Development of a certified reference material for d-phenylalanine with evaluation of enantiomeric purity

• Published in: Analytical and bioanalytical chemistry Vol. 416; no. 23; pp. 5177 - 5189
• Main Authors: Luo, Shiwen, Liu, Yahui, Wang, Xianxia, Wang, Ziliang, Yang, Bin, Wang, Jing, Wu, Liqing
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-09-2024; Springer Nature B.V
• Subjects: Amino acids; Analytical Chemistry; Biochemistry; Calibration; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography; Circular dichroism; Dichroism; Evaluation; Food Science; Impurities; Infrared spectroscopy; Inhomogeneity; International System of Units; Laboratory Medicine; Liquid chromatography; Mass balance; Mass spectrometry; Mass spectroscopy; Monitoring/Environmental Analysis; NMR; Nuclear magnetic resonance; Optical properties; Optical rotation; Organic chemistry; Pharmaceuticals; Phenylalanine; Purity; Quantitation; Raw materials; Reference materials; Research Paper; Rotating liquids; Spectroscopy; Stability tests; Tubes; Mass balance; Purity; Quantitative nuclear magnetic resonance; Phenylalanine; Certified reference material; d-Phenylalanine
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-024-05456-w

d
-Phenylalanine (
d
-Phe) is a small chiral organic molecule that is both an important pharmaceutical intermediate and used as a calibrator for quantifying amino acids in liquid chromatography-circular dichroism. We have developed a process for a national certified reference material (CRM) for
d
-Phe following ISO 17034:2016. The identity of
d
-Phe was confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR), infrared, and ultraviolet (UV) spectroscopy. The absolute optical conformation was also determined using circular dichroism (CD) spectroscopy and optical rotation measurements. Impurities were identified via liquid chromatography (LC) with a UV–Vis detector and a charged aerosol detector (CAD) and LC–MS. Both mass balance and quantitative NMR were employed for value assessment, and the associated uncertainty was evaluated. The certified purity was determined to be 0.995 ± 0.003 g/g, a validation that was confirmed by CD using
l
-Phe CRM as a calibrator. Twenty milligrams of raw material was packed in sealed brown glass tubes for storage, and no inhomogeneity was observed. Stability tests revealed that the
d
-Phe CRM remained stable at −20 °C for at least 26 months, at 4 °C for at least 14 days, and at 25 °C and 60 °C for at least 7 days. The
d
-Phe CRM can be used to ensure the accuracy and reliability of
d
-Phe quantitation in the pharmaceutical field and also as a calibrator to ensure traceability to the International System of Units (SI) for
l
-Phe quantitation and protein purity analysis using LC-CD methods. The approach outlined in this paper also has potential for use in the development of other chiral CRMs.
Graphical Abstract