An improved and fast UHPLC-PDA methodology for determination of <Emphasis Type="Italic">L</Emphasis>-ascorbic and dehydroascorbic acids in fruits and vegetables. Evaluation of degradation rate during storage

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultra-high-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm × 2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intra-day precision. Validation experiments revealed very good recovery rate of 96.6 ± 4.4% for L-AA and 103.1 ± 4.8 % for total vitamin C, good linearity with r ² -values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.     

Kinetic spectrofluorimetric determination of trace ascorbic acid based on its inhibition on the oxidation of pyronine Y by nitrite

A highly sensitive spectrofluorimetric method is proposed for the determination of trace amount of ascorbic acid using a new indication. The method is based on the inhibition of ascorbic acid on the oxidation of pyronine Y (PRY) by nitrite. The detection limit for ascorbic acid is 0.012 microg ml(-1), the linear range of the determination is 0.02-0.36 microg ml(-1). Analytical parameters, such as reagent concentration, pH, reaction temperature and time, were optimized. The relative standard deviations of eleven replication determinations of 0.12 and 0.24 microg ml(-1) ascorbic acid were 1.4 and 0.72%, respectively. This method has been used to determine ascorbic acid in pharmaceuticals, vegetables, fruits and soft drink with satisfactory results.     

Kinetic spectrophotometric determination of ascorbic acid by reduction of toluidine blue

A simple kinetic method is described for the determination of ascorbic acid. The procedure is based on the reduction of toluidine blue with ascorbic acid. The rate of reaction is followed by measuring the decrease in absorbance of toluidine blue (lambda(max) = 600 nm) as a result of its decolorization upon reduction by ascorbic acid. Ascorbic acid in the range of 3-35 microg/ml was determined using slope and fixed time methods of analysis, while the variable time method allowed the determination of 5-50 microg/ml of ascorbic acid. The percent relative standard deviation of the method varied from 0.78 to 1.32% depending on the kinetic method used. The high sensitivity of the method also allows determination of low levels of ascorbic acid in some fruits and vegetables such as dew melon, water melon, parsley and coriander.     

Gold electrodes from compact discs modified with platinum for amperometric determination of ascorbic acid in pharmaceutical formulations

A simple, rapid and precise amperometric method has been developed for quantification of ascorbic acid (AA) in pharmaceutical formulations using flow-injection analysis (FIA). A slice of recordable compact disc (CD) modified by electrodeposition of platinum was employed as the working electrode. The proposed flow system allows determinations in the 1 mumol l(-1) of the analyte and enables 90 determinations per h, employing only 150-mul sample. The method permits the direct quantification of ascorbic acid in many pharmaceutical products, avoiding cumbersome processes as previous separations, solvent extraction or sample filtration. This new procedure was applied to commercial pharmaceutical tablets and the results obtained were identical than the ones obtained by the classical iodometric method. The calibration plots for freshly prepared ascorbic acid standards were highly linear in the concentration range of 1-10 mumol l(-1) with a relative standard deviation (R.S.D.) <1%. For all real samples studied, the deviations were situated between 0.5 and 8.7%.     

Synergistic effect of UV and l-ascorbic acid on the reduction of graphene oxide: Reduction kinetics and quantum chemical simulations

A photochemical strategy for eco-friendly reduction of graphene oxide (GO) was developed by using l-ascorbic acid (L-AA) as a photosensitive reducing agent. L-AA was excited and oxidized with deprotonation by UV irradiation (254 nm) and the proton coupled electron transfer induces chemical reduction of GO. This photochemical process is quite eco-friendly and scalable, and the reduction kinetics and degree of GO were highly enhanced. To understand the improved reduction power by UV light, the redox properties of L-AA in the ground and excited states were characterized by using quantum chemical simulations. Based on the results, we clearly demonstrated the mechanism how UV irradiation considerably enhances the reducing power of L-AA for the reduction of GO.     

Comparison of Two Methods of Ascorbic Acid Determination in Vegetables

Abstract

Vegetables (broccoli, Brussels sprouts, cauliflower, green beans, spinach, and turnip) were analyzed for ascorbic acid using a modified AOAC method and compared to a high performance liquid chromatography (HPLC) method. The HPLC method employed a weak ion exchange μBondapalc NH₂ column and detection at 254 nm. The two methods did not differ in ascorbic acid values for fresh and three-week stored broccoli, cauliflower, green beans, turnip and three-week stored Brussels sprouts and spinach. A higher ascorbic acid content was found for fresh Brussels sprouts and spinach when measured by the HPLC method.     

