Electrochemical determination of the total antioxidant capacity of human plasma

Electrochemical reduction of oxygen at a glassy carbon electrode in a 0.05 mol L^–1 solution of (C₂H₅)₄NI in dimethylformamide leads to generation of the superoxide anion-radical. This product of reversible one-electron oxygen reduction reacts with antioxidants, a process which is based on protonation of the anion-radical by the antioxidant. Rate constants of this interaction have been calculated. Human plasma antioxidants also react with electrochemically generated superoxide anion-radical. A voltammetric method is proposed for estimation of the total antioxidant capacity (TAC) of plasma on the basis of on this reaction. The TAC of plasma was also determined using constant-current coulometry with electrogenerated bromine as the active species. A correlation was observed between TAC data obtained by voltammetry ( , in -tocopherol units) and coulometry (Br₂ as titrant). TAC of plasma from patients with purulent infections was determined. Statistically significant differences were found between TAC of patients and control group. Treatment of purulent infections increases the TAC of plasma. So, use of electrochemical methods (voltammetry and coulometry) for determination of TAC can be used for estimation of the effectiveness of treatment.     

Development and validation of a sensitive LC‐MS/MS method for determination of tacrolimus on dried blood spots

A bioanalytical method for the quantification of tacrolimus (TAC) on dried blood spots (DBS) using liquid chromatography, electrospray ionization coupled with tandem mass spectrometry (LC‐ESI‐MS/MS) was developed and validated. It involves solvent extraction of a punch disk of DBS followed by liquid–liquid extraction. The analyte and the internal standard (IS, ascomycin) were separated on a phenyl column using an isocratic mobile phase elution at a flow rate of 0.3 mL/min. The assay was linear from 1 to 80 ng/mL. The mean recovery of TAC was 76.6%. Intra‐assay, inter‐assay imprecision and biases were all less than 15%. TAC on DBS was stable for at least 10 days at room temperature, and at least 24 h at 50°C. A chromatographic effect of the filter paper (Whatman 903) was not detected. The volume of blood (15–50 μL) and hematocrit of blood (ranging from 23.2 to 48.6%) did not show a significant influence on detection of TAC concentration by DBS‐LC‐MS/MS. Fifty samples from patients were detected by both DBS‐LC‐MS/MS and microparticle enzyme‐linked immunoassay (MEIA). TAC concentrations measured by DBS‐LC‐MS/MS method tended to be lower than those by MEIA. Copyright © 2012 John Wiley & Sons, Ltd.     

First UHPLC‐MS/MS method coupled with automated online SPE for quantification both of tacrolimus and everolimus in peripheral blood mononuclear cells and its application on samples from co‐treated pediatric patients

Tacrolimus (TAC, FK‐506) and everolimus (EVE, RAD001) are immunosuppressors used to treat pediatric patients undergoing liver transplantation. Their hematic TDM by liquid chromatography became standard practice. However, it does not always reflect concentrations at their active site. Our aim was to develop and validate a new method for the simultaneous TAC and EVE quantification into target cells: peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were collected using cell preparation tubes; cells number and mean cell volume were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC–MS/MS coupled with an automated online solid‐phase extraction platform. Chromatographic run was performed on an Acquity UPLC® BEH C18 1.7 μm (2.1 × 50 mm) column at 45 °C, for 6 min at 0.5 ml/min. Mobile phases were water and methanol, both with 2 mm ammonium acetate and 1 ml/l formic acid). XBridge® C8 10 μm (1 × 10 mm) SPE cartridges were used, and the internal standard was ascomycin. Following Food and Drug Administration guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r ² = 0.998) and intra‐day and inter‐day imprecision and inaccuracy were <15%. A reproducible matrix effect was observed, with a good recovery for all compounds. Drug amounts in 15 ‘real’ PBMCs samples from five pediatric patients in co‐treatment resulted within the calibration range (0.039–5 ng). Concentrations from each patient were standardized using their evaluated mean cell volume: intra‐PBMCs concentration was meanly 19.23 and 218.61 times higher than the hematic one for TAC and EVE, respectively. This method might be useful in clinical routine, giving reliable data on drugs concentration at the active site. Copyright © 2017 John Wiley & Sons, Ltd.     

