Department of Food Science and Technology, Food Chemistry Laboratory, BOKU - University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190, Vienna, Austria,
Anal Bioanal Chem. 2013 Oct;405(25):8053-61. doi: 10.1007/s00216-013-6974-2. Epub 2013 Apr 25.
A rapid ultra-high performance liquid chromatography (UHPLC) protocol for the determination of amino acids as their respective 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives was successfully applied for assessing free amino acid levels in commercial cheese samples representing typical product groups (ripening protocols) in cheesemaking. Based on the Waters AccQ.Tag™ method as a high performance liquid chromatography (HPLC) amino acid solution designed for hydrolyzate analyses, method adaptation onto UHPLC was performed, and detection of AQC derivatives was changed from former fluorescence (λ(Ex) 250 nm/λ(Em) 395 nm) to UV (254 nm). Compared to the original HPLC method, UHPLC proved to be superior by facilitating excellent separations of 18 amino acids within 12 min only, thus demonstrating significantly shortened runtimes (>35 min for HPLC) while retaining the original separation chemistry and amino acid elution pattern. Free amino acid levels of the analyzed cheese samples showed a high extent of variability depending on the cheese type, with highest total amounts found for original Italian extra-hard cheeses (up to 9,000 mg/100 g) and lowest for surface mold- or bacterial smear-ripened soft cheeses (200-600 mg/100 g). Despite the intrinsic variability in both total and specific concentrations, the established UHPLC method enabled reliable and interference-free amino acid profiling throughout all cheese types, thus demonstrating a valuable tool to generate high quality data for the characterization of cheese ripening.
成功应用一种快速超高效液相色谱（UHPLC）方案，将氨基酸分别衍生为 6-氨基喹啉基-N-羟基琥珀酰亚胺基碳酸酯（AQC）衍生物，用于评估代表干酪制作中典型产品组（成熟方案）的商业干酪样品中的游离氨基酸水平。以 Waters AccQ.Tag™方法（一种用于水解物分析的高效液相色谱（HPLC）氨基酸溶液）为基础，对方法进行 UHPLC 适应性改造，将 AQC 衍生物的检测由以前的荧光（λ(Ex) 250nm/λ(Em) 395nm）改为紫外（254nm）。与原始 HPLC 方法相比，UHPLC 仅在 12 分钟内即可实现 18 种氨基酸的出色分离，从而证明其具有明显更短的运行时间（HPLC 为>35 分钟），同时保留了原始分离化学和氨基酸洗脱模式。所分析干酪样品的游离氨基酸水平具有高度的可变性，取决于干酪类型，原始意大利硬质干酪（高达 9,000mg/100g）的总含量最高，表面霉菌或细菌擦生软干酪（200-600mg/100g）的总含量最低。尽管总浓度和特定浓度存在内在变异性，但建立的 UHPLC 方法能够在所有干酪类型中可靠且无干扰地进行氨基酸分析，从而为干酪成熟的特征描述生成高质量数据提供了有价值的工具。
