Multi-syringe flow-injection systems improve antioxidant assessment

Conventionally, measuring antioxidant capacity is time consuming, laborious and costly, especially in routine work. These limitations can be overcome using multi-syringe flow-injection analysis (MSFIA), which has succeeded in automating the most widely applied antioxidant-capacity assays that use chromogen radicals or reactive species found in vivo. We highlight the features of MSFIA and improved methodologies in using MSFIA to measure antioxidant capacity.     

Flow injection analysis methods for determination of diffusion coefficients

Two flow injection analyses (FIA) methods for the determination of diffusion coefficients in a straight single tube FIA system were developed. Based on the analytical solution of the convection-diffusion equation, linear relationships of the logarithmic values of the dispersion coefficient (D) and the half-peak width (W_1/2) with the diffusion coefficient (D_m) were obtained. Experiments were designed to verify these methods. For example, for potassium hexacyanoferrate (III) a D_m value of 0.72 × 10⁵ cm² s⁻¹ was found versus a literature value of 0.76 × 10⁵ cm² s⁻¹ (error, 5%). For potassium hexacyanoferrate (II) a D_m value of 0.67 × 10⁵ cm² s⁻¹ was obtained versus a literature value of 0.63 × 10⁵ cm² s⁻¹ (error, 6%). The diffusion coefficients of some important biomedical compounds, such as dopamine, epinephrine, norepinephrine and ascorbic acid, were then determined. The values of 10⁵ D_m/cm² s⁻¹ are 0.60 ± 0.03, 0.44 ± 0.02, 0.60 ± 0.01 and 0.68 ± 0.06, respectively.     

Flow injection determination of histamine with a histamine dehydrogenase-based electrode

A flow injection analysis (FIA) system for the determination of histamine was developed using histamine dehydrogenase (HmDH)-based electrode. Histamine dehydrogenase was immobilized in an osmium-derivatized redox polymer, poly(1-vinylimidazole) complexed with Os(4,4′-dimethylbipyridine)₂Cl₂ (PVI-dmeOs), film on a glassy carbon electrode. As expected from the characteristics of this enzyme in a solution, this electrode exhibits high selectivity to histamine and is not sensitive to other primary amines including common biogenic amines, putrescine, cadaverine and tyramine. The detection limit for histamine was 100 pmol ( μl injection) at a S/N ratio of 3, and response linearity was retained up to 0.6 mM. The FIA system was successfully applied to the determination of histamine in fish samples. The performance of the FIA system is discussed and compared with a high-performance liquid chromatography (HPLC) method which is routinely used for histamine analysis.     

A new approach for the sequential injection spectrophotometric determination of the total antioxidant activity

A sequential injection system based on the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic-acid) methodology was developed. The proposed method, incorporating a mixing chamber in the side port of the selection valve, was evaluated to measure the total antioxidant activity of several beverages and foods.The ABTS⁺ is generated by oxidation of ABTS with potassium persulfate and is reduced in presence of hydrogen-donating antioxidants converting into a colourless product. The applicability of the developed method was tested by measurement of the antioxidant activity of pure compounds as well as by analysing complex food and beverage samples. The antioxidant activity was presented as l(+) ascorbic acid equivalence. The values obtained by this methodology were not significantly different from the results obtained by the original spectrophotometric ABTS assay. For most of the studied antioxidants, antioxidant activity varied with pH and dilution. The proposed SIA system is suitable for screening direct or diluted total antioxidant activity of pure compounds or food samples.     

Sequential injection analysis with electrochemical detection as a tool for economic and rapid evaluation of total antioxidant capacity

This work presents a new flow-based coupled electrochemical technique for evaluation of “total antioxidant capacity (TAC)”. A sequential injection (SI) with amperometric detection was applied to the TAC analysis of commercial instant ginger infusion beverages using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Besides having chromogenic properties, the ABTS reagent behaves as an electroactive species at the glassy carbon electrode in phosphate buffer pH 7.0, the decrease of the cathodic current signal of the ABTS⁺ radical after reaction with antioxidants can be monitored. The SI system, furnished with an in-house electrochemical detection cell (ECD), was optimized with respect to the applied potential, sample and reagent volume, and flow rate to the detector. Gallic acid was used as the standard antioxidant and the capacity was reported as gallic acid equivalent (GAE) unit. TAC measurements of ginger infusions at the optimum condition were performed using the proposed technique and also with the classical batch spectrophotometric ABTS assay. TAC values obtained from our method and the standard method are in good agreement (r² = 0.956). The SI-amperometric technique provided satisfactory precision (4.11% RSD) with rapid sample throughput (40 samples h⁻¹). Also using this method, the consumption of the expensive ABTS reagent was greatly reduced.     