Group-selective determination of total N-nitroso compounds in nitrate-containing human urine samples

A group-selective method for the determination of total N-nitroso compounds (NOC) has been adapted for analysing human urine samples. Nitrate was first removed from urine by an anion-exchange procedure that prevented the significant loss of various added reference NOC and unidentified urinary NOC. The total NOC were then determined by injecting the urine sample (nitrate content less than 1 mmol l-1) or anion-exchange eluate into refluxing ethyl acetate containing either acetic acid for determining heat and acetic acid labile thermal energy analyser responsive compounds (TAC) or into hydrogen bromide for the determination of TAC and NOC. The nitrogen monoxide levels released were measured using thermal energy analysis with chemiluminescence detection, and the differnce between the two determinations represented the concentrations of NOC. The optimum conditions for preventing artefactual nitrosation in urine samples by the addition of sodium hydroxide or sulphamic acid without decomposition of NOC were determined. The influence of time and storage conditions on NOC stability was investigated. Fifteen urine samples collected from volunteers dosed with proline were analysed for total NOC and N-nitrosamino acids revealing a preponderance of unknown NOC. The determination of total NOC in human urine using this group-selective method offers a new approach to the estimation of human exposure to NOC and to isolate hitherto unknown NOC and their metabolites.     

Screening of antinociceptive components in Corydalis yanhusuo W.T. Wang by comprehensive two-dimensional liquid chromatography/tandem mass spectrometry

Formalin-induced pain models were used in rats to evaluate the antinociceptive effect of the total alkaloids of Corydalis yanhusuo (TAC). The results indicated that formalin-evoked spontaneous nociceptive responses (licking behavior) could be inhibited significantly by giving (intragingival) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed. The absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (seven compounds identified) were absorbed into the plasma with appropriate concentrations and about 20 compounds (four compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine, and corydaline) which were reported to possess profound antinociceptive effects exhibited high concentrations in the striatum. This may result from their synergistic effects in regulating the formalin-induced nociception. The results indicated that the comprehensive two-dimensional biochromatography method developed is capable of screening the bioactive components in Corydalis yanhusuo and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.     

Protein–Incorporated Serum Total Antioxidant Capacity Measurement by a Modified CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method

Due to inadequacies in analytical methodology of total antioxidant capacity (TAC) measurement, proteins are initially separated from the human serum matrix by precipitation and are left unmeasured, thereby causing an important “antioxidant gap.” The aim of this work is to measure the TAC of serum with the modified CUPric Reducing Antioxidant Capacity (CUPRAC) method, and to identify the contribution of serum proteins, especially thiol-containing proteins, to TAC. CUPRAC results were statistically compared to those found by reference methods, namely ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric reducing antioxidant power), and Ellman thiols assay. The curves of absorbance vs thiol concentration, as well as of absorbance vs diluted serum (whole serum, trichloroacetic acid (TCA)?precipitated and redissolved serum protein solution, and TCA supernatant fractions) volume, of three distinct serum samples showed excellent linearity and low intercept values only with the modified CUPRAC method. The proposed method will help characterize the “antioxidant gap” of serum TAC originating from protein components which should not be neglected in future antioxidant measurements.     

Direct monitoring of glycohemoglobin A1c in the blood samples of diabetic patients by capillary electrophoresis. Comparison with an immunoassay method

The capillary electrophoresis (CE) study was focused on quantifying glycohemoglobin A1c, HbA1c, in whole blood samples of patients suffering diabetes mellitus. The results showed that dynamic polyionic coating of the capillary made the method very reproducible. The precision evaluation, method comparison and bias estimation in CE were performed during 20 days with patient blood samples and with four control samples. The influence of the storage time and temperature on the glycohemoglobin levels were also tested. High resolution in CE could be used to show evidences of the ageing of the samples stored at -70 degrees C. The results showed that the ageing peak was not originated from HbA1c, because it did not affect the HbA1c level which was in balance with the results of fresh samples measured with immunoassay. The HbA1c % values of blood samples of 105 patients measured with the CE technique varied between 3.6 and 11.8 and they were approximately 2-3% lower than measured with immunoassay. The correlation (R2) of CE results with immunoassay and HPLC results were 0.962 and 0.781, respectively.     

