Institute for Innovation, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan.
Institute for Innovation, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan.
J Chromatogr A. 2019 May 24;1593:91-101. doi: 10.1016/j.chroma.2019.01.075. Epub 2019 Jan 30.
Novel sophisticated derivatizing agents for the efficient enantioselective separation and mass spectrometric detection of d- and l-amino acids have been developed. Two new axially chiral reagents derived from 6,6'-dimethyl-2,2'-biphenyldiamine were synthesized. Their chiral separation and detection abilities were evaluated by derivatizing proteinogenic amino acid standards in reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting diastereomers derived from the developed derivatizing agents and amino acids could be completely separated, because of the effective chiral environment constructed by the axially chiral biphenyl moiety. After optimizing the reactive group, (R)-4-nitrophenyl N-[2'-(diethylamino)-6,6'-dimethyl-[1,1'-biphenyl]-2-yl]carbamate hydrochloride ((R)-BiAC) was found to be the best reagent for highly sensitive simultaneous d,l-amino acid analysis. Using (R)-BiAC, the complete chiral separation of all derivatized proteinogenic amino acids was achieved within 11.5 min with R greater than 1.9, except for certain allo-isomers. An exceptional feature of this reagent was its control of elution order, i.e., it afforded elution of the diastereomers derived from d-amino acids before their l-amino acid counterparts for all 19 proteinogenic amino acids. Sensitive detection was also achieved by introducing a dialkyl amino group and selectively cleaving it at the binding site between the reagent and amino acid. Attomole (amol) detection limits (signal-to-noise ratio = 3) were obtained for the tested d,l-amino acids, in the range 7.0-127 amol. As an example of application, the method was applied to food sample analysis, and detected several d-amino acids. Consequently, the developed method seems likely to facilitate simultaneous determination of enantiomers, including the tiny amounts of d-amino acids found in nature.
已经开发出了用于高效对映选择性分离和质谱检测 d-和 l-氨基酸的新型复杂衍生化试剂。合成了两种新的衍生自 6,6'-二甲基-2,2'-联苯二胺的轴向手性试剂。通过反相液相色谱-串联质谱（LC-MS/MS）将衍生化蛋白质氨基酸标准品评估了它们的手性分离和检测能力。由于轴向手性联苯部分构建了有效的手性环境，因此衍生化试剂和氨基酸衍生的非对映异构体可以完全分离。在优化了反应性基团后，发现（R）-4-硝基苯基 N-[2'-(二乙氨基)-6,6'-二甲基-[1,1'-联苯]-2-基]氨基甲酸盐酸盐（（R）-BiAC）是高度敏感同时分析 d,l-氨基酸的最佳试剂。使用（R）-BiAC，在 11.5 分钟内实现了所有衍生化蛋白质氨基酸的完全手性分离，R 值大于 1.9，除了某些 allo-异构体。该试剂的一个显著特点是它可以控制洗脱顺序，即对于所有 19 种蛋白质氨基酸，它提供了 d-氨基酸衍生的非对映异构体先于其 l-氨基酸对的洗脱顺序。通过引入二烷基氨基基团并选择性地在试剂和氨基酸的结合部位裂解，还实现了灵敏的检测。对于测试的 d,l-氨基酸，获得了纳摩尔（amol）检测限（信噪比=3），范围为 7.0-127 amol。作为应用实例，该方法应用于食品样品分析，并检测到几种 d-氨基酸。因此，所开发的方法似乎很可能有助于同时测定对映体，包括自然界中发现的痕量 d-氨基酸。
