RESUMEN
OBJECTIVE: To develop an ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry method for simultaneous determination of nine trace D-amino acids in thymalfasin, combined with deuterated acid hydrolysis and precolumn-derivatization. METHODS: The sample was hydrolyzed by deuterated acid, followed by precolumn-derivatization with Nα-(2, 4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA). The analysis was then performed on an ACCQ-TAGTM ULTRA C18 column (2.1 mm×100 mm, 1.7 μm), with mobile phase comprising water containing 0.1% formic acid-acetonitrile gradiently eluted at a flow rate of 0.3 mL•min-1. Nine D-amino acids in thymalfasin were correctly quantified using standard curves by Xevo G2-S Q-TOF coupled with electrospray ion source in positive ion mode. RESULTS: The standard curves of the nine D-amino acids derivatives had good linearity in the ranges of the tested concentrations (r>0.991 2). The limits of quantitation of all D-amino acids derivatives were as low as 0.05-0.30 pmol. The precision and recoveries met the requirements of Chinese Pharmacopoeia (2015). The contents of D-amino acids in the samples from five companies were determined to be 25.16-1 322.01 μg•g-1. Among them, D-glutamate, D-aspartate, D-lysine and D-serine had higher contents, which were 1 105.45-1 322.01, 614.15-740.50, 358.06-388.76 and 138.78-291.60 μg•g-1, respectively. CONCLUSION: The method is sensitive, efficient and reliable, working well for simultaneous determination of nine trace D-amino acids in thymalfasin, which provides a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.