Vitamin C is an indicator of the nutritional value of fruits and vegetables. We need an easy and reliable method for quantifying it, especially in lettuce, the most consumed leafy vegetable worldwide. This method prevents vitamin C degradation, and provides an enhanced extraction and a fast, reliable quantification of both ascorbic and dehydroascorbic acids, thanks to it's accuracy and sensitivity.
This method can be used in food technology to create nutritional fact labels, in medicine to study food effects on health and in plant breeding, to increase vitamin C content in different species. Demonstrating the procedure with Ines Medina Lozano, will be Jose Angel Aranjuelo and Arantzazu Castellanos, two analysts from my laboratory. To prepare the plant samples, harvest at least one outer and one inner leaf per plant and immediately freeze the leaves in liquid nitrogen, before placing them in minus 80 degree Celsius storage.
To lyophilize the frozen plant samples, place the uncapped sample tubes on the trays within the freeze dryer chamber of the lyophilizer, and program the lyophilizer as indicated. Maintain the condenser temperature at minus 80.2 degrees Celsius, and the vacuum constant at 112 When the material is completely dry, add 10 millimeter diameter stainless steel balls to the lyophilized samples, in 20 milliliter polypropylene tubes. And use a Multi-Tube Vortexer, at the appropriate time and intensity, to grind the samples into a fine dust.
To prepare ascorbic acid standard stock and dilution solutions, weigh exactly 10 milligrams of ascorbic acid standard on a precision balance. And at 90 milliliters of mobile phase to the sample. Use a magnetic stirrer to dissolve the sample, and use a volumetric flask to accurately measure 100 milliliters of the resulting solution.
Adding additional ultrapure water, acidified at pH two with formic acid, as necessary. Then prepare five dilutions from the stock, to obtain a calibration curve. Working under a low light intensity, add a 50 milligrams of lyophilized ground sample and five milliliters of extraction solvent, to a 15 milliliter polypropylene centrifuge tube and vortex the solution for five seconds.
After shaking the sample for 10 minutes on an orbital shaker at 2000 revolutions per minute, transfer the tube to an ultrasonic bath for 10 minutes, at room temperature and collect the sample by centrifugation. Then strain the supernatant through a 0.22 micron regenerated cellulose filter, into a five milliliter amber vial, to extract both the ascorbic and dehydroascorbic acids in extract one. To prepare the dilution for determining the ascorbic acid concentration in the leaf samples, add 200 microliters of extract one, and 800 microliters of ultrapure water into a two milliliter amber liquid chromatography vial, and close the vial with a PTFE/silicone plug.
For total ascorbic acid extraction, transfer 200 microliters of the extract one, into a two milliliter amber liquid chromatography vial, and add 200 microliters of reducing solution to the vial. Close the vial with a plug and vortex the solution for five seconds. After a 30 minute incubation at room temperature, protected from light, use 200 microliters of 0.4 Molar sulfuric acid, to stop the reaction and to stabilize the ascorbic acid, to an acidic pH.
To prepare the dilution for determining the total ascorbic acid concentration in the samples, add 400 microliters of ultrapure water, to total ascorbic acid extract two. Close the vial with a plug. At least one hour before starting the experiment, confirm that all of the working solutions have been loaded into the UPLC System and switch on the three UPLC modules.
When the internal calibration process has finished, open the software and load the instrumental program. Once the software is loaded click Quaternary Solvent Manager and click the Right Mouse Button to select Launch Console, to access the UPLC management console. Click System, Control and Startup, to proceed to the preparation and stabilization of the UPLC instrument, and click Prime Solvents, QSM, check A, B, C, D, Seal Wash and Duration of Prime, greater than five minutes, to purge all of the UPLC lines, for at least five minutes.
Click SM, check Wash Solvent greater than 45 seconds. Check Purge Solvent, and 35 cycles to purge and clean the injector. To equilibrate the UPLC to method conditions, check Equilibrate to Method and QSM, and set the flow to 0.3 milliliters per minute.
Solvent A to 2%solvent B to 0%solvent C 98%and solvent D to 0%Still in Equilibrate to Method and SM and set the sample to 5 degrees Celsius, and the column to 30 degrees Celsius. Still in Equilibrate to Method and Other, select Lamp On, and click Start. Allow the equipment to stabilize for at least an hour.
To verify the stability, click Launch Console System and QSM and select QSM System Pressure, only to check the pressure in the column. If there are no identifiable trends in the pressure changes, and the delta value is less than 10 pounds per square inch of pressure, in the quick start screen, fill the matrix with the names of the standards and samples to be analyzed. To determine the ascorbic acid concentration in the standards, inject five microliters of each dilution into the UPLC instrument, and carry out the chromatography, starting from most diluted, to the most concentrated solution as indicated in the table.
To determine the ascorbic acid and the total ascorbic acid concentration in the leaf samples, inject five microliters of the diluted extract one and extract two respectively, into the UPLC instrument for chromatographic analysis. To calculate the ascorbic acid and total ascorbic acid concentrations, in the software click Quickstart, Browse Project, Channels, the name of the standard or the sample and PDA channel one, 245 nanometers at 1.2 nanometers. To open the standard and sample chromatograms, click on the starting point of the chromatogram and drag it to the end point, to integrate the corresponding peak in the standards and samples.
Then use the data from the ascorbic acid standard dilutions, to build a calibration curve, to represent the chromatographically determined absorbance values and interpolate the absorbance values of the sample, to obtain the ascorbic acid and total ascorbic acid concentration, according to the formula. Vitamin C quantification in Lactuca matrices, requires the development of a chromatographic approach that can ensure reliable results. Here, a chromatogram resulting from a non-optimized protocol presenting and ascorbic acid peak, together with an unidentified at miner shoulder can be observed.
Nevertheless, after improving the extraction and chromatographic conditions, a resolved ascorbic acid peak without interferences of unknown compounds can be achieved. In addition, the use of UPLC ultraviolet equipment, instead of HPLC ultraviolet, reduces the retention and running times. Interferences in ascorbic acid peaks, consistently result in underestimation of the vitamin C content, due to an insufficient separation during the chromatographic process.
As the overlapping peak areas are integrated by a vertical drop at the deepest point between them. Furthermore, the use of a non-optimized protocol prevents the extraction of any useful conclusion, from the results. As all the samples appear to have a similar vitamin C content, in any of its forms.
In contrast, using the optimized protocol allows the detection of statistically significant differences between the different types of samples. Be sure to collect multiple samples, to preserve the samples correctly, to minimize sample's exposure to oxidizing agents and to quantify both forms of vitamin C.The total vitamin C content can be quantified not only in any plant species but also, in any fruit in any fruit and vegetable, or even processed food with few technique modifications.
