Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS to study the redox state and drug-mediated modulation in cells, worms and animal tissue.

Alterations in reduced and oxidized glutathione (GSH/GSSG) levels represent an important marker for oxidative stress and potential disease progression in toxicological research. Since GSH can be oxidized rapidly, using a stable and reliable method for sample preparation and GSH/GSSG quantification is essential to obtain reproducible data. Here we describe an optimised sample processing combined with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, validated for different biological matrices (lysates from HepG2 cells, C. elegans, and mouse liver tissue). To avoid autoxidation of GSH, samples were treated with the thiol-masking agent N-ethylmaleimide (NEM) and sulfosalicylic acid (SSA) in a single step. With an analysis time of 5 min, the developed LC-MS/MS method offers simultaneous determination of GSH and GSSG at high sample throughput with high sensitivity. This is especially interesting with respect of screening for oxidative and protective properties of substances in in vitro and in vivo models, e.g. C. elegans. In addition to method validation parameters (linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, interday, intraday), we verified the method by using menadione and L-buthionine-(S,R)-sulfoximine (BSO) as well established modulators of cellular GSH and GSSG concentrations. Thereby menadione proved to be a reliable positive control also in C. elegans.

Effect of vitamin K3 supplementation on immunoglobulin G concentration in colostrum of periparturient Holstein dairy cows.

This study was to examine the effects of dietary vitamin K (VK) 3 supplementation on immune-related substances in milk, oxidative stress indices in plasma and VK1, and menaquinone 4 (MK-4) in plasma and milk in periparturient dairy cows. Forty healthy perinatal Holstein-Friesian dairy cows were used in this study. Twenty-one animals were randomly selected and categorized into the VK3 supplemented (50 mg/day/head as VK3) group; the remaining 19 were categorized into the control group. On day 3 after calving, blood and milk were sampled, and their chemical components were determined. The VK3 supplemented group had significantly higher menaquinone 4 levels in plasma and milk on day 3 postpartum than the control group. In addition, there was a significant increase in the immunoglobulin G (IgG) level in milk. VK3 may be absorbed from the gastrointestinal tract and converted to MK-4, the biologically active form of VK, in the mammary gland and other tissues. It was thought that the increase in MK-4 level in plasma and milk induced an increase in the concentration of IgG in milk. VK3 supplementation to periparturient dairy cows may contribute to the production of colostrum with high concentrations of IgG and MK-4.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

Double strand DNA-based determination of menadione using a Fe3O4 nanoparticle decorated reduced graphene oxide modified carbon paste electrode.

In this work an electrochemical label free DNA biosensor (ds-DNA) for the determination of menadione (MD) was developed. The biosensor was constructed using a modified nanocomposite consisting of Fe3O4 nanoparticles decorated reduced graphene oxide (Gr) on a carbon paste electrode (CPE). Scanning electron microscope (SEM), energy dispersive X-ray (EDAX) and Fourier transform infrared (FT-IR) spectroscopy confirmed the structure of the synthesized nanocomposites (electrode composition). The Gr-Fe3O4 nanocomposites formed a sensitive layer with large surface area. Electrochemical studies revealed that modification of the electrode surface with ds-DNA and Gr- Fe3O4 nanocomposite significantly increases the oxidation peak currents and reduces the peak potentials of MD. Under the optimum conditions, calibration curve was linear in the range of 0.3-100.0 nM with a detection limit of 0.13 nM. The relative standard deviation for 50.0 nM was 3.90% (n = 5). The proposed biosensor was successfully applied to the determination of MD.

Determination of Menadione by Liquid Chromatography-Tandem Mass Spectrometry Using Pseudo Multiple Reaction Monitoring.

This study aimed to develop a menadione (MD) determination method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pseudo multiple reaction monitoring (MRM) technique, wherein two quadrupoles are used to monitor the same ion. Detection limits of 40 and 2 pg were obtained for MD and its deuterium-labeled form, respectively, whereas MD intra- and inter-assay coefficient of variation values were determined as 5.4 - 8.2%, with the corresponding recoveries equaling 90.5 - 109.6%. The developed method enables determination of MD in urine, plasma, cell extract, and culture media, demonstrating that pseudo multiple reaction monitoring can achieve quantification of compounds forming no suitable product ions, such as MD.

Comparing monolithic and fused core HPLC columns for fast chromatographic analysis of fat-soluble vitamins.

HPLC stationary phases of monolithic and fused core type can be used to achieve fast chromatographic separation as an alternative to UPLC. In this study, monolithic and fused core stationary phases are compared for fast separation of four fat-soluble vitamins. Three new methods on the first and second generation monolithic silica RP-18e columns and a fused core pentafluoro-phenyl propyl column were developed. Application of three fused core columns offered comparable separations of retinyl palmitate, DL-α-tocopheryl acetate, cholecalciferol and menadione in terms of elution speed and separation efficiency. Separation was achieved in approx. 5 min with good resolution (Rs > 5) and precision (RSD ≤ 0.6 %). Monolithic columns showed, however, a higher number of theoretical plates, better precision and lower column backpressure than the fused core column. The three developed methods were successfully applied to separate and quantitate fat-soluble vitamins in commercial products.

