Fukushima T, Kato M, Santa T, Imai K
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Biomed Chromatogr. 1995 Jan-Feb;9(1):10-7. doi: 10.1002/bmc.1130090103.
The enantiomeric separations of D,L-amino acids derivatized with fluorogenic reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-aminosulphonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) by high-performance liquid chromatography (HPLC) on various Pirkle type chiral stationary phases (CSPs, Sumichiral OA series) with citric acid in methanol as a mobile phase were studied. Since the least retention and no separation was observed for the derivatives of racemic phenylalanine methyl-ester, -amide and a drug without an alpha-carboxyl group, the carboxylic acid group of the amino acid derivatives seemed to contribute to the enantioselective fixation of the derivatives through hydrogen bonding on the N-acyl-amino acid amide moiety of the CSP. The enantioselective retention of the derivatives was attained through the (S) or (R) configuration of valine, phenylglycine, naphthylglycine, naphthylethylamine or the tert-leucine moiety in the CSP. The 2,1,3-benzoxadiazole (benzofurazan) moiety in the derivatives helps the effective fixation of the derivatives through a pi-pi interaction with an aromatic moiety such as a 3,5-dinitrophenyl or naphthyl group in the Pirkle type chiral stationary phases. D-Amino acids in biological samples were easily determined utilizing the present derivatization with NBD-F, enantiomeric separation and fluorometric detection (530 nm em/470 nm ex) following deproteinization of biological samples (serum or brain homogenate) with methanol and centrifugation. The applications of the method were clearly demonstrated by the following results; D-Ala was detected in sera of healthy volunteers at a level of 0.48-3.10 microM. D-Lys was found in the serum of a patient with myeloma and requiring renal dialysis, and D-Ser was found in rat and bovine cerebrum. Peak identification was performed by use of different types of stationary phases especially those bearing the opposite configuration to that of the chiral centre.
研究了用荧光试剂4-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）、4-(N,N-二甲基氨基磺酰基)-7-氟-2,1,3-苯并恶二唑（DBD-F）和4-氨基磺酰基-7-氟-2,1,3-苯并恶二唑（ABD-F）衍生化的D,L-氨基酸在各种Pirkle型手性固定相（CSPs，Sumichiral OA系列）上，以甲醇中的柠檬酸为流动相，通过高效液相色谱（HPLC）进行对映体分离。由于外消旋苯丙氨酸甲酯、酰胺以及一种无α-羧基药物的衍生物保留时间最短且未观察到分离现象，氨基酸衍生物的羧基似乎通过氢键作用对衍生物在CSP的N-酰基-氨基酸酰胺部分的对映选择性固定起作用。衍生物的对映选择性保留是通过CSP中缬氨酸、苯甘氨酸、萘甘氨酸、萘乙胺或叔亮氨酸部分的（S）或（R）构型实现的。衍生物中的2,1,3-苯并恶二唑（苯并呋咱）部分通过与Pirkle型手性固定相中的芳香部分（如3,5-二硝基苯基或萘基）的π-π相互作用，有助于衍生物的有效固定。利用NBD-F进行的本衍生化、对映体分离以及荧光检测（发射波长530 nm/激发波长470 nm），在生物样品（血清或脑匀浆）用甲醇脱蛋白并离心后，可轻松测定生物样品中的D-氨基酸。该方法的应用通过以下结果得到了清晰证明；在健康志愿者血清中检测到D-Ala，水平为0.48 - 3.10 μM。在一名需要肾透析的骨髓瘤患者血清中发现了D-Lys，在大鼠和牛大脑中发现了D-Ser。通过使用不同类型的固定相，特别是那些手性中心构型与目标物相反的固定相来进行峰鉴定。
