Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic.
J Sep Sci. 2010 Mar;33(6-7):765-72. doi: 10.1002/jssc.200900734.
Retention characteristics of ultra-HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC-fluorescence detection and UHPLC-MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin-dihydroneopterin and biopterin-dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8-7.8 within a reasonable analysis time up to 8 min for UHPLC-fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC-MS using lower concentrations of ammonium acetate.
超高效液相色谱（UHPLC）混合固定相（桥连乙基混合（BEH）和 BEH 酰胺）在亲水性相互作用色谱系统中的保留特性在极性碱性蝶呤组中进行了研究（新蝶呤、生物蝶呤、二氢新蝶呤和二氢生物蝶呤）。在 UHPLC-荧光检测和 UHPLC-MS 条件下，详细考察了流动相组成、缓冲液类型、pH 值和浓度对蝶呤保留的影响。考察了选择性、保留特性和柱性能。在任何测试条件下，BEH HILIC 都不能为四种蝶呤的分离提供足够的保留和选择性。BEH 酰胺对所有蝶呤都提供了很强的保留，特别是在 pH 值为 9.8 时。然而，在 pH 值为 9.8 时，新蝶呤-二氢新蝶呤和生物蝶呤-二氢生物蝶呤对的分离选择性大大降低，导致分析时间非常长。在 pH 值为 4.8-7.8 范围内，使用较高浓度的乙酸铵缓冲液进行 UHPLC-荧光检测，在 8 分钟内即可获得四种蝶啶衍生物的最佳分离，而使用较低浓度的乙酸铵进行 UHPLC-MS 则可在 4 分钟内获得最佳分离。