High performance liquid chromatography analysis of aliphatic thiols in alimentary supplements and pharmaceuticals using menadione as a new useful derivatization reagent.

The use of menadione (MD) as a pre-column reagent for high performance liquid chromatography (HPLC) analysis of aliphatic thiols is proposed. The reaction was carried out for 5 min at room temperature and pH 8.5. The developed method was applied to the N-acetylcysteine (NAC) analysis of alimentary supplements and pharmaceutical formulations. The effect of the complex matrix was evaluated by the study of the thiol derivatization reaction both in standard and in placebo solutions. The yield of NAC-MD adduct was found to be quantitative at a reagent to thiol molar ratio of about 4 in comparison with an authentic specimen of synthesized NAC adduct, which was characterized by (1)H NMR, IR and UV. The routine chromatographic separations were performed on a Synergi MAX-RP column using a mobile phase consisting of methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol L(-1)) 70:30 (v/v) at a flow-rate of 0.4 mL min(-1). UV-diode array detection was used setting the wavelength at λ=260 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, selectivity and ruggedness were found to be highly satisfactory. Similar linear responses were observed by standard and placebo solutions (determination coefficient: 0.9996). Limit of detection was about 0.019 µg g(-1). Intra-day precision (relative standard deviation, R.S.D.) was ≤0.81% for NAC to internal standard (IS) peak area ratio, ≤0.28% and ≤0.32%, respectively, for NAC and IS retention times (tR), without significant differences between intra- and inter-day data. NAC recovery studies gave good results (100.12%) with R.S.D.=1.05%.

Dispersive liquid-liquid microextraction for the determination of vitamins D and K in foods by liquid chromatography with diode-array and atmospheric pressure chemical ionization-mass spectrometry detection.

A simple and rapid method was developed using reversed-phase liquid chromatography (LC) with both diode array (DAD) and atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection, for the simultaneous analysis of the vitamins ergocalciferol (D2), cholecalciferol (D3), phylloquinone (K1), menaquinone-4 (K2) and a synthetic form of vitamin K, menadione (K3). The Taguchi experimental method, an orthogonal array design (OAD), was used to optimize an efficient and clean preconcentration step based on dispersive liquid-liquid microextraction (DLLME). A factorial design was applied with six factors and three levels for each factor, namely, carbon tetrachloride volume, methanol volume, aqueous sample volume, pH of sample, sodium chloride concentration and time of the centrifugation step. The DLLME optimized procedure consisted of rapidly injecting 3 mL of acetonitrile (disperser solvent) containing 150 µL carbon tetrachloride (extraction solvent) into the aqueous sample, thereby forming a cloudy solution. Phase separation was performed by centrifugation, and the sedimented phase was evaporated with nitrogen, reconstituted with 50 µL of acetonitrile, and injected. The LC analyses were carried out using a mobile phase composed of acetonitrile, 2-propanol and water, under gradient elution. Quantification was carried out by the standard additions method. The APCI-MS spectra, in combination with UV spectra, permitted the correct identification of compounds in the food samples. The method was validated according to international guidelines and using a certified reference material. The validated method was applied for the analysis of vitamins D and K in infant foods and several green vegetables. There was little variability in the forms of vitamin K present in vegetables, with the most abundant vitamer in all the samples being phylloquinone, while menadione could not be detected. Conversely, cholecalciferol, which is present in food of animal origin, was the main form in infant foods, while ergocalciferol was not detected.

Menadione.

This chapter includes the aspects of Menadione (vitamin K). The drug is synthesized by the use of itaconic acid obtained through Friedel-Craft condensation or by direct oxidation of the 2-methyl-1,4-naphthquinone. Vitamin K generally maintains healthy blood clotting and prevents excessive bleeding and hemorrhage, it is also important for maintaining healthy bone structure and for carbohydrate storage in the body. In addition, it is given to newborn babies born in hospitals to prevent the development of life-threatening bleeding caused by low prothrombin levels. The chapter discusses the drug metabolism and pharmacokinetics and presents various method of analysis of this drug such as compendial tests, electrochemical analysis, spectroscopic analysis, and chromatographic techniques of separation. It also discusses its physical properties such as solubility characteristics, X-ray powder diffraction pattern, and thermal methods of analysis. The chapter is concluded with a discussion on its biological properties such as activity, toxicity, and safety.

Continuous flow-extractive desorption electrospray ionization: analysis from "non-electrospray ionization-friendly" solvents and related mechanism.

