Determination of Free Amino Acids and Total Peptides in Kangfuxin Liquid by HPLC Coupled with AQC Pre-column Derivatization
10.19378/j.issn.1003-9783.2024.06.012
- VernacularTitle:AQC柱前衍生高效液相色谱方法测定康复新液中游离氨基酸和总肽的含量
- Author:
Li QIAO
1
;
Xiaoxiao LIU
;
Fu CHEN
;
Jing HUANG
;
Qunyue WU
;
Zhen LIU
Author Information
1. 广东省药品检验所,广东 广州 510663
- Keywords:
Kangfuxin Liquid;
AQC pre-column derivatization;
HPLC;
free amino acids;
total peptides
- From:
Traditional Chinese Drug Research & Clinical Pharmacology
2024;35(6):870-877
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a method for determining the content of free amino acids and total peptides in Kangfuxin Liquid by HPLC coupled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC)pre-column derivatization,and to evaluate the quality of commercial samples with total peptides as indexes.Methods HPLC separation was performed on Kromasil 100-5 C18 column with mobile phase comprising of 60%acetonitrile(A)and 0.14 mmol·L-1 sodium acetate trihydrate solution(adjusted to pH 5.0 with phosphoric acid)(B)in a gradient elution manner at the flow rate of 1.0 mL·min-1.The column temperature was 39℃.The detection wavelength was 248 nm.Results 14 amino acids(aspartate,glutamic acid,serine,histidine,glycine,arginine,threonine,alanine,proline,valine,lysine,isoleucine,leucine,and phenylalanine)could achieve good separation,and present good linear relationship(r=0.999 5～1.000 0)in the concentration range of 2.0～99.7,3.4～168.5,2.8～139.6,4.0～201.4,4.8～238.1,6.7～336.9,3.3～167.5,9.7～487.3,4.1～202.5,4.4～221.6,5.4～270.5,3.3～166.8,4.8～240.8 and 4.7～236.6 μg·mL-1,respectively.The average recoveries of free amino acids and hydrolyzed amino acids from total peptides were 86.8%～108.1%(n=6)and 83.2%～102.7%(n=6),with RSDs at 2.8%～4.4%and 0.1%～3.1%,respectively.The RSDs of precision,repeatability and stability tests were all lower than 5.0%.Conclusion As compared to ultraviolet spectrophotometry method for the current quality standard,the total amino acids content determined by this method is in consistent to UV.However,this method showed better specificity and reproducibility than UV.When total peptides with biological activity were used as quality control indicators,the pertinence of this method was strengthened.Our study may provide a scientific and reasonable method for the quality control of Kangfuxin Liquid.
