A highly sensitive and selective pre-column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) assay was developed for the determination of the
D
,
L
configurations of 26 free chiral amino acids in industrial products. The samples were dissolved and extracted in 0.1 mol/L HCl solution,and then derivatized by Marfey reagent under alkaline condition at 37 ℃ for 16 hours. Baseline separation of the derivatives was achieved on a Phenomenex Kinetex F5(250 mm ×5 mm,4.6 μm) column. The mobile phase consisted of 10 mmol/L ammonium acetate and acetonitrile by gradient elution. Reverse phase chromatographic resolution of the enantiomers was obtained within 27 min. The derived analytes were sensitively detected using an electrospray ionization source in negative ion mode(ESI-) and multiple reaction monitoring(MRM) mode. The results showed that the derivatized products of 26 chiral amino acids could be effectively separated. The mass concentrations of 26 chiral amino acids showed a good linear relationship with their corresponding peak areas in a corresponding range. The limits of quantification were determined to be 0.5-12.5 ng/mL and correlation coefficients(
r
2
) not less than 0.994 0. The extraction recoveries for all analysts were in the ranges of 80.3%-118% and the relativ
e standard deviations were in the ranges of 0.47%-9.8%. The method was applied to determine chiral amino acids in 180 industrial samples. The results indicated that
D
-amino acids were detected in
L
-type pure amino acid reagent,
L
-type stable isotope-labeled amino acid internal standard reagent,single
L
-amino acid raw material,and composite amino acid raw material powder. It is the first time to evaluate the quality of chiral amino acids in relevant industrial products in China,and it was found that the monitoring and management of chiral configurations need to be strengthened for amino acid complex powders. It also thoroughly investigates the potential impact on the accuracy of test results in the food,environmental,chemical,and pharmaceutical industries sectors when the amino acid purity reagents are used as standards or isotopic internal standards for the detection of relevant samples. With the advantages of rapid,accurate,high sensitivity,and simple sample pretreatment,this method can be an effective technical tool for quality management and health assessment in the fields of research reagents,biopharmaceuticals,chemical food and nutritional health.
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Related Author
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Related Institution
School of Public Health(Shenzhen),Shenzhen Campus of Sun Yat-sen University