Due to their low polarities and dielectric constants, analytes in solvents such as hexane, chloroform, and ethyl acetate exhibit poor electrospray ionization (ESI) efficiency. These are deemed to be "non-ESI-friendly" solvents. Continuous flow extractive desorption electrospray ionization (CF-EDESI) is a novel ambient ionization technique that was recently developed in our group to manipulate protein charge distributions. Here we demonstrate its potential for ionizing analytes from non-ESI-friendly solvents. This feature makes CF-EDESI attractive to the general analytical community due to its apparent potential in lipidomics, normal phase separations, and hyphenation of mass spectrometry with HPLC-NMR systems. In this context, interest was subsequently initiated to discern mechanistic aspects of CF-EDESI. To achieve this, mechanistic experiments associated with a seemingly similar ambient ionization technique, extractive electrospray ionization (EESI), were emulated to compare CF-EDESI and EESI. Analysis of a series of fatty acids in multiple solvents in the negative ionization mode revealed differences between the two techniques. Whereas EESI has been previously shown to operate via extraction of analytes into the spray solvent, data presented here for CF-EDESI point toward a liquid-liquid mixing process to facilitate ionization. Further, a partial factorial design experiment was performed to evaluate the effects of different experimental variables on signal intensity. Sample flow rate was confirmed to be among the most significant factors to affect sensitivity. As a whole, the work presented provides greater insight into a new ambient ionization process, which exhibits expanded capabilities over conventional ESI; in this case, for direct analysis from non-ESI-friendly solvents.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid.

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

Calixarene capped ZnS quantum dots as an optical nanoprobe for detection and determination of menadione.

In this communication we report a p-sulfonatocalix[4]arene coated ZnS quantum dots "cup type" highly stable optical probe for the detection and determination of menadione (VK(3)) with high sensitivity and selectivity. The detection of VK(3) depends on supramolecular host-guest chemistry.

Direct and indirect methods for the determination of vitamin K3 using differential pulse polarography and application to pharmaceuticals.

Two methods for the determination of vitamin K(3) have been developed. Vitamin K(3) in its oxidized form is determined by direct and indirect methods. Its standard solution was prepared by the indirect method using Ti(III) as reducing agent. For this purpose vitamin K(3) (menadion) in a clinical injection solution, which is in its hydroquinone form in the presence of sulfite, is oxidized with oxygen. In 0.2 M HAc and 0.02 M HCl electrolyte vitamin K(3) and Ti(IV) have reduction peaks at -0.58 V at -0.82 V respectively. The reaction between Ti(III) and vitamin takes place quantitatively in a medium of 0.2 M HAc and 0.002 M HCl. After the reduction, the reaction product Ti(IV) is followed from its polarographic peak at about -0.82 V. The most important result in this work is that, with this method vitamin K(3) can be standardized and after standardization this solution can be used for the direct determination in routine analysis with a very simple and fast method, using only the peak at -0.71 V in 0.2 M HAc medium. Both direct and indirect methods have been used for the determination of Vitamin K(3) in a clinical injection solution. The limit of quantification (LOQ) was 1.5x10(-6) M and in both methods the detection limit found was 7x10(-7) M.

Simultaneous determination of five fat-soluble vitamins in feed by high-performance liquid chromatography following solid-phase extraction.

A high-performance liquid chromatography method is developed for the simultaneous determination of menadione, retinyl acetate, cholecalciferol, alpha-tocopherol, and alpha-tocopherol acetate in feed. The present study uses an enzyme to destroy the coating film, ethanol to extract free vitamins, and Oasis HLB cartridges to purify. Vitamins are separated using an Atlantis dC18 column. The mobile phase is methanol-water (98:2 v/v). Detection is performed with a UV-vis detector at 230 and 265 nm. The linearity, accuracy, and repeatability of this method are all satisfactory. Application of the method is suitable for the determination of the fat-soluble vitamins in general feed.

Spectrofluorimetric study on the inclusion interaction between vitamin K3 with p-(p-sulfonated benzeneazo)calix[6]arene and determination of VK3.

The characteristics of host-guest complexation between p-(p-sulfonated benzeneazo) calix[6]arene (SBC6A) and vitamin K3 (VK3) were investigated by fluorescence spectrometry. A 1:1 stoichiometry for the complexation was established and was verified by Job's plot. An association constant of 4.95 x 10(3)L mol(-1) at 20 degrees C was calculated by applying a deduced equation. The interaction mechanism of the inclusion complex was discussed. It was found that the fluorescence of SBC6A could be remarkably quenched by an appropriate amount of VK3 especially when non-ionic surfactant Triton X-100 existed. According to the obtained results, a novel sensitive spectrofluorimetric method for the determination of VK3 based on supramolecular complex was developed with a linear range of 5.0 x 10(-7) -3.0 x 10(-5)mol L(-1) and a detection limit of 2.0 x 10(-7)mol L(-1). The proposed method was used to determine VK3 in commercial preparations with satisfactory results.

