Measurement of ascorbic acid and dehydroascorbic acid in gastric juice by HPLC

New methods are presented for measuring total vitamin C and the ascorbic acid/dehydroascorbic acid ratio in gastric juice. Extracts are prepared from a gastric juice which are suitable for direct injection onto a Waters Nova-pak C18 Radial-pak cartridge for high performance liquid chromatography (HPLC) using ultraviolet absorbance at 270 nm for detection. Both enable removal of interfering mucus and mucopolysaccharide breakdown products in a novel way. The first uses mini-columns of Sephadex G-50, run in acidic conditions to remove large molecular weight material while maintaining the ascorbic acid/dehydroascorbic acid ratio as it was in the fresh sample. Addition of dithiothreitol converts the dehydroascorbic acid quantitatively to ascorbic acid, thus enabling measurement of both components. The second method converts all the dehydroascorbic acid to ascorbic acid at the outset. A perchloric acid extract is neutralized and passed through a Sep-Pak C18. A new internal standard, reductic acid, is introduced for ascorbic acid analysis which behaves identically on Sep-Pak C18. Samples are analysed by ion-pair chromatography using 0.02 M NH4H2PO4 buffer (pH 7.1): methanol (80:20 v/v) containing 0.62 g/L tetrapentylammonium bromide. The detection limit was 1 ng ascorbic acid, and chromatography was completed in 5 min. The values obtained by the two independent HPLC methods were in good agreement with each other and with those obtained by the 2,4-dinitrophenylhydrazine colorimetric method.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric detection

Summary A reproducible method is described for the separation and simultaneous and specific quantitation of ascorbic acid and dehydroascorbic acid by ion-pairing reversed-phase HPLC with fluorometric detection. Copper sulphate and copper acetate were compared as oxidizing reagents for ascorbic acid and 1,2-diaminobenzene dihydrochloride and 1,2-diamino-3,4-dimethylbenzene dihydrochloride as derivatising reagents. The HPLC-method was applied to human plasma. The detection limit reaches 16 ng for ascorbic acid and 3 ng for dehydroascorbic acid. Sample preparation is carried out by solid phase extraction with a recovery of 98%; it is compared with conventional precipitation of plasma proteins by metaphosphoric acid.     

Quantitative Estimation of Dehydroascorbic Acid and Ascorbic Acid by High-Performance Liquid Chromatography—Application to Human Milk,Plasma, and Leukocytes

Abstract

A ‘high-performance’ liquid chromatographic (HPLC) method for quantitation of dehydroascorbic acid and ascorbic acid and its application to protein-free human milk, blood plasma and leukocytes (buffy layer) is described. In the method, DL-homocysteine was used to convert dehydroascorbic acid quantitatively to ascorbic acid that was measured by reversed phase liquid chromatography. Fresh human milk was found to contain ascorbic acid 54.3±6.5 mg/1 (mean±SEM; n=4) and dehydroascorbic acid 21. 0±9.1 mg/1 (mean±SEM, n=4) when stored at +4°C. The concentration of both forms of ascorbic acid was found to detoriate in similar ratios during storage at +4°C, and pasteurization considerably increased the loss of vitamin C. After pasteurization the milk contained ascorbic acid 8.6±3.4 mg/1 (mean±SEM, n=4) and dehydroascorbic acid 6.6±2.4 mg/1 (mean±SEM, n=4). In plasma the dehydroascorbic acid content (0.16±0.03 mg/1, mean±SEM, n=23) was lower than that of ascorbic acid (9.96±0.75 mg/1, mean±SEM, n=23).

The ascorbic acid concentration in the leukocyte mixtures was 0.21±0.04 mg/10⁹ cells (mean±SEM, n=10) and dehydroascorbic acid concentration 0.09±0.03 mg/10⁹ cells (mean±SEM, n=8). A statistically significant (r=0.599, p<0.05) correlation was established between the concentrations of ascorbic acid in plasma and leukocytes.     

HPLC analysis with fluorometric detection of vitamin C in food samples

A high performance liquid chromatographic (HPLC) procedure has been developed for the analysis of ascorbic acid and dehydroascorbic acid in complex matrices. Separation is accomplished with an anion-exchange resin and fluorescent detection is achieved through post-column inline chemistry, involving oxidation of ascorbic acid to dehydroascorbic acid followed by reaction with o-phenylenediamine to form a fluorescent product. Lower limits of detection for both forms of vitamin C are well below the levels found in the usual food sources of this vitamin. The extraction procedures developed yield clean samples for analysis with minimal loss of the vitamers during the analytical procedures. Recoveries are in the range of 90-107%. The results obtained with this HPLC procedure agree well with those obtained with a modified version of the classical procedure of Deutsch and Weeks. A variety of foods including fruit juices, vegetables, and fruits were analyzed.     

