Neocuproine Complex Research Articles

Sequential Injection Analysis Method for the Determination of Glutathione in Pharmaceuticals.

A sequential injection analysis method for the determination of glutathione (GSH) in pharmaceuticals has been developed. It is based on the reduction of the Cu(II)-neocuproine complex by GSH and the formation of an orange-yellow colored Cu(I)-neocuproine complex with maximum absorbance at 458 nm. Under optimal conditions the method is characterized by a linear calibration range of 6.0 × 10-7-8.0 × 10-5 mol L-1 (Amax = 3270 CGSH - 0.0010; R2 = 0.9983), limit of detection of 2.0 × 10-7 mol L-1, limit of quantification of 6.7 × 10-7 mol L-1, repeatability (expressed as relative standard deviation) of 3.8%, and sampling rate of 60 h-1. The newly developed method has been successfully applied to the determination of GSH in pharmaceutical samples with no statistically significant difference between the results obtained and those produced by the standard Pharmacopoeia method.

An efficient protocol for quantifying catalase activity in biological samples

BackgroundCatalase is an important enzyme that helps protect cells against oxidative stress. The current protocol presents a reliable method for measuring catalase (CAT) enzyme activity in biological systems using the CUPRAC-CAT method.MethodsIn the CUPRAC-CAT method, the component of the enzymatic reaction was incubated before adding the Cu(Nc)22+ reagent. The unreacted substrates reduced the Cu(II)-the neocuproine complex, resulting in the highly colored Cu(I)-neocuproine product, which could be detected spectrophotometrically at 450 nm. The negative correlation between catalase activity and the absorbance of the Cu(I)-neocuproine complex was examined. To assess the optimization of Cu(I)-neocuproine complex production, response surface methodology (RSM) was employed, specifically utilizing the Box–Behnken design (BBD). Additionally, the reliability of the newly developed protocol was confirmed through Bland–Altman analysis of catalase activity in paired samples, employing the peroxovanadate method.ResultsThe novel method is just as accurate as the established standard; the correlation between the two methods was 0.99. The CUPRAC-CAT method is stable, sensitive, linear, reproducible, accurate, and selective and can be used for quantifying oxidative stress while measuring catalase activity in liver tissue homogenates.ConclusionsThis study has demonstrated a straightforward and dependable protocol for evaluating catalase activity. The protocol is free from interference and can be easily employed in scientific research, ensuring a high level of accuracy and precision. The CUPRAC-CAT method is an effective technique to monitor bacterial contamination. This method provides quick and reliable results that can help ensure food safety and prevent or address bacterial contamination.Graphical abstract

Efficient and simple method for the quantification of alkyl polyglycosides by hydrolysis and photometric determination of reducing sugars

Alkyl polyglycosides (APG) are important biodegradable surfactants for many applications. The lack of a chromophoric group and the mixture of stereoisomers and oligomers make the quantification by merely chromatic separation difficult. A simple and efficient quantification assay was developed based on the acid hydrolysis of alkyl polyglycoside and subsequent quantification of the hydrolytically cleaved glucose using the neocuproine assay. Complete hydrolysis of alkyl polyglycoside is achieved with 16 wt% sulfuric acid at 100 °C after 30 min and the separation of fatty alcohol and clouding of the reaction mixture is prevented by adding 1,2-propylene-glycol. Hydrolytically cleaved sugars reduce the Cu (II) -neocuproine complex, leading to a color shift which permits the spectrophotometric quantification at 452 nm. A linear correlation between the absorbance of reduced Cu (II) -neocuproine complex and glucose or alkyl polyglycoside concentration is observed up to an analyte concentration of 25 mg/L. Statistical analysis according to one-tailled T-test (S’ 95%), has shown that the lower detection limit for alkyl polyglycosides is 2.97 mg/L and the limit of quantification 8.24 mg/L, respectively. Apart from the simple execution, the assay is reliable and can be utilized both as scientific technique to quantify APG as well as for process and quality control in the industry.