Identification and quantitation of vitamins K1 and K3 in cosmetic products for facial skin protection.

A simple and rapid analytical method was developed for the determination of vitamins K1 and K3 in facial anti-rash creams. The procedure is based on an ultrasonic extraction of the cosmetic sample with dimethylacetamide, in the presence of an internal standard, followed by HPLC separation. HPLC was performed using a C18 column and spectrophotometric detection at 333 nm. A linear gradient elution was carried out starting with 50% acetonitrile-methanol (75:25 v/v) and water up to 100% acetonitrile-methanol for 5 min. Linearity was established over the concentration range from 0.2 to 1.0 mg/ml for vitamin K1 and from 0.02 to 0.1 mg/ml for vitamin K3, with LOD values of 100 ng and 20 ng injected, respectively. The accuracy was verified by spiking experiments on model cosmetic samples. The proposed method has been successfully applied for the analysis of commercial samples of creams.

Vitamin K deficiency of germfree mice caused by feeding standard purified diet sterilized by gamma-irradiation.

Germfree mice died when they were fed a purified diet of AIN-76 formula sterilized by gamma-irradiation. Vitamin K deficiency was suspected and this study was performed to confirm the cause of the death. Germfree mice were fed purified diets of AIN-76 or AIN-93M formula, which were pelleted and sterilized by gamma-irradiation at a dose of 50 kGy. One half of the mice fed the AIN-76 diet died within two weeks and the surviving animals were also in poor health, while 91% of mice fed the AIN-93M diet survived. No hemorrhage was observed grossly in any organs of the surviving animals. Histologically, degeneration with inflammatory cell infiltration was observed as well as hemorrhage and fibrosis in the heart muscles of mice fed the AIN-76 diet. No microscopic lesions were observed in the other organs. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were extremely prolonged when mice were fed the AIN-76 diet. The animals totally recovered when they were intragastrically administered 1 microg/day of vitamin K(3) from the third day of feeding of the AIN-76 diet, except for PT and APTT which were still slightly longer than in mice fed the AIN-93M diet. The concentration of vitamin K(3) supplied in the AIN-76 diet decreased to an undetectable level after gamma-irradiation, while the AIN-93M diet contained 240 microg/kg of vitamin K(1). These results indicate that the deaths of the germfree mice fed the gamma-irradiated AIN-76 diet were caused by vitamin K deficiency. Vitamin K deficiency may cause fatal degeneration of cardiac muscle cells.

Electrochemical evaluation of the inhibitory effects of weak acids on Zygosaccharomyces bailii.

The changes in intracellular redox activity or in mitochondrial electron transport could be taken as indications of the changes in the physiological state of living cells, based on which a mediated electrochemical method was purposed to evaluate the inhibitory effects of weak acids on Zygosaccharomyces bailii, a known food spoilage yeast. The dual mediator systems menadione/ferricyanide and 2,6-dichlorophenolindophenol/ferricyanide were employed as probes to detect the variance in intracellular redox activity and in mitochondrial electron flux, respectively. Measurements were made with a microelectrode voltammetric method to assay the ferrocyanide accumulations arising from menadione- or 2,6-dichlorophenolindophenol-mediated reduction of ferricyanide by Z. bailii suspensions by the presence or absence of increasing concentrations of weak acids. The results obtained from 2 h of incubation showed that the variance in electrochemical response revealed some physiological information underlying the inhibitory effects of weak acid on the yeast. For the first time, it was shown that the mediated electrochemical method provides an adjunct to the conventional method based on respiration inhibition for establishing levels for the utilization of preservatives in the food industry.

Spectrophotometric methods for the rapid determination of menadione and menadione sodium bisulphite and their application in pharmaceutical preparations.

Two simple, rapid and sensitive spectrophotometric determination of menadione and its sodium bisulphite derivative (MSB) have been carried out. The first method involves the reaction of menadione and its sodium bisulphite derivative with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) is sodium hydroxide medium to give blue coloured product having maximum absorption at 625 nm and the coloured species is stable for more than 1 h. The Beer's law is obeyed in the range 0.4-16 microg ml(-1). The second method proposes the reaction of menadione and its sodium bisulphite derivative with resorcinol in concentrated sulphuric acid medium to give red coloured product having maximum absorption at 520 nm and is stable for 3 h. The Beer's law was obeyed in the range of 1-24 microg ml(-1). Molar absorptivity for the above two methods were found to be 7.6 x 10(3) and 4.5 x 10(3) l mol(-1) cm(-1), respectively. All the measurements were carried out at room temperature. These two methods have been successfully applied for menadione and its sodium bisulphite derivatives in injections and tablets of pharmaceutical formulations. The results compare favourably with official method.