amino acids and proteins. Thus, development the high accurate method for essential pure amino acids can provide the key base for the quantification of amino acids and proteins in the life sciences and food security.National Metrology Institute of Japan (NIMJ) has recently produced four pure reference materials of amino acids. National Institute of Standards and Technology (NIST) has also developed a standard reference material (SRM)2389a-seventeen amino acids in0.1mol/L hydrochloric acid. In China, there are some certified reference materials of second-class amino acids with the uncertainties greater than or equal to1.5%. Either derivitization or non-aqueous titration methods were applied as the methods for the purity certification. There may have a positive error caused by the interferences of the related structure impurities when using the non-aqueous titration method. As for the derivatization method, due to the incomplete derivatization reaction or by-products produced in the process of derivatization, the purity certification results may also higher than the real value. In order to develop the high-level pure ceritified reference materials of amino acids, it is imperative to establish a highly efficient, reliable and accurate method for the purity assessment to ensure the accuracy and traceability of measurement results.In this study, the reliable and accuracy methods are described for the purity assessment of four essential amino acids including valine, phenylalanine, leucine and isoleucine, which are closely related to protein quantification and clinical testing, and also widely used for the medicine and feeds industry. The methods of HPLC-CAD, derivatization and HPLC-IT-TOF-MS were applied for the identification of the related structure impurities whose content were more than0.1%. An accuracy and direct method of HPLC-MS/MS was developed for the quantification of the related structure impurities. The residual organic solvents (VOCs) were analized using headspace gas chromatography. The water content was determined using Karl Fischer coulometric method and confirmed by thermogravimetry (TGA) method. The content of the non-volatiles/inorganics was carried out by inductively coupled plasma mass spectroscopy (ICP-MS) and verified by ash residue method. Finally, the mass balance method was used to certify the purity property value and confirmed by quantitative NMR method. The Consultative Committee for Amount of Substance (CCQM) had organized international key comparison CCQM-K55c:Characterization of Organic Substances for Chemical Purity of L-valine. National Institute of Metrology (NIM) took part in this comparison using the developed method and achieved very good results according to the final report published by CCQM. Five related structure impurities were identified and quantified. Also, the uncertainty for this key comparison was relative small among these twenty-five participants.Our results acquired the corresponding international mutual recognition. Our research has built an important basis for the high-level certified reference materials of amino acids.