Comparison of three amino acid analysis methods and their application to the amino acid impurity analysis for the development of high-purity amino acid certified reference materials

The determination of amino acid (AA) impurity is important for the development of AA high-purity certified reference materials (CRMs), which play a key role in metrological traceability. We performed three analytical methods for the determination of AAs, such as postcolumn method derivatized with o -phthalaldehyde (OPA method), precolumn method derivatized with 6-aminoquinolyl- N -hydroxylsuccinimidyl carbamate (AQC method), and liquid chromatography – mass spectrometry combined with ion-pairing reverse-phase chromatography (IP-RP-LCMS method), comparing their separation and sensitivity. We also applied them onto the AA impurity analysis in the candidate CRMs of l -isoleucine (Ile) and l -valine (Val), comparing their qualitative and quantitative performance. The OPA method was superior to others in separation and sensitivity, but several methods seemed to be necessary for the identification of the majority of AA impurities. In relation to the quantitative performance, the analytical results obtained by the different methods were equivalent within their expanded uncertainty for the detected AA impurities. On the other hand, the OPA method could detect the largest number of AA impurities and, consequently, obtained the largest sum of the mass fraction of AA impurities. The expanded measurement uncertainty of the OPA method was sufficiently smaller than the target standard uncertainty, 600 mg kg −1 , where the expanded uncertainty ( k = 2) of the assigned value of high-purity AA CRM should be approximately <0.002 kg kg −1 .