Determination of D-amino acids in thymalfasin by GC-MS based on multi-step derivatization
10.16438/j.0513-4870.2020-0558
- VernacularTitle:基于多步衍生化的GC-MS法测定胸腺法新中D型氨基酸
- Author:
Jie-hong LIN
1
;
Hong-rui YIN
2
;
Hong SHAO
2
;
Gang CHEN
2
;
Mei LIN
2
Author Information
1. Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China; Shanghai Institute for Food and Drug Control, NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai 201203, China
2. Shanghai Institute for Food and Drug Control, NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai 201203, China
- Publication Type:Research Article
- Keywords:
multi-step derivatization;
GC-MS;
thymalfasin;
italic>D-amino acid;
racemic peptide
- From:
Acta Pharmaceutica Sinica
2020;55(10):2435-2441
- CountryChina
- Language:Chinese
-
Abstract:
We utilized a multi-step derivatization gas chromatography-mass spectrometry method for the determination of common amino acid enantiomers, combined with deuterated hydrochloric acid hydrolysis, to identify nine trace D-amino acids in thymalfasin. We optimized the conditions for multi-step derivatization, the volume of reagent for redissolving samples, and the conditions for chromatography and mass spectrometry with isopropanol and trifluoroacetic anhydride as derivatization reagents, and validated the procedure, including sensitivity, linear range, precision, accuracy and recovery. Sixteen pairs of D/L-amino acids and glycine derivatives were separated within 29 min, with the limit of quantification as low as 0.09-2.79 μmol·L-1. Nine amino acid derivatives of thymalfasin showed a good linear relationship within the concentration range examined (r2>0.992 3). The precision results showed that RSD values were less than 10.90%. Accuracy test results of a reference substance ranged from 76.69% to 128.18%. Average recoveries of spiked samples ranged from 70.41% to 125.39%. For the nine D-amino acids assayed, D-Asp and D-Glu content in six batches of thymalfasin were highest, ranging 0.41%-0.49% and 0.25%-0.33%, respectively, with others less than 0.25%. The method is sensitive, efficient and reliable, available for seventeen common amino acids and their enantiomers, and works well with simultaneous determination of nine trace D-amino acids in thymalfasin, providing a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.