固相萃取–气相色谱–负化学源质谱法检测水果和蔬菜中的毒杀芬

采用改进的分散固相萃取(QuEchERs)法对样品进行前处理,建立水果和蔬菜中毒杀芬残留的气相色谱–负化学电离源质谱(GC–NCI–MS)检测方法。样品中的毒杀芬由正己烷提取,经吸附剂PSA+GCB净化,在GC–NCI–MS的选择离子扫描模式下进样分析。毒杀芬的色谱保留时间在12.5~18.0 min区间内,采用面积归一化法积分,外标法定量。毒杀芬质量浓度在0.050~2.000 mg/L范围内与色谱峰面积呈良好的线性关系,相关系数r=0.999 1。分别以蓝莓、黄桃、菠菜为基质,在0.025,0.050 mg/kg添加水平下,毒杀芬的回收率为107.2%~118.1%,测定结果的相对标准偏差为5.5%~8.8%(n=6),定量限为0.025 mg/kg。该方法检测快速,适用于水果和蔬菜中毒杀芬残留的日常检测。     

Simultaneous determination of ascorbic acid and acetylsalicylic acid in pharmaceutical formulations

A direct, simple, and practical first-derivative spectrophotometric method is described for simultaneous determination of ascorbic acid and acetylsalicylic acid. The effects of the solvent, excipients, and spectral variables on the analytical signal were investigated. The drugs were determined simultaneously with a 0.01 M methanolic hydrochloric acid solution as the solvent, and the signals were evaluated directly by using the zero-crossing method at 245.0 and 256.0 nm for acetylsalicylic acid and ascorbic acid, respectively. The method allows the simultaneous determinations of acetylsalicylic acid and ascorbic acid in the ranges of 6.6 x 10(-6) to 1.5 x 10(-4)M and 3.4 x 10(-6) to 2.0 x 10(-4)M, respectively, with standard deviation of <2.0%. The proposed method was applied to determinations of these drugs in tablets.     

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.     

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.     

Magnetic ionic liquid assisted single‐drop microextraction of ascorbic acid before its voltammetric determination

For the first time, we coupled a microextraction technique using a magnetic ionic liquid with voltammetric determination. A hydrophobic magnetic ionic liquid that contains the tetrachloromanganate(II) anion, namely, aliquat tetrachloromanganate(II), was synthesized and used for the extraction of ascorbic acid from aqueous solutions followed by voltammetric determination. The extraction procedure was carried out using a single drop microextraction technique in which the ascorbic acid containing magnetic ionic liquid was separated with a magnet and then cast onto the surface of a carbon paste electrode modified with TiO₂ nanoparticles for the voltammetric quantification of the extracted ascorbic acid. Electrochemical quantification was carried out in a blank phosphate buffer solution. After optimizing different experimental conditions, a linear concentration range of 1.50–40.0 nM with a detection limit of 0.43 nM was obtained for the determination of ascorbic acid. The presented approach was successfully applied to the determination of ascorbic acid in samples of vitamin C effervescent tablets and orange juice.     

Spectrophotometric determination of ascorbic acid in pharmaceutical preparations and fruit juices

Conditions were established for the determination of ascorbic acid using phsophovanadotungstic acid as reagent. The method was applied to the determination of ascorbic acid in pure form, pharmaceutical preparations and fruit juices. The method is sensitive (2-24 micrograms ml-1 of ascorbic acid) and rapid and tolerates the presence of common ingredients usually found in fruit juices. The results obtained with the proposed method showed good agreement with those given by the standard method.     

Sequential injection redox or acid-base titration for determination of ascorbic acid or acetic acid

Two sequential injection titration systems with spectrophotometric detection have been developed. The first system for determination of ascorbic acid was based on redox reaction between ascorbic acid and permanganate in an acidic medium and lead to a decrease in color intensity of permanganate, monitored at 525 nm. A linear dependence of peak area obtained with ascorbic acid concentration up to 1200 mg l⁻¹ was achieved. The relative standard deviation for 11 replicate determinations of 400 mg l⁻¹ ascorbic acid was 2.9%. The second system, for acetic acid determination, was based on acid–base titration of acetic acid with sodium hydroxide using phenolphthalein as an indicator. The decrease in color intensity of the indicator was proportional to the acid content. A linear calibration graph in the range of 2–8% w v⁻¹ of acetic acid with a relative standard deviation of 4.8% (5.0% w v⁻¹ acetic acid, n=11) was obtained. Sample throughputs of 60 h⁻¹ were achieved for both systems. The systems were successfully applied for the assays of ascorbic acid in vitamin C tablets and acetic acid content in vinegars, respectively.     

A spectroscopic study on applicability of spectral analysis for simultaneous quantification of l-dopa, benserazide and ascorbic acid in batch and flow systems

The usefulness of derivative spectrophotometry for simultaneous assay of l-dopa, benserazide and ascorbic acid in pharmaceuticals was studied. The parameters of derivatisation depends on composition of solution in which particular compound was determined. For quantification of l-dopa in mixtures with benserazide or ascorbic acid the first derivative was used. Its determination in ternary mixture (l-dopa+benserazide+ascorbic acid) is possible by third derivative spectra. Benserazide was assayed in presence of l-dopa using first derivative while in ternary mixture by third derivative. Direct determination of ascorbic acid is possible applying first derivative only in presence of l-dopa. The elaborated derivative spectrophotometric methods were used for assaying of l-dopa and benserazide in their commercial form "Madopar". The proposed spectrophotometric derivative method of simultaneous determination of l-dopa and benserazide was combined with FIA technique.     