Flow injection analysis of DPPH radical based on electron spin resonance

In order to construct a rapid and selective determination system of free radicals, we developed an FIA system using an electron spin resonance (ESR) spectrophotometer (flow injection spin analysis) equipped with a flow-through flat cell. In the present investigation, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used as a model free radical. Using a single line flow system, 0.5 mM DPPH was repetitively injected. When the magnetic field was fixed at 335.3 mT, the largest change in the ESR signal was observed and obtained peak height was proportional to the concentration of DPPH radical. A double line flow system was constructed in which a carrier stream containing 0.15 mM DPPH was fed into the flat cell after confluence with a sample stream. When ascorbic acid was injected as a typical DPPH radical scavenger, a negative peak appeared in proportional to the concentration. Lower detection limit of ascorbic acid was 0.01 mM (S/N=4), sampling frequency was 13 samples per h, and a satisfactory reproducibility (CV=3.2%, 0.1 mM, n=5) was obtained. The present system was also applied to estimate the DPPH radical-scavenging activity of other substances and food samples.     

Spectrophotometric FIA methods for determination of hydrogen peroxide: Application to evaluation of scavenging capacity

The determination of hydrogen peroxide (H₂O₂) and the evaluation of scavenging capacity against this species were performed using five colorimetric reactions, which were adapted to flow injection analysis. The reactions chosen were based on the oxidation of iodide (I⁻ method), on the formation of titanium-peroxide complex (TiP method), on the formation of titanium-xylenol orange-peroxide complex (TiXoP method), on the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB method) and on the co-oxidation of phenol-4-sulfonic acid and 4-aminoantipyrine (PSA/4-AAP method). The operational conditions were studied in order to improve the sensitivity of each method. Concerning to the method sensitivity, the ranking order was TMB method > I⁻ method > TiXoP method ∼PSA/4-AAP method > TiP method. All methods showed an excellent repeatability (RSD < 2%) and, except for I⁻ method, relative deviations from the reference method were <1.9%. The FIA manifolds were adapted to perform the determination of scavenging capacity against H₂O₂ and glutathione (GSH) was applied as model compound. TiP and TiXoP methods were not suitable as no inhibition or an increase of analytical signal was attained. PSA/4-AAP method was chosen for further application to dietary phenolics and pharmaceutical compounds, providing IC₅₀ values for those compounds that are fast reacting antioxidants.     

A microfluidic system for evaluation of antioxidant capacity based on a peroxyoxalate chemiluminescence assay

A microfluidic system incorporating chemiluminescence detection is reported as a new tool for measuring antioxidant capacity. The detection is based on a peroxyoxalate chemiluminescence (PO-CL) assay with 9,10-bis-(phenylethynyl)anthracene (BPEA) as the fluorescent probe and hydrogen peroxide as the oxidant. Antioxidant plugs injected into the hydrogen peroxide stream result in inhibition of the CL emission which can be quantified and correlated with antioxidant capacity. The PO-CL assay is performed in 800-μm-wide and 800-μm-deep microchannels on a poly(dimethylsiloxane) (PDMS) microchip. Controlled injection of the antioxidant plugs is performed through an injection valve. Of the plant-food based antioxidants tested, β-carotene was found to be the most efficient hydrogen peroxide scavenger (SA _HP of 3.27 × 10⁻³ μmol⁻¹ L), followed by α-tocopherol (SA _HP of 2.36 × 10⁻³ μmol⁻¹ L) and quercetin (SA _HP of 0.31 × 10⁻³ μmol⁻¹ L). Although the method is inherently simple and rapid, excellent analytical performance is afforded in terms of sensitivity, dynamic range, and precision, with RSD values typically below 1.5%. We expect our microfluidic devices to be used for in-the-field antioxidant capacity screening of plant-sourced food and pharmaceutical supplements. Figure Assembled PDMS microchip sandwiched between two glass plates with the top plate containing capillary reservoirs     

