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.     

Determination of ascorbic acid, dehydroascorbic acid, and isoascorbic acid in blood

Summary High-performance liquid chromatography with UV-detection at 254 nm was used for determining ascorbic acid, dehydroascorbic acid, and isoascorbic acid in plasma and erythrocytes obtained from healthy volunteers and from patients with coronary infarct, acute lymphatic leukemia (ALL), and acute myeloic leukemia (AML). Deproteinisation was done by a simple ultrafiltration technique. Dehydroascorbic acid (DHA) was determined after reduction to ascorbic acid with dithiothreitol. The separation system chosen allows the search for the stereoisomer isoascorbic acid. To exclude interfering substances which could coelute with ascorbic or isoascorbic acid, several other compounds present in plasma were investigated including e.g. the aromatic amino acids, glutathione, cysteine, and uric acid. The results reveal a drastic decrease in total vitamin C concentration in plasma and erythrocytes with a concomitant increase in rel. DHA concentration in patients with AML. In ALL patients, both the total vitamin C and rel. DHA concentrations were increased.

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Bestimmung von Ascorbinsäure, Dehydroascorbinsäure und Isoascorbinsäure in Blut

Zusammenfassung High-performance-liquid-Chromatographie mit UV-Nachweis bei 254 nm wurde zur Bestimmung von Ascorbinsäure, Dehydroascorbinsäure und Isoascorbinsäure in Plasma und Erythrocyten von gesunden Probanden und von Patienten mit Herzinfarkt, akuter lymphatischer Leukämie (ALL) und akuter myeloischer Leukämie (AML) benutzt. Deproteinisierung erfolgte mittels einer einfachen Ultrafiltrationstechnik. Dehydroascorbinsäure wurde nach Reduktion mit Dithiothreitol als Ascorbinsäure bestimmt. Die benutzte Methode erlaubt auch die Bestimmung des Stereoisomers Isoascorbinsäure. Der mögliche Einfluß von im Plasma vorkommenden Substanzen auf die Bestimmung des Vitamin C wurde untersucht. Die Ergebnisse zeigen eine drastische Abnahme der Gesamt-Vitamin C-Konzentration in Plasma und Erythrocyten mit einer parallelen Zunahme der relativen DHA-Konzentration in AML-Patienten. In ALL-Patienten sind sowohl die Gesamt-Vitamin C- als auch die relative DHA-Konzentrationen erhöht.

     

Amperometric microsensor for direct probing of ascorbic acid in human gastric juice

This article reports on a novel microsensor for amperometric measurement of ascorbic acid (AA) under acidic conditions (pH 2) based on a carbon fiber microelectrode (CFME) modified with nickel oxide and ruthenium hexacyanoferrate (NiO-RuHCF). This sensing layer was deposited electrochemically in a two-step procedure involving an initial galvanostatic NiO deposition followed by a potentiodynamic RuHCF deposition from solutions containing the precursor salts. Several important parameters were examined to characterize and optimize the NiO-RuHCF sensing layer with respect to its current response to AA by using cyclic voltammetry, and scanning electron microscopy-energy dispersive X-ray spectroscopy methods. With the NiO-RuHCF coated CFME, the AA oxidation potential under acidic conditions was shifted to a less positive value for about 0.2 V (E_p of ca. 0.23 V vs. Ag/AgCl) as compared to a bare CFME, which greatly improves the electrochemical selectivity. Using the hydrodynamic amperometry mode, the current vs. AA concentration in 0.01 M HCl, at a selected operating potential of 0.30 V, was found to be linear over a wide range of 10-1610 μM (n = 22, r = 0.999) with a calculated limit of detection of 1.0 μM. The measurement repeatability was satisfactory with a relative standard deviation (r.s.d.) ranging from 4% to 5% (n = 6), depending on the AA concentration, and with a sensor-to-sensor reproducibility (r.s.d.) of 6.9% at 100 μM AA. The long-term reproducibility, using the same microsensor for 112 consecutive measurements of 20 μM AA over 11 h of periodic probing sets over 4 days, was 16.1% r.s.d., thus showing very good stability at low AA levels and suitability for use over a prolonged period of time. Moreover, using the proposed microsensor, additionally coated with a protective cellulose acetate membrane, the calibration plot obtained in the extremely complex matrix of real undiluted gastric juice was linear from 10 to 520 μM (n = 14, r = 0.998). These results demonstrated the unique featuring of the proposed NiO-RuHCF microsensor under acidic conditions with enhanced sensitivity and stability and proved its promising potentiality for direct amperometric probing of AA at physiological levels in real gastric juice environments.     