抗坏血酸在PPy-HEImTfa/Pt电极上的电催化氧化及其机理

以铂为基底电极,在1-乙基咪唑三氟乙酸盐(HEImTfa)离子液体中电化学合成导电聚吡咯(PPy),制得PPy-HEImTfa/Pt电极;采用循环伏安法研究了PPy-HEImTfa/Pt电极对抗坏血酸的电催化氧化性能.结果表明:PPy-HEImTfa/Pt电极对0.1mo·lL-1抗坏血酸具有较高的电催化氧化活性,与相同条件下硫酸溶液中在铂表面修饰的聚吡咯(PPy-H2SO4/Pt)电极和裸铂电极相比,其氧化峰电位分别降低了0.10和0.19V,氧化峰电流分别增加了3.0和3.6mA.同时采用原位傅里叶变换红外(insitu FTIR)光谱技术对抗坏血酸在PPy-HEImTfa/Pt电极上的电氧化机理进行了研究,结果表明:抗坏血酸在PPy-HEImTfa/Pt电极上首先被氧化为脱氢抗坏血酸,在水溶液中脱氢抗坏血酸迅速发生水合作用形成水合脱氢抗坏血酸,它进一步水解并发生内酯开环反应生成2,3-二酮古洛糖酸;在较高电位下,部分抗坏血酸最终被氧化成CO2.     

Monitoring of vitamin C species in aqueous solution by flow injection analysis coupled with an on-line separation with reversed-phase column

Two vitamin C species of ascorbic acid and dehydroascorbic acid in aqueous solution were monitored by flow injection analysis. Ascorbic acid and dehydroascorbic acid were resolved by a reversed-phase column, and dehydroascorbic acid was reduced to ascorbic acid by an on-line post-column reaction with dithiothreitol. Both natural and reduced ascorbic acids were photometrically detected at 260 nm, and the two vitamin C species were simultaneously determined. The determination range was from 0 to 8 × 10⁻⁵ M with a limit of detection of 1.7 × 10⁻⁶ M. The proposed method was applied to the conversion monitoring of ascorbic acid and dehydroascorbic acid in weakly acidic to weakly alkaline aqueous solutions, as well as to the determination of the vitamin C in some beverage samples.     

Applications of a simultaneous assay of ascorbic acid, dehydroascorbic acid and ascorbic sulphate in biological materials

A modified spectrophotometric assay for ascorbic acid and its derivatives based on their reaction with 2,4-dinitrophenylhydrazine (DNPH) is described. Using standard ascorbic acid or ascorbic sulphate solutions, together with animal tissue or compound diet extracts, the conditions for ascorbic acid degradation were determined. For the differential measurement of reduced ascorbic acid (AA), dehydroascorbic acid (dAA) and ascorbic sulphate (AS), five series of simultaneous determinations were performed. These included the use of (1) KBrO3 for the hydrolysis of AS, (2) 2,6-dichlorophenolindophenol as an oxidant, (3) DNPH to form a hydrazone derivative with dAA and (4 and 5) two blanks (where ascorbate was degraded) to correct for interfering substances. A variety of vertebrate and invertebrate tissues were examined for their ascorbate content, and the advantages of the modified procedure over currently available assays are discussed. The results suggest that the Artemia cyst is a unique material in which ascorbic sulphate is present in large amounts whereas fish tissues do not contain this form of vitamin C.     

Über das Verhalten von Ascorbinsäure gegenüber Hydroxylamin,ein Beitrag zur Analytik des Vitamins C

A spectrometric method is presented for the quantitative determination of 0.5–5 mg/100 ml of ascorbic and dehydroascorbic acids. It is based on the formation of hydroxamates by reaction with hydroxylamine. The conditions and the application of this method are discussed. Colouring matter and phenolic compounds, which can present difficulty in the photometric measurement, are eliminated by treatment with polyamide powder. Furthermore, the separation of the hydroxamic acid derivatives of ascorbic and dehydroascorbic acids by thinlayer chromatography on cellulose is described.     

