D-amino acids are currently paid attention to as new physiologically active substances. Foodstuffs and beverages containing D-amino acids are a matter of interest. Until now, the profiles of D-amino acids have not been reported in natural and fermented teas except theanine. In this study, an off-line 2D-HPLC method combining a Gemini C18 column and a CHIRALPAK® IC-3 column or a self-prepared poly(MQD-co-HEMA-co-EDMA) monolithic capillary column was employed for the separation and detection of D-amino acids in tea samples with the co-existence of a large amount of L-amino acids. The free amino acid fractions in longjing, black, oolong, and pu-erh tea samples were separated and collected after pre-column derivatization using 9-fluorenylmethoxycarbonyl (FMOC) chloride in the reversed-phase mode, and then were concentrated and separated to D- and L-forms on a chiral column. Among them, the D-form of isoleucine (Ile) (1.0–1.6%), alanine (Ala) (0.1–0.8%), phenylalanine (Phe) (0.1–0.4%), valine (Val) (0.2–0.3%), serine (Ser) (0.1–0.2%), aspartic acid (Asp) (0.2%), and proline (Pro) (0.1%) were detected in longjing and oolong teas. Differences between natural tea and fermented tea in the profiles of D-amino acids as well as the total amino acids were also observed. The results provided useful information for the bio-function research of D-amino acids in tea.

D-amino acids are currently paid attention to as new physiologically active substances. Foodstuffs and beverages containing D-amino acids are a matter of interest. Until now, the profiles of D-amino acids have not been reported in natural and fermented teas except theanine. In this study, an off-line 2D-HPLC method combining a Gemini C18 column and a CHIRALPAK® IC-3 column or a self-prepared poly(MQD-co-HEMA-co-EDMA) monolithic capillary column was employed for the separation and detection of D-amino acids in tea samples with the co-existence of a large amount of L-amino acids. The free amino acid fractions in longjing, black, oolong, and pu-erh tea samples were separated and collected after pre-column derivatization using 9-fluorenylmethoxycarbonyl (FMOC) chloride in the reversed-phase mode, and then were concentrated and separated to D- and L-forms on a chiral column. Among them, the D-form of isoleucine (Ile) (1.0–1.6%), alanine (Ala) (0.1–0.8%), phenylalanine (Phe) (0.1–0.4%), valine (Val) (0.2–0.3%), serine (Ser) (0.1–0.2%), aspartic acid (Asp) (0.2%), and proline (Pro) (0.1%) were detected in longjing and oolong teas. Differences between natural tea and fermented tea in the profiles of D-amino acids as well as the total amino acids were also observed. The results provided useful information for the bio-function research of D-amino acids in tea.

•A new carbamoylated quinidine based monolith was prepared and applied in chiral micro-LC.•This monolith demonstrated good permeability, efficiency and reproducibility.•The monolithic column was successfully applied to the enantioseparation of a wide range of N-derivatized amino acids.•By using a self-assembled LIF detector, this monolithic column can be used for determining trace d-amino acids (fmol level) in the presence of large amounts of their l-forms.A novel carbamoylated quinidine based monolith, namely poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate (poly(MQD-co-EDMA)), was prepared for the micro-LC enantioseparation of N-derivatized amino acids. The influence of the mobile phase composition, including the organic modifier proportion, the apparent pH and the buffer concentration, on the enantioresolution of N-derivatized amino acids was systematically investigated. Satisfactory column performance in terms of permeability, efficiency and reproducibility was obtained in most cases. The majority of the enantiomers of the tested N-protected amino acids, including 3,5-DNB, 3,5-DClB, FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B derivatives, could be baseline separated on the poly(MQD-co-EDMA) monolithic column within 25 min. A self-assembled laser induced fluorescence (LIF) detector was employed to improve sensitivity when analyzing 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives of amino acids. Ten NBD-derivatized amino acids, including arginine and histidine whose enantioseparation on quinidine carbamate based CSPs has not been reported so far, were enantioresolved on the poly(MQD-co-EDMA) monolith column. It is worth noting that the d-enantiomers of NBD-derivatized amino acids eluted first, except in the case of glutamic acid. The LOD values obtained with the LIF detector were comparable to those reported using conventional LC-FL methods. The prepared poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace d-amino acids in biological samples.Figure optionsDownload full-size imageDownload as PowerPoint slide

The physiological and bio-marker function of D-acidic amino acids is now becoming the focus in metabolomics study and new drug discovery. A fully automated two-dimensional high performance liquid chromatography (2D-HPLC) was established by using silica-based monolithic ODS column as the first dimension column with acetonitrile-trifluoro acetic acid-water (9:0.05:91, V/V) as the mobile phase, micro Chiralpak QD-1-AX column as the enantiomer separation column with 10 mM citric acid in methanol- acetonitrile (50:50, V/V) as the mobile phase for the second dimension separation, and 4-fluoro-7-nitro-2,1,3- benzoxadiazole (NBD-F) as the fluorometrical derivative reagent. The method has a higher separation efficiency (Rs > 2.5) and a higher detection sensitivity (LOD = 1 fmol) than that of existing methods in the determination of acidic amino acids enantiomers, meanwhile, an online confirmation of the enantiomers amounts were achieved by this method. The recoveries were around 97%–104%, and the RSDs of intra-day and inter-day were less than 5%. Furthermore, by analyzing the aging model senescence accelerated mouse prone 1 (SAMP1) mice which have low immunocompetence, the amounts of D-aspartic acid in thymus and spleen were determined as (206 ± 18) nmol g−1 and (264 ± 21) nmol g−1, respectively. In the experiment, it was found for the first time that there was an obvious increasing trend of D-aspartic acid (p < 0.01) in thymus and spleen of SAMP1 mice compared to senescence accelerated mouse resistant 1 (SAMR1) mice.In this 2D-HPLC system, NBD-Asp and NBD-Glu were firstly separated in a reversed-phase model, their fractions were collected in the different loops of multi-loop valve, respectively, then switched to the QD-1-AX column immediately for the enantiomeric separation at different time. NBD-Asp and NBD-Glu enantiomers were completely separated (Rs > 2.5) without interference.