Fluorescence ascorbic acid determination

Schmid et al. used the same principle to develop sensors to be incorporated into FI systems for the determination of ascorbic acid in fruit juices [38] and that of lactic acid in dairy products [39]. The membrane used in both applications consisted of decacyclene dissolved in silicone rubber that was treated similarly as the membrane in glucose sensors (Fig. 3.4.B). The oxygen optrode was coated with a sheet of carbon black as optical insulation in order to protect it from ambient light or intrinsic sample fluorescence. Ascorbic acid oxidase or lactic acid oxidase was immobilized by adsorbing it onto carbon black and cross-linking it with glutaraldehyde. The FI system automatically buffered and diluted the food samples, thereby protecting the biosensor from a low pH and interferents. 

Chromatographic methods, notably hplc, are available for the simultaneous determination of ascorbic acid as well as dehydroascorbic acid. Some of these methods result in the separation of ascorbic acid from its isomers, eg, erythorbic acid and oxidation products such as cHketogulonic acid. Detection has been by fluorescence, uv absorption, or electrochemical methods (83—85). Polarographic methods have been used because of flieti accuracy and their ease of operation. Ion exclusion (86) and ion suppression (87) chromatography methods have recently been reported. Other methods for ascorbic acid determination include enzymatic, spectroscopic, paper, thin layer, and gas chromatographic methods. Excellent reviews of these methods have been published (73,88,89). 

FIGURE 10.13 The TLC profiles of labeled peaks isolated from [U- C]ascorbic-acid-modified calf lens protein obtained from Bio-Gel P-2 chromatography. Peaks 2 to 7 were spotted on a preparative silica gel TLC plate and developed with ethanol/ammonia (7 3, v/v). The fluorescence in each lane was detected by irradiation with a Wood s lamp at 360 nm, and the pattern of radioactivity was determined by scanning the plate with AMBIS imaging system. (Reprinted with permission from Cheng, R. et al., Biochim. Biophys. Acta, 1537, 14-26, 2001. Copyright (2001) Elsevier.)... 

Separation, identification and determination of ascorbic acid, ascorbyl palmitate and sodium ascorbate were performed by TLC on layers of Kieselgel HF254. The determination was based on the UV absorption of the compounds and on the fluorescence of the layer. When the layer is excited with UV radiation of 254 xm, the fluorescence is quenched in the zones corresponding to the UV absorbing compounds. A direct measure of the concentration of the separated substances is therefore possible. ... 

Amplihcation factors of 8 to 12 were claimed for the determination of phenol in a FLA system by a cyclic process depicted in equations 5 (Section VI.A. 1). Phenol is converted to o-benzoquinone in contact with immobilized tyrosinase held in a fixed bed reactor the quinone reacts with ascorbic acid (91) to yield catechol and dehydroascorbic acid (136) catechol can be enzymatically oxidized again to o-benzoquinone and so forth. The accumulated dehydroascorbic acid forms with o-phenylenediamine (137) a highly fluorescent product (kex 345 nm, kfl 410 nm). LOD was ca 0.02 (xM for phenol and catechol the linear range for phenol was 0.1 to 2 p,M and for catechol 0.02 to 2... 

NACE with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (p/linear plot -log[(mu 0/mu) - 1] versus pH, using 20% isopropanol and 40% ACN as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 MC540) asfluorophores, has been performed for the analysis of AA and its stereoisomer, isoascorbic acid (lAA), and the limits of detection of AA and lAA are 0.30 and 0.17 pM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with lAAasthe internal standard in less than three minutes, and its concentration is 76.7 + 0.4 mM. 

An alternative approach is to utilise the fluorescence which is exhibited by the product of the condensation ofdehydroascorbic acid with o-phenylenediamine. The quinoxaline produced fluoresces at 427 nm when irradiated at 350 nm. Generally the procedure consists of oxidising any L-ascorbic acid present to dehydroascorbic acid, so that the total may be determined spectrofluorimetrically. 

As shown in Fig. 1.16a, upon addition of CATl the fluorescence of P6 is efficiently quenched. The ( )-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) is added and allowed to equilibrate for 35 min, which reverses the quenching due to the scavenging of nitroxide radicals via hydrogen transfer from trolox to CATl. The fluorescence recovery depends on the concentration of trolox (Fig. 1.16b). The trolox can be detected in the range of 10 100 pM. The same probe can also be used to detect the capabilities of a variety of antioxidants. Sensing for ascorbic acid can be accomplished with high sensitivity and selectivity because of its excellent antioxidant capabilities. The ascorbic acid concentration can be determined in the 50 nM to 200 pM range (Fig. 1.17a). Control experiments were also done with a nonspecific quencher. A , A -dimethyl-d, 4 -bipyridinium (MV +) for ascorbic acid. As shown in Fig. 1.17b, upon addition of MV +, the fluorescence of P6 is... 

