A systematic procedure for the determination of purity values of amino acid reference materials was developed by use of mass balance method where four categories of impurities (related structure impurities (RSIs), water, organic solvent residue (OSR), and non-volatile residue (NVR)) were quantified separately. The amount of RSIs was determined using a combination of three quantification methods. To ensure metrological traceability in the determination of RSIs, at least one such impurity in each candidate amino acid reference material was quantified using liquid chromatography–isotope dilution tandem mass spectrometry (LC-IDMS/MS). Other RSIs were determined using external calibration liquid chromatography–tandem mass spectrometry (LC-MS/MS) or o-phthaldialdehyde (OPA) derivatization, followed by liquid chromatography–ultraviolet (LC-UV) measurement. As the UV absorption of most RSIs came basically from the same chromophore after OPA derivatization, a relative peak area approach was used in the LC-UV method to quantify the amount of RSIs by comparing their peak areas with that of a reference RSI. The reference RSI was pre-selected and the amount determined by LC-IDMS/MS separately. The absence of D-amino acids was confirmed using Marfey’s reagent derivatization, followed by LC-UV analysis. The amounts of water, OSR, and NVR were measured using Karl Fischer coulometry, gas chromatography–mass spectrometry (GC-MS) and thermogravimetry, respectively. By using this procedure, four amino acid (L-valine, L-leucine, L-isoleucine, and L-phenylalanine) certified reference materials (CRMs) were developed from the candidate materials. The homogeneity and stability of the CRMs were demonstrated by use of LC-IDMS/MS or OPA-LC-UV method, following the principles in ISO 17034 and ISO Guide 35.
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The authors are grateful to the Health Sciences Authority, Singapore, for the support of this project. The authors would also like to thank Dr. Lingkai Wong for valuable discussion.
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Liu, H., Cheow, P.S., Yong, S. et al. Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities. Anal Bioanal Chem 412, 8023–8037 (2020). https://doi.org/10.1007/s00216-020-02936-7