Comparative evaluation of Fe(III) reducing power-based antioxidant capacity assays in the presence of phenanthroline, batho-phenanthroline, tripyridyltriazine (FRAP), and ferricyanide reagents

The chemical diversity of antioxidants in complex matrices such as plant extracts makes it difficult to separate and quantify antioxidants from these solutions. Therefore it is desirable to establish methods that can measure the total antioxidant capacity (TAC) levels directly from plant extracts. Iron(III)-based TAC assays, especially the most widely used FRAP (ferric-reducing antioxidant power), play an important role in this regard. However, many problems have been reported in the application of the FRAP assay, the most serious one being the incomplete oxidation of a number of antioxidants during the time protocol of the assay. Thus, six different ferric ion-based total antioxidant capacity (TAC) assays have been comparatively tested, modified, and improved so as to obtain more sensitive and precise results for complex mixtures, namely: 1,10-phenanthroline (o-phen) method (with incubation), batho-phenanthroline method (with incubation), original FRAP method, modified FRAP method (with incubation), original ferricyanide method, and modified ferricyanide method (with incubation). Two new assays in this regard (i.e., o-phen and batho-phen) have been established, and the existing assays (FRAP and ferricyanide) have been modified so as to let the oxidation reactions of antioxidants reach completion. The molar absorptivity for a variety of antioxidants was highest for modified FRAP, batho-phen, and original FRAP methods. The absorption maximum wavelength shifted batochromically to a higher extent for modified ferricyanide, FRAP, and batho-phen procedures, decreasing the possibility of interferences due to organics absorbing in the near-UV range of the visible spectrum where most antioxidant assays are performed. The linear concentration ranges were shown to be further extended and linear correlation coefficients improved with respect to the most widely used ferric-based assay, FRAP. Of the six assays tested and developed, only the modified ferricyanide procedure gave high intercept values and low addivitity of TAC values of constituents in complex mixtures, requiring further attention of method optimization. Thus, it was shown that the most widely used FRAP could be effectively modified, and o-phen, batho-phen, and ferricyanide methods constitute cheaper alternatives to FRAP under certain conditions, with partly improved molar absorptivity (and thus sensitivity) for antioxidants, lower intercept values (and higher precision), broader linear range (and higher flexibility), and better additivity of TAC values of antioxidant constituents in mixtures.     

Simple and rapid method for the determination of uric acid-independent antioxidant capacity

Determination of the relative contribution of uric acid level increases to the total measured antioxidative activity could be very useful for testing antioxidative products and their effect on human health. The aim of this report is to present a simple spectrophotometric method that combines the measurement of total antioxidative capacity of a sample by ferric reducing/antioxidative power (FRAP) assay, with the uricase-reaction (specific elimination of uric acid), in order to establish and correct for the contribution of uric acid in FRAP values. We measured FRAP values, with (uric acid-independent antioxidant capacity, TAC-UA) and without (total antioxidant capacity, TAC) uricase treatment, and expressed it as μmol/L of uric acid equivalents. In such way, it was possible to determine both total and uric acid-independent antioxidant capacity, plasma uric acid (UA, as the difference between TAC and TAC-UA), and the ratio of the uric acid in total antioxidant capacity (UA/TAC).     

Simple Rapid Validated RP-LC Method for the Estimation of Flurbiprofen in Rabbit Serum and Aqueous Humor

Abstract

New reversed-phase liquid chromatographic methods, with UV detection, were developed for the quantitative estimation of flurbiprofen in rabbit blood serum and aqueous humor. The mobile phase and other chromatographic conditions were optimized to minimize interference from biological matrix and at the same time provide sufficient sensitivity for the method to be adopted for in vivo studies of ophthalmic formulations of flurbiprofen. Acetonitrile was used to precipitate proteins from serum or aqueous humor during sample preparation. A mobile phase of methanol: acetonitrile: phosphate buffer pH 5.6 (40:20:40) was employed with UV detection at 248 nm for estimation of drug in both the biological matrix. The retention time and asymmetry factor for the proposed method of estimation in serum and aqueous humor was found to be 3.1312±0.0101 min and 1.1310±0.0091 respectively. The linear regression equations obtained by least square regression method, were Area (µV sec) = 52.27 × Conc. (in ng/ml)–1618.70 in serum and Area (µV sec) = 61.79 × Conc. (in ng/ml) ? 783.24 in aqueous humor. The results of analysis were treated statistically, as per ICH guidelines for validation of analytical procedures, USP-2003, and by recovery studies. The results were found to be accurate, reproducible and free from interference. The developed methods were further used for estimation of flurbiprofen in rabbit serum and aqueous humor following single topical administration of in-house aqueous drop and market formulation to rabbit eye.     