Reversed flow injection and sandwich sequential injection methods for the spectrophotometric determination of copper(II) with cuprizone

Two new flow methods, flow injection analysis (FIA) and sequential injection analysis (SIA), for the spectrophotometric determination of Cu(II) in water at trace levels have been developed and optimised. Both methods are based on the reaction with oxalic acid bis(cyclohexylidene hydrazide) (cuprizone) in alkaline media. The two procedures have been developed for the final aim to compare their performances and to offer new rapid heavy metals analysis tools, avoiding the use of extraction steps. A detailed study of the physico-chemical parameters affecting the systems performances has been carried out. The reversed FIA and sandwich SIA approaches offered the best sensitivity. In both cases, an extremely good linearity has been obtained within the range 0.06-4 μg ml⁻¹ (correlation coefficient r=0.9999), whereas the observed detection limits were 0.013 and 0.004 μg ml⁻¹, for FIA and SIA, respectively. Furthermore, due to the great similarity of the diffusion zones in the reaction slugs, our approach offers the opportunity to compare the two methods in analogous conditions. This SIA method, besides keeping its typical reagent saving features, offered analytical performances equivalent to those of FIA. To obtain these results, an original “stop-flow like” method was successfully employed in the SIA approach. Both methods were validated by analysis of real water samples, after copper addition, and certified reference samples of fortified and waste waters.     

流动注射光度法测定盐酸普鲁卡因

在盐酸介质中,利用盐酸普鲁卡因－亚硝酸钠－α萘胺的重氨化－偶合反应,建立了流动注射光度法测定盐酸普鲁卡因的新方法。利用控制加 权形心单纯形优化法选择最佳实验条件,测定的线性范围为0－30μg/mL,该法已用于药物中盐酸普鲁卡因含量的测定。     

流动注射电化学发光法测定半胱氨酸和谷胱甘肽

采用流动注射分析技术研究了半胱氨酸和谷胱甘肽对鲁米诺微弱电化学发光的增敏行为。对影响电化学发光的各因素进行了试验和探讨 ,提出了可能的反应机理 ,并建立一种电化学发光测定半胱氨酸和谷胱甘肽的新方法。半胱氨酸和谷胱甘肽的浓度在 1 .0× 1 0 - 6 mol/L～ 5 .0× 1 0 - 5 mol/L和 1 .0× 1 0 - 6mol/L～ 2 .0× 1 0 - 5 mol/L之间呈良好的线性关系 ,相关系数分别为 0 .993和0 998,检出限分别为 0 .67μmol/L和 0 .72 μmol/L。对 1 .0× 1 0 - 5mol/L的半胱氨酸和谷胱甘肽进行 1 1次平行测定 ,相对标准偏差分别为 4.5 %和 3.7%。     

Flow injection system for hydrolysable tannin determination

A flow injection system was proposed to evaluate the transient product of a colorimetric reaction between hydrolysable tannin and potassium iodate (KIO₃) solution. The system optimization was accomplished by using statistical methods based on experimental design. Flow rate of KIO₃ solution, sample volume, carrier flow rate, and reaction coil were the selected factors for evaluation. On screening step, complete factorial 2⁴ was used and two levels for each selected factor were studied. For the optimization phase, a centered face composite design 2² + star was employed to evaluate sample volume and flow rate of KIO₃ solution, which were the factors identified in the screening phase as having more influence on the absorbance signal. After optimization, the proposed system was compared with batch determination. Some characteristics, such as analytical frequency, reagent consumption and chemical residues generation presented better results by the use of the proposed system if compared with batch method. The system presented good repeatability with standard deviation lower than 3%, for n = 10, linearity (R² = 0.9974) for tannic acid standard, analytical frequency of 15 injections h^− 1 and limit of quantification of 24 mg L^− 1 of tannic acid. Good results were obtained when the proposed system was applied to hydrolysable tannin determination in Stryphnodendron barbatimão, Eucalyptus citriodora and Phyllanthus niruri, samples of plants commonly used in popular medicine.     