Total ascorbic and dehydroascorbic acid in human serum has been determined by Iodine oxidation followed by reaction of dehydroascorbic acid with l,2-diamino-4,5-dimethoxy-benzene the Intensely fluorescent product permitted detection down to 5 ng of ascorbic acid with samples as small as 4 yl of serum. 

Vitamins are the foodstuff components most often quantified using fluorimetric means. There are several official fluorimetric methods for the determination of three water-soluble vitamins vitamin Bi (thiamine) (AOAC 942.23 and 957.17), B2 (rib-oflavine) (AOAC 970.65 and 981.15), and C (ascorbic acid) (AOAC 984.26). Thiamine is determined by oxidation to fluorescent thiochrome with alkaline hexacyanoferrate(III) or an alternative oxidant (Figure 1). The method is quite simple, reproducible, and selective and provides good recoveries. Many LC methods for thiamine determination in foods have... 

Vitamin C status can be assessed by measuring plasma levels or urinary excretion. However, due to practical disadvantages, e.g., quantitative sampling of urine and instability of vitamin C, determination of vitamin C in urine has been mainly replaced by determination of plasma levels. Methods include direct determination by FIPLC, or automated assays based on a derivatization of ascorbic acid forming colored or fluorescent derivatives. While a plasma concentration <11.4p.molH is widely used to characterize deficiency, literature data signifying adequate status vary from >17 to 28.4p.moll. ... 

Various carbonyl compounds and decomposition products of methyl linolenate hydroperoxides were tested for their interaction with DNA by measuring fluorescence in the presence of ferric chloride and ascorbic acid. 2,4-Alkadienals and 2,4,7-decatrienals were among the most active decomposition products of linolenate hydroperoxides (Table 5.8). To determine the type of reactive species involved in fluorescence formation with DNA, the effect of free radical antioxidants and a singlet oxygen quencher were examined. )3-Carotene, a-tocopherol and phenolic antioxidants strongly inhibited DNA fluorescence formed by decomposition of linolenate hydroperoxides in the presence of ferric chloride and ascorbic acid (Table 5.9). These results indicate that singlet oxygen and free radical species are important intermediates in the interaction of linolenate hydroperoxides with DNA in the presence of iron and ascorbic acid. 

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. 

A reliable simultaneous reversed-phase h.p.l.c. assay for ascorbic acid and dehydroascorbic acid in biological systems utilized sequential elecmx hemical and u.v. detection. The determination of these acids in dairy foods was achieved by derivatization with 4-methoxy- or 4-ethoxy-1,2-phenylenediamine to form quinoxaline derivatives, reversed-phase chromatography and fluorescence detection. Detection limits were 50 and 70 fmol per 5 pi injection, respectively. Simultaneous determination of ascorbic acid and sulphite in beer was achieved using a strong acid resin colunui, acid eluant, and pulsed amperometric detection. ... 

Vitamin C is another soluble vitamin frequently determined in foodstuffs using fluorimetric detection. It is most often oxidized to dehydroascorbic acid with HgCl2 or 2,6-dichloroindophenol, which is subsequently reacted with p-phenylenediamine to form a fluorescent quinoxaline derivative (Figure 1). The method can be implemented in an automated fashion. The determination of total vitamin C (ascorbic and dehydroascorbic acid) in foods (vegetables and fruits) is also of great interest. The recommended method uses reversed-phase ion-pair LC and fluorimetric detection. Total vitamin C and total isovitamin C (erythorbic and dehydroerythorbic acids) can be determined similarly by prior extraction with trichloroacetic acid and precolumn derivatization, wherein ascorbic and erythorbic acids are oxidized enzymatically and total dehydroascorbic and dehydroerythorbic acids are treated with o-phenyl-enediamine. 

MS Ah, ET Phillippo. Simultaneous determination of ascorbic, dehydroascorbic, isoascorbic and dehydroascorbic acids in meat-based food products by liquid chromatography with postcolumn fluorescence detection a method extension. J AOAC Int 79 803-808, 1996. 

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