Enhancement on the degradation of naproxen in Cu0 activated peroxymonosulfate system by complexing reagents

In recent years, there has been growing interest in the mechanism (radical or nonradical) of persulfate activation processes. In this study, the enhancement of naproxen (NPX) degradation in a Cu0/peroxymonosulfate (PMS) system by complexing reagents was investigated. Surprisingly, neocuproine (NCP) alters the nature of reactive species in the Cu0/PMS system. A high-valent copper species, Cu(III)-NCP, was found to dominate NPX degradation rather than radicals under acid conditions for the first time. Moreover, systematically designed experiments revealed that the Cu(III)-NCP complex was a strong selective oxidant that reacted with organics through a single electron transfer pathway. Meanwhile, the degradation efficiency of NPX was highly dependent on the solution pH and dosage of Cu0 and NCP, but was irrelevant to the concentration of NPX. Additionally, the enhancement of NCP on other copper based PMS activation systems (i.e., Cu2+/HA/PMS and Cu0/HA/PMS systems) was investigated. Considering that the released copper can be removed by a simple precipitation method to meet the effluent standards, the new complex-enhanced Cu0/PMS system provided a new method to enhance the degradation efficiencies of pollutants by a copper-catalyzed Fenton-like system.

A quantitative method for the assessment of homocysteine thiolactonase enzyme activity

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

Determination of penicillamine, tiopronin and glutathione in pharmaceutical formulations by kinetic spectrophotometry.

A novel and simple method for the determination of penicillamine (PEN), tiopronin (mercaptopropionyl glycine, MPG) and glutathione (GSH) in pharmaceutical formulations by kinetic spectrophotometry has been developed and validated. It is based on the redox reaction where the thiol compound (RSH) reduces CuII-neocuproine complex to CuI-neocuproine complex. The non-steady state signal of the formed CuI- neocuproine complex is measured at 458 nm. The initial rate and fixed time (at 1 min) methods were validated. The calibration graph was linear in the concentration range from 8.0 × 10‒7 to 8.0 × 10‒5 mol L-1 for the initial rate method and from 6.0 × 10‒7 to 6.0 × 10-5 mol L-1 for the fixed time method, with the detection limits of 2.4 × 10-7 and 1.4 × 10‒7 mol L-1, resp. Levels of PEN, MPG and GSH in pharmaceutical formulations were successfully assayed by both methods. The advantages of the presented methods include sensitivity, short analysis time, ease of application and low cost.

A novel digital image method for determination of reducing sugars in aged and non-aged cachaças employing a smartphone

For the first time, a method is proposed for colorimetric determination of reducing sugars in cachaça employing digital image and a smartphone as detector. The method was based on the reduction of Cu(II) to Cu(I) by sugars and followed by the formation of a colored Cu(I)-Neocuproine complex. A calibration curve was linear from 0.1 to 15 g L−1 for glucose and fructose with limits of detection of 0.012 g L−1 and 0.010 g L−1, respectively. It was observed that the non-aged cachaças, known for having inferior flavors and aromas, had a reducing sugar content three times higher than the aged cachaças, once a common practice among producers is to add sugar to adjust sensory deficits in the final product. Furthermore, the method is simple, does not require complex technical knowledge and it could be used as a tool to check possible fraud, adulteration or non-compliance to the law.

Development and In Vivo Application of a Water-Soluble Anticancer Copper Ionophore System Using a Temperature-Sensitive Liposome Formulation.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.

Improved in vitro antioxidant properties and hepatoprotective effects of a fermented Inula britannica extract on ethanol-damaged HepG2 cells

This study aimed to improve antioxidant effect and hepatoprotective effect of Inula britannica using fermentation. Epigallocatechin gallate (EGCG) in an I. britannica extract was found to be upregulated from 2.06 to 10.28μg/mg during fermentation (p < 0.001). After fermentation, DPPH radical-scavenging ABTS radical-scavenging, and superoxide anion-scavenging abilities increased to 92.65%, 694.25μM Trolox/mL, and 86.38%, respectively, at 500μg/mL (p < 0.05). Cupric-ion-reducing capacity with formation of the Cu+-neocuproine complex increased by 5.88%, 6.38%, 3.24%, and 8.55% at 62.5 to 500μg/mL. Ferric-ion-reducing capacity of the fermented extract increased by 20%, 7.16%, 3.85%, and 5.45% at each concentration (p < 0.05). Unfermented extracts yielded cell viability of 91.42%, 90.59%, 88.38%, and 79.17%, whereas the fermented extract yielded 100.28%, 99.66%, 96.15%, and 89.90%, respectively, at each concentration in ethanol-damaged HepG2 cells (p < 0.05). Additionally, the fermented extract decreased alanine transaminase activity from 117.2 to 61.7 U/mL in the ethanol-damaged HepG2 cell line (p < 0.01). Overall, both antioxidant and hepatoprotective effect increased by fermentation in I. britannica extract. These properties are expected to lead to new antioxidant agents via production of EGCG by fermentation.