Determination of Vitamin C in Plasma and Dialysate from Uremia Patientsby High Performance Liquid Chromatography with Electrochemical Detection

 Abstract：Aconvenientandvalidmethodforthedeterminationofascorbicacid(AA)anddehydroascorbicacid(DHAA)inplasmaanddialysatefrompatientswithuremiabyhighperformanceliquidchromatographywithelectrochemicaldetectionisdescribedAmixtureof08g/Lmetaphosphoricacidand     

Determination of glutathione, glutathione disulphide, ascorbic acid and dehydroascorbic acid in tissues by reversed-phase liquid chromatography with electrochemical detection

Summary High-performance liquid chromatography with electrochemical detection was applied to the estimation of glutathione, glutathione disulphide, ascorbic acid and dehydroascorbic acid in various tissues of man, animal, and plant. The simultaneous determination of glutathione and ascorbic acid in tissues was done by a coulometric method. Separation of glutathione and ascorbic acid and unequivocal substance identifications were performed on a 100×4.6 mm RP-18 Spheri 5 column. As mobile phase 0.015 mol/l o-phosphoric acid, pH 2.3 was used. Retention time of ascorbic acid was 5.0 min and of glutathione 10.0 min. Dehydroascorbic acid was determined after reduction to ascorbic acid with dithiothreitol. Glutathione disulphide was reduced at pH 7.5 by -nicotinamide-dinucleotide phosphate and glutathione reductase, EC 1.6.4.2., to regenerate glutathione. To exclude interfering substances, several other compounds present in tissues and foods were investigated. This coulometric method is highly sensitive, specific and simple. Very low concentrations of ascorbic acid, glutathione, dehydroascorbic acid, and glutathione disulphide (<500 pg/injection) could be analysed using this HPLC-ECD method.(on leave to Mexico)     

Simple in-line postcolumn oxidation and derivatization for the simultaneous analysis of ascorbic and dehydroascorbic acids in foods

A new analytical procedure for the simultaneous determination of L-ascorbic acid (AA), isoascorbic acid (IAA), L-dehydroascorbic acid (DHAA), and isodehydroascorbic acid (IDHAA) in food by high-performance liquid chromatography (HPLC) is developed. After separation on an HPLC column, an in-line oxidation of AA and IAA to DHAA and IDHAA, respectively, is performed on a short column of activated charcoal. The dehydroascorbic acids are derivatized with a 1,2-phenylenediamine solution in a heated capillary Tefzel reactor into fluorescent quinoxaline compounds and monitored fluorometrically. The chromatographic method provides good separation of LAA, LDHAA, and their diastereoisomers in a relatively short time (-10 min). After optimization of postcolumn derivatization conditions, calibration runs and recovery tests are performed. The fluorescent response in terms of peak area is highly proportional to the concentration of all derivatives examined over a range of 0.1 to 100 microg/mL solution for LAA, LDHAA, IAA, and IDHAA. Recoveries were in the range of 97 to 103%. The detection limit is 0.1 mg of each ascorbic acid derivative per 100 g food. A wide variety of foods (fruits, fruit juices, vegetables, vegetable products, milk, liver, and sausage) are analyzed by the developed procedure. The Vitamin C (LAA and LDHA) contents determined according to the present analytical method are in the same order of magnitude as the result of precolumn derivatization and the fluorometric methods. The described method is a highly specific procedure for determining Vitamin C in food. It is simple to handle, only slightly susceptible to disturbance, perfectly suitable for serial determinations, and yields reproducible results.     

Electrochemical Behaviors of Ascorbic Acid and Uric Acid in Ionic Liquid

Cyclic voltammetric measurements at platinum electrode have been carried out to investigate the electrochemical oxidation of ascorbic acid and uric acid in ionic liquid, [bmim][BF₄]. It is important that a typical redox couple of ascorbic acid was obtained and it is oxidized to dehydroascorbic acid in [bmim][BF₄]. However, there is no electron-transfer for uric acid and no electrochemical oxidation carried out in the same ionic liquid. It provides a new way to eliminate the interfering between ascorbic acid and uric acid in the study of the electrochemical behaviors for them.     