Chiral recognition of octadentate Na complex with tetra-armed cyclen by molecularly imprinted polymers

Chiral indanyl substituted tetra-armed cyclens (TAC) formed octadentate complexes with Na⁺. Since their four side arms stand up and are bundled to form quadruplicated helical structures, they can have Δ- or Λ-types enantiomers based on complex helicity. In this study, TAC-imprinted polymers were prepared using an ion-pair complex of the sodium salt of (S)-indanyl substituted TAC (TAC(S)) and 2-sulfoethyl methacrylate as a template, and ethylene glycol dimethacrylate as a cross-linker. Affinity of the obtained polymers for the TAC Na⁺ complexes was evaluated chromatographically and the imprinted polymer gave longer retention time for the template than that for its antipode where the separation factor was given to be 1.29-1.45 under the NaOH concentrations of 10-25 mM in the eluent. These results indicate that the imprinted polymer could discriminate helix structures of TAC Na⁺ complexes.     

Expedient synthesis of a novel asymmetric selectively deprotectable derivative of the ATAC scaffold

An efficient multigram scale synthesis of a new asymmetric triazacyclophane scaffold, the ATAC (Asymmetric-TAC) scaffold, bearing three selectively removable groups is described. This scaffold is slightly more rigid than our frequently used TAC (TriAzaCyclophane) scaffold. The synthesis of the required triamine is very high yielding without difficult steps or purifications and was also applied to a much improved synthesis of our original TAC scaffold. Especially the tedious first reaction step, that is, mono-oNBS-protection of a triamine could be omitted. The rigidity of the triazacyclophane ring in both TAC- and ATAC scaffolds has also been investigated using variable temperature ¹H NMR experiments.     

Comparison of three colorimetric methods for the determination of manganese in freshwaters

Three calorimetric methods for the determination of manganese in freshwaters have been investigated and compared with the atomic-absorption spectrometry (AAS) method. Two of the methods, viz. the formaldoxime (FAD) and pyridylazonaphthol (PAN) methods were found to yield poor calibration curves upon standardization, and to have high average errors for the estimation of manganese concentrations in standard solutions. The FAD method, when applied to freshwater samples, was found to be subject to an interfering UV absorbance which could not be negated successfully by a reagent blank; the method thus tended to give high estimations at low concentrations of manganese, but the results obtained also appear to be very sensitive to the nature of the freshwater matrix. On the other hand the PAN method was found to give very high estimations for the concentration of manganese, in both standard solutions and in freshwater samples, owing to the nature of the absorbance of the ligand used for manganese determination. This method also appears to be affected by the freshwater matrix, leading to gross overestimation of manganese levels. A third method, based on a porphyrin ligand [T-(4-CP)P], was found to give results comparable with those of the AAS method in estimating the soluble manganese content in freshwaters.     

The use of 2-2-thiazolylazo-p-cresol to minimize the interference of Ni and Cu for the bismuth determination in alloys by hydride generation atomic absorption spectrometry

Thiazolylazo p-cresol (TAC) was studied as a masking agent for the determination of Bi in rich Ni and Cu alloys by hydride generation atomic absorption spectrometry (HGAAS). In the presence of TAC, Bi/Ni and Bi/Cu ratios up to 1:160 000 and 1:16 000, respectively, were found to be tolerable. No buffering was necessary once the masking agent was effective even in very acid medium. Limits of detection at the low mug g(-1) range were reached and the accuracy of the procedure was confirmed by the good concordance between found and certified values in the analysis of certified reference materials (CRM).     

Photocrosslinking of polyethylene. II. Properties of photocrosslinked polyethylene

Some properties of photocrosslinked high density polyethylene (2 mm thick sheets) have been studied. The homogeneity of the network is greatly improved by the application of triallylcyanurate (TAC) as crosslinking agent. The role of TAC in promoting the crosslinking is discussed. The crosslinked PE is found to be durable towards immersion in boiling water. Oxygen permeability increases while density and melting point decrease with increasing degree of crosslinking. The network chain density is obtained using conventional swelling method in boiling xylene, which together with data from melting point depression show that contribution of chain entanglement is insignificant to the gel formation of PE in this work.     

Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods

Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC_FRAP value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-l-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.     

Sequential flow injection determination of chlorine species using a triiodide-selective electrode detector.