Novel optical sensor-based method for determining total tocopherol content in serum

Lipid-soluble endogenous antioxidants, such as tocopherols, protect biological structures from oxidative damage through their chain-breaking ability and thus the development of a simple method for tocopherol determination is important. In this study, a novel optical sensor-based method was proposed for determining total tocopherol content (TTC) of serum, with selectivity for $\alpha $-tocopherol over $\beta $-carotene. The proposed method is based on the use of a copper(II)-neocuproine complex (Cu(II)-Nc) reagent immobilized onto a Nafion membrane, and the absorbance changes associated with the formation of Cu(I)-Nc chelate on the membrane strip as a result of reaction with serum tocopherols were measured at 450 nm. The calibration graph of $\alpha $-tocopherol was linear with a slope of 1.96 x 10$^{4}$ L mol$^{-1}$ cm$^{-1}$. The limit of detection and limit of quantification for tocopherol in the proposed method were found as 0.50 and 1.67 μM, respectively. The TTC value of synthetic serum samples was determined with the use of the proposed method, and the obtained results were comparable to those of the CUPRAC spectrophotometric method. The proposed method enables simple, rapid, and in situ determination of total tocopherol content of serum by taking advantage of optical sensors.

Glutathione Peroxidase Activity of Biological Samples Using A Novel Microplate-Based Method

Background: Glutathione peroxidase (GSH-Px) is one of the major enzyme in the antioxidative defense mechanism present in the cells. Methods: In the present study a novel and simple microplate-based method was developed, using the cupric neocuproine complex (Cu(Nc)2 2+) as a chromogenic oxidizing reagent, for the assessment of GSH-Px activity of biological samples for the first time. In this GSH-Px enzyme activity measurement, the GSH-Px catalyzed oxidation of Reduced Glutathione (GSH) gives rise to oxidized form of GSH (GSSG). The recommended method was based on the reduction of Cu(Nc)2 2+ to highly colored Cu(I)- neocuproine complex (Cu(Nc)2 +) by the unconsumed GSH, and measurement spectrophotometrically at 450 nm, the difference being correlated to GSH-Px activity of the analytes. Results: Under the optimum conditions, a linear calibration graph was obtained in the range of 18.5- 92.5 µM of GSH with Limit Of Detection (LOD) of 1.03 µM. Application of developed method to tissue homogenates (n = 9) provided GSH-Px activity values in agreement with those of the reference GSH-Px-5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) method. Conclusion: Cupric Ion Reducing Antioxidant Capacity (CUPRAC) antioxidant method was implemented in a microformat (96 well plates) and the reaction time of original CUPRAC method was significantly shortened from 30 to 4 min. Multi-sample can be simultaneously detected thanks to a novel microplate method. Keywords: GSH-Px activity, microplate based method, modified CUPRAC method, biological samples, neocuproine complex, reduced glutathione (GSH).

SPECTROPHOTOMETRIC DETERMINATION OF REDUCING SUGAR IN WINES EMPLOYING IN-LINE DIALYSIS AND A MULTICOMMUTED FLOW ANALYSIS APPROACH

A multicommuted flow procedure for the spectrophotometric determination of reducing sugars in wine has been developed. A dialysis unit was incorporated in the system to minimize matrix interferences and eliminate the colored compounds of the wine. The oxidation-reduction reaction in an alkaline medium between reducing sugars and the Cu2+-Neocuproine complex was employed. The compound formed was monitored at 460 nm. In the proposed procedure, there was a linear response between 0.25 and 4.0 g L-1 reducing sugars (y = (0.36 ± 0.02) + (0.31 ± 0.01) * x, R = 0.9975, n = 6), a coefficient of variance of 2.6% for a reducing sugar solution of 2.0 g L-1 (n = 7), a limit of detection estimated at 0.03 g L-1, reagent consumption per determination of 0.330, 0.128 and 0.160 mg for neocuproine, CuSO4 and NaOH, respectively. A sampling throughput of 45 determinations per hour was achieved. The proposed system was applied to wine samples and comparing the results with an AOAC official method no significant difference at a 95% confidence level.

