Medina-Lozano Inés, Bertolín Juan Ramón, Zufiaurre Raquel, Díaz Aurora
Unidad de Hortofruticultura, Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA); Instituto Agroalimentario de Aragón - IA2 (CITA-Universidad de Zaragoza).
Unidad de Producción y Sanidad Animal, Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA); Instituto Agroalimentario de Aragón - IA2 (CITA-Universidad de Zaragoza).
J Vis Exp. 2020 Jun 30(160). doi: 10.3791/61440.
Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized. Samples were collected to represent the entire plant, frozen at -80 °C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.
维生素，尤其是维生素C，是存在于水果和蔬菜中的重要微量营养素。维生素C也是它们抗氧化能力的主要贡献者。生菜是全球消费者最喜爱的蔬菜之一。准确测定生菜及其他相关物种中维生素C含量的方法至关重要。我们在此描述一种使用超高效液相色谱 - 紫外（UPLC - UV）技术的方法，其中对样品制备、维生素提取和色谱条件进行了优化。采集样品以代表整株植物，在 -80°C下冷冻并冻干，以防止不必要的氧化并便于操作。维生素C的提取在酸性介质中进行，这也有助于其稳定性。由于维生素C可以以两种不同的可相互转化形式存在，即抗坏血酸（AA）和脱氢抗坏血酸（DHAA），为了准确定量，这两种化合物都应进行测定。DHAA在还原为AA后间接定量，因为在光谱的紫外范围内，AA的吸光度高于DHAA。从同一提取物中进行两次测量，一次在还原反应之前，一次在还原反应之后。在第一种情况下，我们定量AA含量，在第二种情况下，我们以AA的形式定量AA和DHAA的总和（TAA：总抗坏血酸）。然后，通过从TAA中减去第一次测量得到的AA来间接获得DHAA的量。它们通过UPLC - UV测定，使用市售的AA标准品建立校准曲线并优化色谱程序，以在短时间内获得完全分离的AA峰。该方法稍作改动或无需改动就能轻松外推至任何其他植物材料。其准确性揭示了否则难以察觉的统计学显著差异。本文稿更深入地讨论了其他优点和局限性。