流动注射双安培法测定柚皮苷

基于柚皮苷的不可逆氧化和氧化铂的不可逆还原构成双安培检测体系应用于柚皮苷的直接检测。使用两支经阳极化预处理的铂电极,在外加电压为0 V时,通过偶合柚皮苷在一支电极上的氧化和氧化铂在另一支电极上的还原两个不可逆电极过程,构成流动注射双安培检测体系。实验发现,在pH 9.62BR(Briton Robinson)缓冲溶液中,测得柚皮苷氧化电流与其浓度在6.0×10-5~1.0×10-3mol/L范围内呈良好的线性关系(r=0.9953,n=8),检出限为1.0×10-5mol/L。连续20次测定6.0×10-4mol/L柚皮苷,其峰电流相对标准偏差(RSD)为3.3%。常用药物赋形剂和无机离子均不干扰柚皮苷的测定。方法用于模拟样品中柚皮苷的测定。     

Flow injection analysis coupled with HPLC and CE for monitoring chemical production processes

Summary In chemical and pharmaceutical production plants process control is often performed by plant personal near the process (at-line). Alternatively, spectroscopic procedures like near infrared may be coupled to the process using in-line or on-line interfaces. When the analytical problem cannot be solved by these established approaches chromatographic techniques can be directly coupled to the production process. An application of chemical reaction monitoring which is difficult to perform is reported. The analytical problems are solved by HPLC and CE. Both techniques in the application presented need an on-line derivatization step prior to the analysis. Flow Injection Analysis (FIA) was applied for this sample preparation step. For the online coupling to the commercially available CE-instrument a special sample vial was developed. The application shows sufficient reproducibility and analysis speed for near real-time monitoring. of the process. Dedicated to Professor Dr. Heinz Engelhardt on the occasion of his 65^th birthday.     

流动注射双安培法测定环境水样中苯酚

基于苯酚在铂电极上的氧化和不可逆电对的双安培检测原理,建立了流动注射双安培测定环境水样中苯酚的新方法。通过偶合苯酚在一支铂电极上的氧化和KMnO4在另一支铂电极上的还原这两个不可逆电对,构建双安培检测体系,在外加电位差为0V时,苯酚的氧化电流与其浓度在1.0×10-6～1.0×10-4mol/L范围内呈线性关系,检出限为5.0×10-7mol/L,连续30次测定2.0×10-5mol/L苯酚,电流值RSD为1.4%。用本方法分别对几种含酚废水样品中苯酚含量进行测量,并与4 氨基安替比啉比色法进行了对照。     

Electrochemical study and flow injection analysis of paracetamol in pharmaceutical formulations based on screen-printed electrodes and carbon nanotubes

Acetaminophenol or paracetamol is one of the most commonly used analgesics in pharmaceutical formulations. Acetaminophen is electroactive and voltammetric mechanistic studies for the electrode processes of the acetaminophenol/N-acetyl-p-quinoneimine redox system are presented. Carbon nanotubes modified screen-printed electrodes with enhanced electron transfer properties are used for the study of the electrochemical-chemical oxidation mechanism of paracetamol at pH 2.0.Quantitative analysis of paracetamol by using its oxidation process (in a Britton-Robinson buffer solution pH 10.0) at +0.20 V (vs. an Ag pseudoreference electrode) on an untreated screen-printed carbon electrode (SPCE) was carried out. Thus, a cyclic voltammetric based reproducible determination of acetaminophen (R.S.D., 2.2%) in the range 2.5 × 10⁻⁶ M to 1 × 10⁻³ M, was obtained. However, when SPCEs are used as amperometric detectors coupled to a flow injection analysis (FIA) system, the detection limit achieved for paracetamol was 1 × 10⁻⁷ M, one order of magnitude lower than that obtained by voltammetric analysis. The repeatability of the amperometric detection with the same SPCE is 2% for 15 successive injections of 10⁻⁵ M acetaminophen and do not present any memory effect.Finally, the applicability of using screen-printed carbon electrodes for the electrochemical detection of paracetamol (i.e. for quality control analysis) was demonstrated by using two commercial pharmaceutical products.     