Experimental IR and Raman spectra and quantum chemical studies of molecular structures, conformers and vibrational characteristics of L-ascorbic acid and its anion and cation

IR and spectra of the L-ascorbic acid (L-AA) also known as vitamin C have been recorded in the region 4000-50 cm(-1). In order to make vibrational assignments of the observed IR and Raman bands computations were carried out by employing the RHF and DFT methods to calculate the molecular geometries and harmonic vibrational frequencies along with other related parameters for the neutral L-AA and its singly charged anionic (L-AA(-)) and cationic (L-AA(+)) species. Significant changes have been found for different characteristics of a number of vibrational modes. The four ν(O-H) modes of the L-AA molecule are found in the order ν(O(9)-H(10))>ν(O(19)-H(20))>ν(O(7)-H(8))>ν(O(14)-H(15)) which could be due to complexity of hydrogen bonding in the lactone ring and the side chain. The CO stretching wavenumber (ν(46)) decreases by 151 cm(-1) in going from the neutral to the anionic species whereas it increases by 151 cm(-1) in going from the anionic to the cationic species. The anionic radicals have less kinetic stabilities and high chemical reactivity as compared to the neutral molecule. It is found that the cationic radical of L-AA is kinetically least stable and chemically most reactive as compared to its neutral and anionic species.     

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.     

Electrochemical behavior of the bis(2,2'-bipyridyl)copper(II) complex immobilized on a self-assembled monolayer modified electrode for L-ascorbic acid detection

Modification of a gold electrode has been achieved by immobilizing a bis(2,2'-bipyridyl)copper(II) complex in a self-assembled monolayer (SAM) of 3-mercaptopropionic acid. The electrostatic interaction of the negatively charged SAM with a di-positive copper complex allowed the attachment. The modified electrode exhibited excellent redox behavior. The dependence of the modified electrode response was investigated in terms of pH, supporting electrolyte and ionic strength. Moreover, it showed good electrocatalytic activity for ascorbic acid oxidation, allowing convenient quantification at levels down to 8.1 x 10(-8) mol l(-1). The [Cu(bipy)2]/SAM modified electrode under optimized operational conditions (PIPES buffer 0.01 mol l(-1) at pH 6.8 and 200 mV vs. SCE) presented a linear response range between 1.0 micromol l(-1) and 100.0 micromol l(-1) for ascorbic acid. This modified electrode also presented an excellent repeatability, showing a relative standard deviation of 2.1% for a series of 12 successive measurements of a 5.0 micromol l(-1) ascorbic acid solution. Furthermore, the electroactivity was maintained over a long period (e.g., 92% after 100 determinations).     

Evaluation and optimization of high-throughput enzymatic assays for fast l-ascorbic acid quantification in fruit and vegetables

In this paper, we compare and evaluate the applicability of three UV-VIS absorbance based assays for high-throughput quantification of ascorbic acid in horticultural products. All the methods involve the use of a common enzyme (ascorbate oxidase) in combination with a different indicator molecule. The three methods were retrieved from literature: a direct oxidase-method, an OPDA coupled oxidase-method and a PMS-method, which is commercially available. The analysis in high-throughput context involved the analysis in microplates in combination with the use of an automated liquid handling system. We checked (i) the performance factors of the selected methods on standard solutions, (ii) the applicability of the defined methods in high-throughput context, and, (iii) the accuracy of the methods on real samples using HPLC as a reference technique. The OPDA-method was found to be the most appropriate method for the quantification of ascorbic acid in high-throughput context with a linear range between 7.0 and 950 mg L⁻¹ and excellent correlation parameters (slopes close to 1, intercepts close to 0, R² > 0.91) with the reference technique when real samples were analyzed. Finally, this method was optimized for assay cost and assay time. Hereto the enzymatic reaction was mathematically described using a model for enzyme kinetics, which was then used to calculate the optimal concentrations of ascorbate oxidase and OPDA. As a result of the modeling the amount of enzyme in the assay could be reduced with a factor 2.5 without affecting significantly the reaction time. In a last step the optimal concentrations were used for a successful validation with the HPLC-reference technique.     

Comparison of ascorbic and citric acid contents inRosa canina L. fruit growing in the Central Asian region

The evaluation ofRosa canina L. fruits and their products is depended in part on their organic acid contents. In this study, ascorbic and citric acid contents inRosa canina L. fruits collected from five different regions in Central Asia were investigated. Amounts of acids determined with the HPLC method were found to be 0.048–0.1143 g/100 g for ascorbic acid and 5.9–7.5 g/100 g for citric acid. Presented at the Third International Symposium on the Chemistry of Natural Products (Bukhara, Uzbekistan, October 19–22, 1998). Deceased. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 768–771, November–December, 1998.     

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.