The methaemoglobin forming and GSH depleting effects of dapsone and monoacetyl dapsone hydroxylamines in human diabetic and non-diabetic erythrocytes in vitro

The respective methaemoglobin forming and GSH depleting capabilities of monoacetyl dapsone hydroxylamine (MADDS-NHOH) and dapsone hydroxylamine (DDS-NHOH) were compared in human diabetic and non-diabetic erythrocytes in vitro with a view to select the most potent agent for future oxidative stress and antioxidant evaluation studies. Administration of both metabolites to non-diabetic erythrocytes over the 20 min period of the study resulted in significantly more methaemoglobin formation at all four time points compared with the diabetic erythrocytes (P<0.0001). At all four time points, significantly more methaemoglobin was formed in response to MADDS-NHOH in non-diabetic cells compared with the effects of DDS-NHOH on diabetic erythrocytes (P<0.0001). At the 5 and 10 min time points, significantly more methaemglobin was formed in non-diabetic cells in the presence of MADDS-NHOH compared with DDS-NHOH (P<0.05). At the 5 min time point only, significantly more methaemoglobin was formed in the presence of MADDS-NHOH in diabetic cells compared with that of DDS-NHOH (P<0.01). However, compared with diabetic control GSH levels, the presence of DDS-NHOH caused a significant depletion in GSH at 5, 10 and 20 min time points in diabetic cells (P<0.001). In addition, the presence of DDS-NHOH caused a significant reduction in GSH levels in diabetic cells in comparison with those of non-diabetics at the 5, 10 and 20 min, (P<0.005). DDS-NHOH was also associated with a significant depletion of GSH levels in diabetic cells compared with those of non-diabetic control erythrocytes (P<0.0001). The presence of MADDS-NHOH in diabetic erythrocytes led to a significant reduction in GSH levels at the 20 min time point compared with those of non-diabetics (P<0.001), but there were no significant differences at the 5, 10 and 15 min points. Due to its greater GSH-depleting action, DDS-NHOH will be selected for future use in the oxidative stress assessment in diabetic erythrocytes. © 2004 Elsevier B.V. All rights reserved.

Effects of dihydrolipoic acid (DHLA), α-lipoic acid. N-acetyl cysteine and ascorbate on xenobiotic-mediated methaemoglobin formation in human erythrocytes in vitro

α-Lipoic acid, dihydrolipoic acid (DHLA), N-acetyl cysteine and ascorbate were compared with methylene blue for their ability to attenuate and/or reduce methaemoglobin formation induced by sodium nitrite, 4-aminophenol and dapsone hydroxylamine in human erythrocytes. Neither α-lipoic acid, DHLA, N-acetyl cysteine nor ascorbate had any significant effects on methaemoglobin formed by nitrite, either from pre-treatment, simultaneous addition or post 30 min addition of the agents up to the 60 min time point, although N-acetyl cysteine did reduce methaemoglobin formation at 120 min (P<0.05). In all three treatment groups at 30, 60 and 120 min, there were no significant effects mediated by DHLA or N-acetyl cysteine on 4-aminophenol (1 mM)-mediated haemoglobin oxidation. Ascorbate caused marked significant reductions in 4-aminophenol methaemoglobin in all treatment groups at 30-120 min except at 30 min in the simultaneous addition group (P<0.0001). Neither α-lipoic acid, nor N-acetyl cysteine showed any effects on hydroxylamine-mediated methaemoglobin formation at 30 and 60 in all treatment groups. In contrast, DHLA significantly reduced hydroxylamine-mediated methaemoglobin formation at all three time points after pre-incubation and simultaneous addition (P<0.001), while ascorbate was ineffective. Compared with methylene blue, which was effective in reducing methaemoglobin formation by all three toxins (P<0.01), ascorbate was only highly effective against 4-aminophenol mediated methaemoglobin, whilst the DHLA-mediated attenuation of dapsone hydroxylamine-mediated methaemoglobin formation indicates a possible clinical application in high-dose dapsone therapy. © 2003 Elsevier B.V. All rights reserved.