Construction of a novel highly selective potentiometric perchlorate sensor based on neocuproine–Cu(II) complex formed in situ during the conditioning period

A potentiometric perchlorate-selective electrode based on a phenanthroline derivative, neocuproine (2,9-dimethyl-1,10-phenanthroline), was developed. An electroactive substance, a neocuproine complex of Cu2+, providing a selective response to perchlorate was formed in situ in the electrode membrane structure during the conditioning process. Membrane optimization studies showed that the most suitable membrane composition was 1.0% (w/w) neocuproine, 69.0% (w/w) o-nitrophenyloctylether (o-NPOE) and 30.0% (w/w) poly(vinyl chloride) (PVC) in terms of potentiometric performance characteristics. For the proposed electrode, the linear range, slope, detection limit, pH working range and response time were determined as 2.5 × 10–7–1.0 × 10–1 M, –53.01 mV/decade, 1.0 × 10–7 M, 4.0–12.5 and ≈3 s, respectively. The proposed sensor was successfully used in the determination of perchlorate contents of some perchlorate spiked tap, river and dam water samples.

Self-assembled nanocontainer mediated oxidation of Fe(ii) by Cu(ii)–neocuproine complex: a model system to emulate electron transfer proteins

Catalysis of coordination inspired Fe(ii) oxidation by Cu(ii)–neocuproine complex.

Development of flow methods for the determination of N-acetyl-L-cysteine in pharmaceutical formulations

Background: N-acetyl-L-cysteine (NAC) is a synthetic aminothiol antioxidant that has been in clinical use for more than 40 years, primarily as a mucolytic agent and in the management of paracetamol (acetaminophen) poisoning. There is a need for a new, robust, inexpensive, and rapid method of determination of NAC in pharmaceutical formulations, in order to assure proper quality control (QC). Patient safety during therapy, on the other hand, is indirectly linked to proper QC of the medication. Novel flow methods for the determination of NAC, such as the flow-injection method (FIA) or the sequential-injection method (SIA), have been developed and validated. Both methods are kinetic methods, which means that the measurement of the analytical signal is made under dynamic conditions in which the concentrations of reactants and products are changing as a function of time. The proposed flow methods of analysis (FIA and SIA) are interesting alternatives in NAC determinations instead of conventional batch methods and chromatography with different detectors. The advantages afforded by the flow methods of analysis are high sample frequency, low consumption of sample and reagents, low contamination risks, and significant reproducibility that provides high precision and enhanced selectivity as a result of the kinetic nature of the recorded analytical signal. Furthermore, the proposed flow methods of analysis require very limited laboratory bench space and necessary instrumentation. Methods: The proposed methods are based on the reduction of Cu(II)-neocuproine reagent to Cu(I)-neocuproine with the analyte, in a Britton-Robinson buffer solution (pH 3.0). The non-steady-state absorbance of the formed yellow Cu(I)-neocuproine complex is measured at 458 nm. For the flow-injection method the three-line manifold with one reaction coil was used. Optimization of manifold parameters and experimental conditions were carried out by means of univariate method. The sequential-injection manifold consisted of a Cheminert® M50 pump (VICI Valco), a syringe-free stepper motor-driven pump, a 10-port selection valve model (C25-3180D) with a multiposition actuator control module (EMHCA-CE ; VICI Valco). Both flow systems use a spectrophotometric detector. Results: Using a flow-injection method of analysis, a linear calibration curve is established in a concentration range of 6 × 10−7 to 4 × 10−5 mol/l NAC with a detection limit of 9.4 × 10−8. On the other hand, using the sequential-injection method of analysis, linearity was obtained in the concentration range of 4 × 10−6 to 3 × 10−4 mol/l. The detection limit was found to be 1.2 × 10−6 mol/l. The proposed methods are simple, rapid, sensitive and reproducible (FIA: RSD 0.9 %, n = 100 ; SIA: RSD 1.9 %, n = 100). In addition, the proposed methods are sensitive enough to enable determination of near-nanomole amounts of NAC without expensive instruments with an analytical frequency of 120 / h (FIA) and 60 / h(SIA). Discussion: The proposed methods can be applied for the determination of NAC in pharmaceutical preparations. Therefore, they could be useful for QC of NAC-containing medications, indirectly improving patient safety during therapy (e. g. by reducing the risk of under- or over-dosage).

Flow Injection Analysis of N-acetyl-L-cysteine Based on the Reduction of Copper(II)-neocuproine Reagent

ABSTRACTA novel flow-injection analysis spectrophotometric procedure for the determination of N-acetyl-L-cysteine in pharmaceutical formulations is reported. The method is based on the reduction of Cu(II)-neocuproine to Cu(I)-neocuproine by the sulfhydryl group of N-acetyl-L-cysteine in aqueous solution at pH = 3. The absorbance of the yellow Cu(I)-neocuproine complex was measured at 458 nm. The primary chemical and instrumental variables were examined thoroughly. The method was validated in terms of linear dynamic range, limit of detection, limit of quantitation, selectivity, precision, and accuracy. Measurements confirmed its suitability for the reliable determination of N-acetyl-L-cysteine in pharmaceutical preparations at a sampling rate of 180 analyses per hour.