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.     

New method for the spectrophotometric determinations of ascorbic acid and dehydroascorbic acid

Summary The condensation of dehydroascorbic acid with o-phenylene diamine or its 4,5-dimethyl derivative yields quinoxaline derivatives which can be measured by photometry. Ascorbic acid can be determined after preliminary oxidation with mercury(II) chloride in the same way. This highly specific method has been employed for determinations in pharmaceutical formulations (coefficient of variation ± 1–1.5%).

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Neue spektrophotometrische Methode zur Bestimmung von Ascorbinsäure und Dehydroascorbinsäure

Zusammenfassung Die Kondensation von Dehydroascorbinsäure mit o-Phenylendiamin oder 4,5-Dimethyl-o-phenylendiamin führt zur Bildung von spektrophotometrisch auswertbaren Chinoxalinderivaten. Ascorbinsäure kann nach vorangehender Oxidation mit Quecksilberchlorid auf die gleiche Weise bestimmt werden. Die Methode wurde zu Bestimmungen in pharmazeutischen Präparaten verwendet (Variationskoeffizient ± 1–1,5%).

The author thanks Mrs. S.V. Ramakrishnan and Miss K. Mihályfi for their technical assistance.     

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)     

Rapid determination of ascorbic acid, dehydroascorbic acid, and total vitamin C by electrochemiluminescence with a thin-layer electrochemical cell

This paper reports on a rapid and sensitive method for the simultaneous determination of ascorbic acid (H₂A), dehydroascorbic acid (DHA), and total vitamin C by electrochemiluminescence (ECL) using a thin-layer electrochemical cell. Significant ECL signals can be generated by the anodic oxidation of Ru(bpy)₃ ²⁺ in the presence of H₂A or DHA in pH 8.8 phosphate buffer solution. Because of the extremely small dead volume of the thin-layer cell (approximately 1.5 μL), almost all amount of H₂A is assumed to be completely oxidized to DHA with a short pre-electrolysis step. As a result, it is possible to determine the reductive vitamin C (H₂A) by square wave voltammetry before the pre-electrolysis step, while total vitamin C (sum of H₂A and DHA) is able to be determined at a subsequent ECL step. The method was employed for the determination of vitamin C in commercial beverages with the analytical results in good agreement with the certified values.

Rapid and sensitive determination of dehydroascorbic acid in addition to ascorbic acid by reversed-phase high-performance liquid chromatography using a post-column reduction system

An improved procedure for the direct determination of l-dehydroascorbic acid (DHAA), in addition to l-ascorbic acid (AA), has been developed. The two biologically active forms of vitamin C were separated using reversed-phase high-performance liquid chromatography. DHAA was reduced to AA with dithiothreitol (DTT) in a post-column reaction system. Complete conversion was achieved at 50 degrees C using a 1-ml reaction coil. Recoveries were in the range of 95-99% and both forms could be detected spectrophotometrically at 267 nm with high sensitivity. Reproducible results [relative standard deviations 2.4% (DHAA) and 1.0% (AA), n = 5] were obtained when the method was applied to the analysis of rose hip samples.     

Routine analysis of ascorbic acid in citrus juice using capillary electrophoresis.

A procedure to monitor citrus juice samples was established to quantitate vitamin C by capillary electrophoresis using a previously developed method. Dilution and filtration were the only preparation requirements and separation was achieved with an uncoated capillary using a 35mM sodium borate buffer (pH 9.3) containing 5% (v/v) acetonitrile at 21 kV and 23 degrees C. Detection was performed by high speed scanning between 200 and 360 nm. From the multiwave length scan, the electropherogram at 270 nm was extracted and used to quantitate ascorbic acid. The ascorbic acid concentration was calculated with an internal standard method, with ferulic acid as internal standard. The level of ascorbic acid during analysis was stabilized with ethylenediaminetetraacetic acid and dithiothreitol was used to reduce dehydroascorbic acid to ascorbic acid to estimate the total vitamin C level. Results were similar to those obtained by liquid chromatography and the method is now used to determine routinely the level of ascorbic acid in citrus juices.     