A preliminary evaluation of a novel method to monitor a triple antioxidant combination (vitamins E, C and alpha-lipoic acid) in diabetic volunteers using in vitro methaemoglobin formation

Eight otherwise healthy diabetic volunteers took a daily antioxidant supplement consisting of vitamin E (200 IU), vitamin C (250 mg) and α-lipoic acid (90 mg) for a period of 6 weeks. Diabetic dapsone hydroxylamine-mediated methaemoglobin formation and resistance to erythrocytic thiol depletion was compared with age and sex-matched non-diabetic subjects. At time zero, methaemoglobin formation in the non-diabetic subjects was greater at all four time points compared with that of the diabetic subjects. Resistance to glutathione depletion was initially greater in non-diabetic compared with diabetic samples. Half-way through the study (3 weeks), there were no differences between the two groups in methaemoglobin formation and thiol depletion in the diabetic samples was now lower than the non-diabetic samples at 10 and 20 min. At 6 weeks, diabetic erythrocytic thiol levels remained greater than those of non-diabetics. HbA1c values were significantly reduced in the diabetic subjects at 6 weeks compared with time zero values. At 10 weeks, 4 weeks after the end of supplementation, the diabetic HbA1c values significantly increased to the point where they were not significantly different from the time zero values. Total antioxidant status measurement (TAS) indicated that diabetic plasma antioxidant capacity was significantly improved during antioxidant supplementation. Conversion of α-lipoic acid to dihydrolipoic acid (DHLA) in vivo led to potent interference in a standard fructosamine assay kit, negating its use in this study. This report suggests that triple antioxidant therapy in diabetic volunteers attenuates the in vitro experimental oxidative stress of methaemoglobin formation and reduces haemoglobin glycation in vivo. © 2003 Elsevier Science B.V. All rights reserved.

Risk factors for childhood myopia:findings from the NICER study

PURPOSE. We explored risk factors for myopia in 12- to 13-year-old children in Northern Ireland (NI). METHODS. Stratified random sampling was performed to obtain representation of schools and children. Cycloplegia was achieved using cyclopentolate hydrochloride 1%. Distance autorefraction was measured using the Shin-Nippon SRW-5000 device. Height and weight were measured. Parents and children completed a questionnaire, including questions on parental history of myopia, sociodemographic factors, childhood levels of near vision, and physical activity to identify potential risk factors for myopia. Myopia was defined as spherical equivalent ≤0.50 diopters (D) in either eye. RESULTS. Data from 661 white children aged 12-to 13-years showed that regular physical activity was associated with a lower estimated prevalence of myopia compared to sedentary lifestyles (odds ratio [OR] = 0.46 adjusted for age, sex, deprivation score, family size, school type, urbanicity; 95% confidence interval [CI], 0.23–0.90; P for trend = 0.027). The odds of myopia were more than 2.5 times higher among children attending academically-selective schools (adjusted OR = 2.66; 95% CI, 1.48–4.78) compared to nonacademically-selective schools. There was no evidence of an effect of urban versus nonurban environment on the odds of myopia. Compared to children with no myopic parents, children with one or both parents being myopic were 2.91 times (95% CI, 1.54–5.52) and 7.79 times (95% CI, 2.93– 20.67) more likely to have myopia, respectively. CONCLUSIONS. In NI children, parental history of myopia and type of schooling are important determinants of myopia. The association between myopia and an environmental factor, such as physical activity levels, may provide insight into preventive strategies.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Microporous polycaprolactone matrices for drug delivery and tissue engineering:the release behaviour of bioactives having extremes of aqueous solubility

Microporous polycaprolactone (PCL) matrices loaded with hydrophobic steroidal drugs or a hydrophilic drug - pilocarpine hydrochloride - were produced by precipitation casting using solutions of PCL in acetone. The efficiency of steroid incorporation in the final matrix (progesterone (56 %) testosterone (46 %) dexamethasone (80 %)) depended on the nature of the drug initially co-dissolved in the PCL solution. Approximately 90 % w/w of the initial load of progesterone, 85 % testosterone and 50 % dexamethasone was released from the matrices in PBS at 37°C over 8 days. Pilocarpine hydrochloride (PH)-loaded PCL matrices, prepared by dispersion of powder in PCL solution, released 70-90 % of the PH content over 12 days in PBS. Application of the Higuchi model revealed that the kinetics of steroid and PH release were consistent with a Fickian diffusion mechanism with corresponding diffusion coefficients of 5.8 × 10-9 (progesterone), 3.9 × 10 -9 (testosterone), 7.1 × 10-10 (dexamethasone) and 22 × 10-8 cm2/s (pilocarpine hydrochloride). The formulation techniques described are expected to be useful for production of implantable, insertable and topical devices for sustained delivery of a range of bioactive molecules of interest in drug delivery and tissue engineering.