Stopped-flow method for assessment of pH and timing effect on the ABTS total antioxidant capacity assay

The pH and timing effect on the relative capacity of antioxidant compounds to scavenge the ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation (ABTS⁺) expressed as the trolox equivalent antioxidant capacity (TEAC) is assessed for a wide range of antioxidants using a modification of the original ABTS assay at different pHs (4.6, 5.4 and 7.4).To study both fast and slow reacting antioxidants, a stopped-flow method based on a low cost laboratory-made system was used. For most of the tested antioxidants, total antioxidant capacity values were found to be a function of pH and time allowed for the reaction.Structurally similar compounds have the same time and pH-dependent behaviour even if they have significant differences in total antioxidant capacity values. The ABTS⁺ scavenging reaction rate was found to depend strongly on pH for p-coumaric acid, glutathione, BHT and albumin. Quercetin, gallic acid, (+)-catechin and (−)-epicatechin showed the higher activities at all pHs.The stopped-flow method can be utilized for screening of antioxidant compounds of unknown kinetics towards ABTS⁺ at different pHs and the results can be used to predict the total antioxidant capacity of structurally related compounds.     

Flow injection titration basing on the merging-zone technique

The proposed approach relies on titration of a sample that is gradually diluted in strictly controlled way in the flow injection system developed. On each step of sample dilution equal volumes of the sample and titrant solutions are simultaneously injected into two carrier streams and the zones are merged with each other. Then, they are mixed completely in the mixing chamber, merged with a stream of indicator and directed to a detector. It has been revealed that the method provides the results with accuracy better than ±3.3% (RE) and with mean repeatability lower than 1.0% (RSD). When the analyte concentration in a sample is too low to be determined directly, the procedure of titration with standard addition is exploited. The proposed approach has been successfully applied to the determination of total acidity in vinegars and magnesium and calcium in pharmaceutical products. The results obtained were comparable with those provided by the reference methods. The proposed procedure is characterized by low consumption of sample (usually less than 2 mL), titrant (about 3 mL) and indicator (about 0.6 mL). Average time of a single analysis is similar to time of traditional batch analysis.     

流动注射双安培法测定橙皮甙

基于橙皮甙的不可逆氧化和氧化铂的不可逆还原体系构成了双安培法直接检测橙皮甙的新方法。在外加电压为0.2 V时,通过偶合橙皮甙在一支电极上的氧化和氧化铂在另一支电极上的还原两个不可逆电极过程,构成流动注射双安培检测体系。在pH 8.95的B-R缓冲溶液中,测得橙皮甙的氧化电流与其浓度在6.0×10-6~8.0×10-4mol/L范围内呈良好的线性关系(r=0.9997,n=11),检出限为1.0×10-6mol/L。连续30次测定6.0×10-4mol/L橙皮甙,其峰电流相对标准偏差(RSD)为3.2%,常用药物赋形剂和无机离子均不干扰橙皮甙的测定。方法已用于江中健胃消食片中橙皮甙含量的测定。     

Flow injection potentiometric sensor for determination of phenylpropanolamine hydrochloride

A new polymeric membrane electrode has been constructed for the determination of phenylpropanolamine hydrochloride. The electrode was prepared by solubilizing the phenylpropanolamine-phosphomolybdate ion associate into a polyvinyl chloride matrix plasticized by dibutylphthalate as a solvent mediator. The electrode showed near-Nernstian response over the concentration range of 1 × 10^?5–1 × 10^?2 M with low detection limit of 6.3 × 10^?6 M. The electrode displays a good selectivity for phenylpropanolamine with respect to a number of common inorganic and organic species. The electrode was successfully applied to the potentiometric determination of phenylpropanolamine ion in its pure state and its pharmaceutical preparation in batch and flow injection conditions.