ALTERNATIVE CALIBRATION SYSTEMS FOR THE SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF COMPOUNDS WITH OVERLAPPING ABSORPTION SPECTRA

Two simple and efficient procedures have been developed for the rapid simultaneous determination of compounds with mutual spectral interference (rifampicin (RIF) and isoniazid (INH)). The first method was based on the UV–Vis spectral signal (190–600 nm) of synthetic RIF and INH aqueous solutions, whereas the second method involved the visible spectral signal registered between 350 and 800 nm after the reaction of INH with a Cu2+/neocuproine complex. Both multivariate spectrophotometric methods show excellent prevision capacity, providing results that are statistically equivalent with those provided by the standard chromatographic procedure. The methods were validated according to criteria established by ANVISA, showing precision, accuracy and robustness compatible with the requirements for new analytical methods, additionally allowing the reduction of waste generation.

Automatic flow injection analysis (FIA) determination of total reducing capacity in serum and urine samples.

Automation of total antioxidant capacity assessment can substantially increase the determination throughput, allowing large scale studies and screening experiments. Total reducing capacity evaluation can be implemented under different chemistries, including the CUPRAC-Cupric Ion Reducing Antioxidant Capacity -assay. This assay is based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex by reducing (antioxidant) components of biological samples. In this chapter, we propose an automatic flow injection method for evaluation of total reducing capacity in serum and urine samples, attaining end-point data within 4 min using a kinetic matching strategy.

A novel differential pulse voltammetric (DPV) method for measuring the antioxidant capacity of polyphenols-reducing cupric neocuproine complex.

A novel differential pulse voltammetric (DPV) method is presented, using a chromogenic oxidizing reagent, cupric neocuproine complex (Cu(Nc)2(2+)), for the assessment of antioxidant capacity of polyphenolic compounds (i.e., flavonoids, simple phenolic acids, and hydroxycinnamic acids), ascorbic acid, and real samples for the first time. The electrochemical behavior of the Cu(Nc)2(2+) complex was studied by cyclic voltammetry at a glassy carbon (GC) electrode. The electroanalytical method was based on the reduction of Cu(Nc)2(2+) to Cu(Nc)2(+) by antioxidants and electrochemical detection of the remaining Cu(II)-Nc (unreacted complex), the difference being correlated to antioxidant capacity of the analytes. The calibration curves of individual compounds comprising polyphenolics and vitamin C were constructed, and their response sensitivities and linear concentration ranges were determined. The reagent on the GC electrode retained its reactivity toward antioxidants, and the measured trolox equivalent antioxidant capacity (TEAC) values of various antioxidants suggested that the reactivity of the Cu(II)-Nc reagent is comparable to that of the solution-based spectrophotometric cupric ion reducing antioxidant capacity (CUPRAC) assay. This electroanalytical method better tolerated sample turbidity and provided higher sensitivity (i.e., lower detection limits) in antioxidant determination than the spectrophotometric assay. The proposed method was successfully applied to the measurement of total antioxidant capacity (TAC) in some herbal tea samples such as green tea, sage, marjoram, and alchemilla. Results demonstrated that the proposed voltammetric method has precision and accuracy comparable to those of the spectrophotometric CUPRAC assay.

Kinetic Spectrophotometric Determination of N-acetyl-L-cysteine Based on the Reduction of Copper(II)-neocuproine Reagent

A novel simple kinetic spectrophotometric method for the determination of N-acetyl-L-cysteine (NAC) has been developed and validated. The proposed method is based on the reduction of Cu II - neocuproine reagent to Cu I -neocuproine with the analyte, in a Britton-Robinson buffer solution (pH = 3.0). The non-steady state absorbance of the formed Cu I -neocuproine complex is measured at 458 nm. The initial rate and fixed time (at 1 min) methods were utilized for constructing the calibration graphs. The calibration curves for both methods were linear in concentration range from 6.0  10 -7 to 8.0  10 -5 mol L -1 with the limit of detection 1.7  10 -7 mol L -1 . The slope of the initial method calibration curve (1.0181) confirmed the first order reaction. Both proposed methods were successfully applied for the de- termination of NAC in its commercial pharmaceutical formulations. (doi: 10.5562/cca2161)