Application of a New Dehydroascorbic Acid Reducing Agent in the Analysis of Vitamin C Content in Food

The analysis of total vitamin C content in food is most frequently performed by reducing dehydroascorbic acid to ascorbic acid, which is then assayed with the technique of high-performance liquid chromatography combined with spectrophotometric detection. Tris(2-carboxyethyl)phosphine is currently the only agent in use that efficiently reduces dehydroascorbic acid at pH < 2. Therefore, there is a continued need to search for new reducing agents that will display a high reactivity and stability in acidic solutions. The objective of the study was to verify the applicability of unithiol and tris(hydroxypropyl)phosphine for a reducing dehydroascorbic acid in an extraction medium with pH < 2. The conducted validation of the newly developed method of determining the total content of vitamin C using tris(hydroxypropyl)phosphine indicates its applicability for food analysis. The method allows obtaining equivalent results compared to the method based on the use of tris(2-carboxyethyl)phosphine. The low efficiency of dehydroascorbic acid reduction with the use of unithiol does not allow its application as a new reducing agent in vitamin C analysis.     

Fluorimetric determination of total ascorbic acid by a stopped-flow mixing technique

A simple, rapid and automatic fluorimetric method for the determination of total ascorbic acid is described. The method makes use of the stopped-flow mixing technique in order to achieve the rapid oxidation of ascorbic acid by dissolved oxygen to dehydroascorbic acid, which then reacts with o-phenylenediamine to form a fluorescent quinoxaline. The initial rate and fluorescence signal of this system are directly proportional to the ascorbic acid concentration. The calibration graph was linear over the range 0.1-30 microg ml(-1) (kinetic method) and 0.25-34 microg ml(-1) (equilibrium method). The precision (% RSD) was close to 0.5%. The method has been used for the determination of ascorbic acid in pharmaceutical formulations, fruit juices, soft drinks and blood serum.     

Flow injection spectrophotometric determination of ascorbic acid in soft drinks and beer

Two spectrophotometric methods, a photochemical and a non-photochemical, for the determination of ascorbic acid in soft drinks and beer using a flow-injection system are proposed. The non-photochemical method is based on the redox reaction that takes place between ascorbic acid and Fe(III), yielding dehydroascorbic acid and Fe(II). Fe(II) reacts with 1,10-phenantroline, originating the reddish orange Fe(phen)3(2+) complex (ferroin). This complex is spectrophotometrically monitored at 512 nm, and the signal is directly related to the concentration of ascorbic acid in the sample. The photochemical method has the same basis, nevertheless, uses the irradiation with visible light to enhance the redox reaction and so achieve higher sensitivities in the analysis. The non-photochemical method shows a linear range between 5 and 80 microg mL(-1), with a relative standard deviation of 1.6% (n = 11), a detection limit of 2.7 microg mL(-1) and a sample throughput of 60 samples h(-1). The photochemical method shows a linear range between 1 and 80 microg mL(-1), with a relative standard deviation of 1.0% (n = 11 ), a detection limit of 0.5 microg mL(-1) and a sample throughput of 40 samples h(-1).     

Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables

A liquid chromatographic method has been described for the determination of total vitamin C, ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables. The complete separation of AA and DHAA could be achieved on a C18 column using 0.2 M KH2PO4 (pH adjusted to 2.4 with H3PO4) as the mobile phase at a flow-rate of 0.5 ml/min. Since the detection sensitivity was poor for DHAA even at 210 nm, it was estimated as the difference between the total AA after DHAA reduction and AA content of the original sample, using dithiothreitol (DTT) as the precolumn reductant. The reaction times for the complete conversion of DHAA to AA at room temperatures were 150, 120, 90 and 75 min for 1, 2, 4 and 8 mmol DTT per mmol of DHAA, respectively. The percentage recovery ranged from 81.7 to 105.9. AA contents of some selected fruits and vegetables were analyzed comparatively by liquid chromatography and enzymatic assay to validate the method.     

Atomic absorption spectrometry for the automatic indirect determination of ascorbic acid based on-the reduction of manganese dioxide.

