Tojo Yosuke, Hamase Kenji, Nakata Minako, Morikawa Akiko, Mita Masashi, Ashida Yutaka, Lindner Wolfgang, Zaitsu Kiyoshi
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Nov 1;875(1):174-9. doi: 10.1016/j.jchromb.2008.06.025.
A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.
已开发出一种全自动二维高效液相色谱系统，该系统采用微径ODS柱和窄径对映体选择性柱，用于同时测定哺乳动物体内脯氨酸、反式-4-羟基脯氨酸和顺式-4-羟基脯氨酸的对映体。在第一维中，内径为0.53 mm的整体式ODS柱的理论塔板数比填充颗粒ODS柱大3至6倍，在第二维中，内径为1.5 mm的Chiralpak QN-2-AX柱实现了最佳对映体分离（分离因子：1.44 - 1.83）。日内和日间精密度的相对标准偏差值小于5.8%，D-对映体的定量下限为1 fmol。该方法成功应用于血清和富含胶原蛋白的皮肤组织中脯氨酸和羟脯氨酸对映体的测定。在血清（1.57±0.19 nmol/mL）和皮肤（0.093±0.015 nmol/mg蛋白质）中均检测到少量D-脯氨酸，而D-羟基脯氨酸的含量低于定量下限。
