Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)
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Date
2020
Authors
Medina Lozano, InésBertolín Pardos, Juan Ramón
Zufiaurre, Raquel
Díaz Bermúdez, Aurora
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Abstract
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.
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Bibliographic citation
JoVE (Journal of Visualized Experiments), num. 160, (2020)
AGROVOC subjects
LactucaLactuca sativa
Valor nutritivo
Vitamina C
Técnicas analíticas
Sponsorship
This work was funded by the projects RTA2017-00093-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and LMP164_18 from the Government of Aragón; and by the Operational Programme FEDER Aragón 2014-2020 and the European Social Fund from the European Union [Grupos Consolidados A12-17R: “Grupo de investigación en fruticultura: caracterización, adaptación y mejora genéica” and A14-17R: “Sistemas agroganaderos alimentarios sostenibles” (SAGAS)]. I. M.L. was supported by a predoctoral contract for training doctors from the Spanish Ministry of Science, Innovation and Universities (MCIU) and the Spanish State Research Agency (AEI).