Jin Yueying,Xu Chun-Yan,Wang Jing,Man Xiaoxi,Shi Qing,Nie Fenghuan,Nan Jun,Min Jun Zhe
A new simple and sensitive method for the simultaneous detection of free DL-amino acids (DL-AA) in natto was established by UPLC-fluorescence (FL) using the newly developed fluorescence derivatization reagent DBD-trans-2-methyl-L-proline (DBD-M-Pro) by precolumn derivatization. DBD-M-Pro reacts with DL-amino acids at 60 °C for 60 min. Using BEH C18 (2.1 mm × 100 mm, 1.7 μm) column gradient analysis for 20 min, the resolution of diastereomers after the derivatization of four DL-amino acids was 1.65–3.50. The detection limit for amino acids was 0.25–2.5 pmol. The average recovery (%) of DL-amino acids was 86.65–118.70%. The method was applied to the detection of chiral amino acids in natto beans and mucus, and four chiral amino acids, DL-Gln, DL-Asp, DL-Glu, and DL-Ala, were simultaneously detected. At the same time, the DL-amino acid content and D/L ratio of three different manufacturers of natto were compared quantitatively.
Songze Wang,Xin Zhou,Xin Wang,Shengyu Cheng,Xi-Ling Li,Jun Nan,Jun Zhe Min
Human hair is a non-invasive biological sample that is easy to collect and store and can reflect long-term body health. However, the correlation between DL-amino acids and metabolic diseases in hair samples has not been studied. Therefore, we propose a novel UHPLC-HRMS method for analyzing seven free chiral amino acids (DL-Thr, DL-Glu, DL-Ala, DL-Val, DL-Pro, DL-Leu, and DL-Phe) simultaneously in hair samples by derivatization of chiral probe 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-trans-2-methyl-L-proline (DBD-M-Pro) labeled with targeted amino functional groups. Gradient elution was carried out using an ACQUITYTM BEH C18 (100×2.1 mm,1.7 μm) column with a mobile phase of 0.15 % formic acid (FA) in 10 mM ammonium acetate (CH 3 -COONH 4 ) and 0.2 % FA in acetonitrile. The labelled DL-amino acid diastereoisomers could be completely separated, with a resolution (Rs) of 1.59–11.44. These amino acids show a strong linear correlation within the range of 3.1–99.2 pmol (R 2 ≥ 0.9990). Intraday and interday precision was 1.87 %–14.87 %. The average recovery was 96.12 %–105.33 %. The limit of detection (LOD) ranged from 0.29 to 2.11 pmol. We then employed the method to determine the concentration of free chiral amino acids in hair samples from 30 healthy volunteers (HVs) and 30 diabetes patients (DPs). Male diabetes patients had significantly higher levels of L-Thr, L-Val, L-Leu ( p < 0.05), and D-Ala ( p < 0.01) in their hair samples than male healthy volunteers and female diabetes patients had significantly higher levels of D-Ala ( p < 0.05) in their hair samples than female healthy volunteers. This is the first study to confirm the feasibility of using free DL-amino acids in human hair as potential biomarkers for diabetes.
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