A flow injection analysis (FIA) method for the determination of four residual chlorine species, namely combined available chlorine (CAC), free available chlorine (FAC), total available chlorine (TAC) and chlorite (ClO2-) was developed using a flow-through triiodide-selective electrode as a detector. An important strategy of speciation studies utilized the kinetic discrimination of reactions between the CAC and FAC with Fe2+, which was applied to the speciation of FAC, CAC and TAC. The speciation of available chlorine species and chlorite (an oxychlorine species) was achieved by using the same set-up, but using flow streams of different pH. The effects of the pH of the carrier stream, the flow rate and the sample volume were studied. The method exhibited linearity from 2.8 x 10(-6) to 2.8 x 10(-4) M active chlorine (expressed as OCl-) with a detection limit of 1.4 x 10(-6) M. The selectivity of the method was studied by examining the minimum pH for the oxidation of iodide by other oxidants, and also by assessing the potentiometric selectivity coefficients. The proposed method was successfully applied to the determination of chlorine species in tap water, and disinfecting formulations where good agreement occurred between the proposed and standard methods were found.     

In situ AFM studies on self-assembled monolayers of adsorbed surfactant molecules on well-defined H-terminated Si(111) surfaces in aqueous solutions

The formation of self-assembled monolayers (SAMs) of adsorbed cationic or anionic surfactant molecules on atomically flat H-terminated Si(111) surfaces in aqueous solutions was investigated by in situ AFM measurements, using octyl trimethylammonium chloride (C8TAC), dodecyl trimethylammonium chloride (C12TAC), octadecyl trimethylammonium chloride (C18TAC)) sodium dodecyl sulfate (STS), and sodium tetradecyl sulfate (SDS). The adsorbed surfactant layer with well-ordered molecular arrangement was formed when the Si(111) surface was in contact with 1.0x10(-4) M C18TAC, whereas a slightly roughened layer was formed for 1.0x10(-4) M C8TAC and C12TAC. On the other hand, the addition of alcohols to solutions of 1.0x10(-4) M C8TAC, C12TAC, or SDS improved the molecular arrangement in the adsorbed surfactant layer. Similarly, the addition of a salt, KCl, also improved the molecular arrangement for both the cationic and anionic surfactant layers. Moreover, the adsorbed surfactant layer with a well-ordered structure was formed in a solution of mixed cationic (C12TAC) and anionic (SDS) surfactants, though each surfactant alone did not form the well-ordered layer. These results were all explained by taking into account electrostatic repulsion between ionic head groups of adsorbed surfactant molecules as well as hydrophobic interaction between their alkyl chains, which increases with the increasing chain length, together with the increase in the hydrophobic interaction or the decrease in the electrostatic repulsion by incorporating alcohol molecules into the adsorbed surfactant layer, the decrease in the electrostatic repulsion by increasing the concentration of counterions, and the decrease in the electrostatic repulsion by alternate arrangement of cationic and anionic surfactant molecules. The present results have revealed various factors to form the well-ordered adsorbed surfactant layers on the H-Si(111) surface, which have a possibility of realizing the third generation surfaces with flexible structures and functions easily adaptable to circumstances.     

Measurement of237Np in the marine environment of coastal nuclear sites in India

Nepturium-237 levels in sea water and sediments from Indian coastal waters receiving radwaste discharges have been investigated. The method used for the estimation is neutron activation and using this method,²³⁷Np activity as low as 10^–7 Bq/l in sea water could be measured. Neptunium-237 levels found are compared with the values reported for other areas.     

A validated liquid chromatographic method for estimation of solasodine in various Solanum species and market formulations

This paper describes an improved liquid chromatographic (LC) method involving a prechromatographic derivatization step for the estimation of solasodine from berries of various Solanum species, market samples of Solanum xanthocarpum herb, extract, and its market formulations. Solasodine has heterocyclic nitrogen but has no conjugated double bonds in its structure. However, in all reported LC methods, detection was made in the ultraviolet range of 200-213 nm. In the present study, a prechromatographic derivatization of solasodine was done by forming an ion-pair complex of the heterocyclic nitrogen using the acidic dye methyl orange and acetate buffer of pH 4.7. Detection could be made in the visible range at 530 nm in this method. The method was validated and successfully applied to determine solasodine content in various plant samples and polyherbal formulations. The relative standard deviation was found to be 0.025% for system precision, and 0.8% for the linearity of the method, and the correlation coefficient was 0.999. Plant samples and market formulations were analyzed and found to contain solasodine in the range of 0.113-0.227% (w/w) on a fresh weight basis in berries; 0.3-1.278% (w/w) and 0.412% (w/w) on a dry weight basis in S. xanthocarpum herb powder and extract, respectively; and 0.245-0.525% (w/w) on dry weight basis in formulations containing S. xanthocarpum herb powder. No matrix interference was encountered. The method was found to be accurate, with a mean recovery of 100.5 +/- 0.83%. The method has good reproducibility and was found to be suitable for estimation of solasodine.