Ascorbic acid, determination

Substances easily oxidized and indirectly determined (ascorbic, acid, carboxylic acids, amines, etc.)... 

LOZANO A, ALEGRIA A, BARBERA R, FARRE R and LARGARDA M J (1998), Evaluation of methods for determining ascorbic acid, citric acid, lactose and maltodextrins in infant juices and formulas , Alimentaria, 298, 95-99. 

L. A. Pachla, D. L. Reynolds, and P. T. Kissinger, Analytical methods for determining ascorbic acid biological samples, food products, and pharmaceuticals, J. Assoc. Off. Anal. Chem., 68 1 (1985). 

TLC has been widely used to determine ascorbic acid concentrations in foods,pharmaceutical preparations,and biological materials.Isomers of ascorbic acid and their oxidation product, dehydroascor-bic acid, were separated by TLC on sodium borate-impregnated silica gel and cellulose plates. This TLC method has been adapted to separate and identify ascorbic acid and dehydroascorbic acid in fresh orange and lime juices, pharmaceutical preparations (ascorbic acid), and guinea pig tissues (liver, kidney, and eye lens) and fluids (plasma and urine). 

At this time, the selection of the method for determining ascorbic acid content requires some knowledge of the forms of vitamin C likely to be present in a given food product, the number of assays to be performed, and the spectrum of foods being assayed. In addition, the presence of interfering substances must be assessed. 

Challenge Problem. Verdini and Lagier developed an iodimetric titration procedure for determining ascorbic acid in vegetables and fruits. They compared the results of their titration experiments with similar results from an HPLC method... 

These workers also carried out a recovery study in which they determined ascorbic acid in samples, then spiked the samples with additional ascorbic acid and redetermined the mass of the analyte. Their results are shown in the following table. 

Applications. The proposed method was applied to determine ascorbic acid in a sample of Yinqiao vitamin C tablets (0.0500 g ground up tablets (20) dissolved in water and filtered and diluted to 500 mL). The recovery rates of the method is 96.6% and 96.3 % RSD was < 3.00% which indicated the results were quantitative and /-tests assumes there was no significant differences between recovery efficiency and 100 % at confidence level of 95 %. 

HPLC with UV-based diode array detection (DAD-UV) or electrochemical detection is normally used to determine ascorbic acid. Many types of electrochemical determinations of ascorbic acid have been proposed. Although the electrochemical determinations using enzyme-based biosensors exhibited high specificity and sensitivity, these methods suffer in the fabrication of the electrodes and in automatic analysis. Recently, chemically modified screen-printed electrodes have been constructed for the determination of ascorbic acid. This is one of the most promising routes for mass production of inexpensive, reproducible, and reliable electrochemical sensors. 

TLC has been used to determine ascorbic acid in foods, pharmaceutical preparations, and biological fluids. Paper chromatography was used to separate ascorbic and dehydroascorbic acid in plant extracts, and the spots were visualized using 2,5-dichlorophenol indophenol (Bui-Nguyen, 1985). This procedure is probably applicable to TLC on cellulose. 

Esteve et al. described a method for determining ascorbic acid from blood plasma and serum at the wavelength of 254 nm (Fig. 5). They used 4-hydroxya-cetanilide as an internal standard and deproteinized sample with 10% MPA (1 + 1). The sensitivity of the detection was 31 ng/mL. They tested the possible effect of hemolysis on the chromatogram and found no interference with the ascorbic acid peak (77). Tanishima and Kita used the wavelength of 265 nm that was also suitable for detecting maleic acid that was added as internal standard for ascorbic acid extraction and chromatography (74). 

In addition to ascorbic acid, uric acid is an important antioxidant in blood plasma (9). To assess the antioxidative capacity of plasma it is useful to determine ascorbic acid and uric acid in a single chromatographic run. Ascorbic acid and uric acid have been measured simultaneously from heparinized plasma by UV detection (64). To optimize the detection during method setup, the sample was dissolved in the mobile phase, pH 5.5, where ascorbic acid had the absorbance maximum at 262 nm and uric acid at 285 nm. The wavelength of 262 nm was chosen because ascorbic acid is present at a lower concentration than uric acid in plasma. The same mobile-phase pH (5.5) and UV detection at 280 nm for... 

Combined UV and electrochemical detection was used for determining ascorbic acid and DHA in small animal and brain tissues. Ascorbic acid was detected amperometrically and DHA at the wavelength of 215 nm in a single run. However, quantitative measurement of DHA might be difficult with this method, because of several interfering compounds absorbing at this wavelength (86). 

Sodium hydroxide together with sodium borohydride were used for oxidizing ascorbic acid and DHA to products that have the absorbance maximum at higher wavelength than the native compounds. The derivatives could not be identified due to their unstable chemical characters, but their spectra were identical. The method was adapted for determining ascorbic acid and DHA in tomato juice (91) and in fish tissues (45). 

Ktnetec (48) determined ascorbic acid, DHA, acetylsalicylic acid, and its degradation product, salicylic acid, from pharmaceuticals. He used UV detection for the measurement of ascorbic acid, acetylsalicylic acid, and fluorescence detection with the exitation wavelength set at 350 nm and emission wavelength set at 430 nm for measurement of the quinoxaline derivative of o-phenylenediamine and DHA. The o-phenylenediamine reagent was eluted as an unretained compound in the amino column thus causing no interference with other compounds in either UV or fluorescence detection. The detection limit was about 0.002 mg/ mL of sample, the injection volume was 20 pL. 

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