A new and simple flow injection method followed by atomic absorption spectrometry has been developed for the indirect determination of ascorbic acid. The proposed method is based on oxidation of ascorbic acid to dehydroascorbic acid using a solid-phase manganese dioxide (30% m/m suspended on silica gel beads) reactor. The flow of the sample through the column reduces the MnO2 to Mn(II) in an acidic carrier stream of 6.3 mM HNO3 (pH 2.2) with flow rate of 4.0 ml/min at room temperature; Mn(II) is measured by atomic absorption spectrometry. The absorbance of Mn(II) is proportional to the concentration of ascorbic acid in the sample. The calibration curve was linear up to 30 mg/L, with a detection limit of 0.2 mg/L for a 220 microL injected sample volume. The developed procedure was found to be suitable for the determination of AsA in pharmaceuticals and foods with a relative standard deviation better than 1.09% and a sampling rate of about 95 h(-1). The results exhibit no interference from the presence of large amounts of organic compounds. The reliability of the method was established by parallel determination against the 2,6-dichlorophenol-indophenol methods.     

Sensitive spectrophotometric determination of ascorbic acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP)

A simple and very sensitive method has been developed for the determination of ascorbic acid based on the oxidation of ascorbic acid to dehydroascorbic acid by iron(III), followed by a complexation of iron(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol(Br-PADAP). The iron(II) complex is formed immediately, with absorption maxima at 560 and 748 nm and a molar absorptivity of 1.31 × 10⁵ l mole^–1cm^–1 and 5.69 × 10⁴ l mole^–1cm^–1, respectively. The ascorbic acid determination is possible with a linear range up to 2.4 μg ml^–1, a calibration sensitivity of 0.744 ml μg^–1 at 560 nm and 0.323 ml μg^–1 at 748 nm, and a detection limit of 15 ng ml^–1 and 44 ng ml^–1, respectively. The procedure was used for the ascorbic acid determination in several fruit juices and pharmaceutical formulations. The results demonstrated a good precision (R.S.D. < 1%) and are in agreement with those obtained with others methods. The Br-PADAP method proposed is six times more sensitive than the method using the iron(II)-1,10-phenanthroline system. Received: 7 May 1996 / Revised: 1 July 1996 / Accepted: 8 August 1996     

Sensitive spectrophotometric determination of ascorbic acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP)

A simple and very sensitive method has been developed for the determination of ascorbic acid based on the oxidation of ascorbic acid to dehydroascorbic acid by iron(III), followed by a complexation of iron(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol(Br-PADAP). The iron(II) complex is formed immediately, with absorption maxima at 560 and 748 nm and a molar absorptivity of 1.31 × 10⁵ l mole^–1cm^–1 and 5.69 × 10⁴ l mole^–1cm^–1, respectively. The ascorbic acid determination is possible with a linear range up to 2.4 μg ml^–1, a calibration sensitivity of 0.744 ml μg^–1 at 560 nm and 0.323 ml μg^–1 at 748 nm, and a detection limit of 15 ng ml^–1 and 44 ng ml^–1, respectively. The procedure was used for the ascorbic acid determination in several fruit juices and pharmaceutical formulations. The results demonstrated a good precision (R.S.D. < 1%) and are in agreement with those obtained with others methods. The Br-PADAP method proposed is six times more sensitive than the method using the iron(II)-1,10-phenanthroline system. Received: 7 May 1996 / Revised: 1 July 1996 / Accepted: 8 August 1996     

Anion-exchange high-performance liquid chromatographic determination of ascorbic acid and hexavalent chromium in rat lung preparations after treatment with sodium chromate in vitro and in vivo

Simultaneous analysis of ascorbic acid and chromium (VI) in soluble fractions and bronchoalveolar lavage fluids of rat lungs treated with sodium chromate in vitro and in vivo was performed by anion-exchange high-performance liquid chromatography coupled to a photodiode-array detector. Absorbances at 265 and 370 nm were used for the determination of ascorbic acid and chromium (VI), respectively. The calibration graphs of standard solutions were linear in the test ranges of ascorbic acid an chromium (VI) (below 10 and 8 ppm, respectively). The detection limits of ascorbic acid and chromium (VI) were 1 and 0.5 ng, respectively. The recovery of ascorbic acid from lung tissues homogenized at pH 7.4 was 99%, and that of chromium (VI) was 96%, when tissues were homogenized under alkaline conditions (pH 11.4). Using this method, ascorbic acid levels in the soluble fractions and lavage fluids of normal rat lungs were determined. In the lung of a rat intratracheally injected with a saline solution of sodium chromate, ascorbic acid decreased to 80% of the normal level, and ca. 90% of the chromium (VI) was reduced within 4 min after injection, indicating that the ascorbic acid-related reduction of chromium (VI) is very rapid. The present method will be useful for studies of the reduction of chromium (VI) by ascorbic acid in biological systems.