Assay for both ascorbic and dehydroascorbic acid in dairy foods by high-performance liquid chromatography using precolumn derivatization with methoxy- and ethoxy-1,2-phenylenediamine

A procedure for the simultaneous determination of both ascorbic and dehydroascorbic acid in dairy foods by high-performance liquid chromatography using precolumn derivatization with 4-methoxy- and 4-ethoxy-1,2-phenylenediamine is presented. The derivatives are isolated by solid-phase extraction and analysed by fluorescence detection on a resin-type reversed-phase column at pH 9. Retention times are 2 and 3.2 min for the derivatives of ascorbic and dehydroascorbic acid, respectively. Relative standard deviations of the within- and between-assay tests are 7.1 and 5.5%, respectively, for ascorbic and 11 and 9%, respectively, dehydroascorbic acid. The limits of detection are 50 and 70 fmol per 5-microl injection for ascorbic and dehydroascorbic acid, respectively.     

Determination of bile acids and lysolecithin in artificial and natural gastric juice by HPLC for characterization of antacida

Work-up procedures and HPLC separation systems for determination of taurocholic, glycocholic, chenodeoxycholic, and cholic acids and lysolecithin in artificial and natural gastric juice are described. These compounds are used for testing the binding capacity of antacida to the individual analytes. Work-up is simple, no extraction or filtration being required. The optimized combinations of stationary and mobile phases allow selective and sensitive determination of the respective bile acids in gastric juice. For optimization, the capacity factors of two related bile acids were evaluated for numerous commercial stationary phases. The mechanism of retention is different for free and conjugated bile acids. Special aspects of routine analysis are discussed.     

Measurement of ascorbic acid in single human neutrophils by capillary zone electrophoresis with electrochemical detection

A method has been developed for the determination of ascorbic acid (AA) in individual human neutrophils by capillary zone electrophoresis with electrochemical detection at a carbon fiber microdisk bundle electrode. The natively easily oxidized substances such as glutathione, dopa, dopamine, serotonin, epinephrine do not interfere with the determination of ascorbic acid. A procedure of treating capillaries, which can overcome the influence of the adsorption of the substances in cells on the inner surface wall of the capillary on the migration time and the number of theoretical plates of interests, has been described. The average amount of AA in an individual neutrophil is 0.557 fmol, which is consistent with the literature value.     

Voltammetric determination of 5-hydroxydole-3-acetic acid in human gastric juice

A method for the determination of the major serotonin metabolite — 5-hydroxyindole-3-acetic acid (5-HIAA) in human gastric juice by cyclic voltammetry was described. The measurement conditions were investigated. The potential window was chosen from +0.1 to +0.9 V, the supporting electrolyte was 0.025 M PBS solution (pH 2.0). The method allowed determination in the concentration range from 2.0 ×10⁻⁷ to 2.0 ×10 ⁻⁵ M and a detection limit of 80 nM. When samples of gastric juice were analyzed with the method, we obtained the mean content of 5-HIAA in the gastric juice. Meanwhile, interference from other ions and substances were examined. The experimental results indicate that the method for the determination of gastric juice samples is successful.     

高效液相色谱法测定浓缩胡萝卜汁中辛硫磷残留量

建立了浓缩胡萝卜汁中辛硫磷残留量的高效液相-二极管阵列检测方法.样品经乙酸乙酯提取,硅胶柱净化,采用反向C18色谱柱,流动相为V（甲醇）：V（水）=7：3,波长为285 nm.结果表明：在0.1～10.0μg/mL范围内回归方程为：Y=48.676ρ-1.4548,r=0.99985,呈良好的线性关系,方法检出限为0.02 mg/kg,平均回收率为76.5%～89.3%,相对标准偏差为2.8%～4.5%.