System-on-a-Chip (SOC) based environmental monitoring platform for the detection of hazardous materials

The purpose of this research is design considerations for environmental monitoring platforms for the detection of hazardous materials using System-on-a-Chip (SoC) design. Design considerations focus on improving key areas such as: (1) sampling methodology; (2) context awareness; and (3) sensor placement. These design considerations for environmental monitoring platforms using wireless sensor networks (WSN) is applied to the detection of methylmercury (MeHg) and environmental parameters affecting its formation (methylation) and deformation (demethylation). ^ The sampling methodology investigates a proof-of-concept for the monitoring of MeHg using three primary components: (1) chemical derivatization; (2) preconcentration using the purge-and-trap (P&T) method; and (3) sensing using Quartz Crystal Microbalance (QCM) sensors. This study focuses on the measurement of inorganic mercury (Hg) (e.g., Hg2+) and applies lessons learned to organic Hg (e.g., MeHg) detection. ^ Context awareness of a WSN and sampling strategies is enhanced by using spatial analysis techniques, namely geostatistical analysis (i.e., classical variography and ordinary point kriging), to help predict the phenomena of interest in unmonitored locations (i.e., locations without sensors). This aids in making more informed decisions on control of the WSN (e.g., communications strategy, power management, resource allocation, sampling rate and strategy, etc.). This methodology improves the precision of controllability by adding potentially significant information of unmonitored locations.^ There are two types of sensors that are investigated in this study for near-optimal placement in a WSN: (1) environmental (e.g., humidity, moisture, temperature, etc.) and (2) visual (e.g., camera) sensors. The near-optimal placement of environmental sensors is found utilizing a strategy which minimizes the variance of spatial analysis based on randomly chosen points representing the sensor locations. Spatial analysis is employed using geostatistical analysis and optimization occurs with Monte Carlo analysis. Visual sensor placement is accomplished for omnidirectional cameras operating in a WSN using an optimal placement metric (OPM) which is calculated for each grid point based on line-of-site (LOS) in a defined number of directions where known obstacles are taken into consideration. Optimal areas of camera placement are determined based on areas generating the largest OPMs. Statistical analysis is examined by using Monte Carlo analysis with varying number of obstacles and cameras in a defined space. ^

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

Novel analytical methodologies for the monitoring of traditional and non-traditional pollutants in different environmental compartments of South Florida

Routine monitoring of environmental pollution demands simplicity and speed without sacrificing sensitivity or accuracy. The development and application of sensitive, fast and easy to implement analytical methodologies for detecting emerging and traditional water and airborne contaminants in South Florida is presented. A novel method was developed for quantification of the herbicide glyphosate based on lyophilization followed by derivatization and simultaneous detection by fluorescence and mass spectrometry. Samples were analyzed from water canals that will hydrate estuarine wetlands of Biscayne National Park, detecting inputs of glyphosate from both aquatic usage and agricultural runoff from farms. A second study describes a set of fast, automated LC-MS/MS protocols for the analysis of dioctyl sulfosuccinate (DOSS) and 2-butoxyethanol, two components of Corexit®. Around 1.8 million gallons of those dispersant formulations were used in the response efforts for the Gulf of Mexico oil spill in 2010. The methods presented here allow the trace-level detection of these compounds in seawater, crude oil and commercial dispersants formulations. In addition, two methodologies were developed for the analysis of well-known pollutants, namely Polycyclic Aromatic Hydrocarbons (PAHs) and airborne particulate matter (APM). PAHs are ubiquitous environmental contaminants and some are potent carcinogens. Traditional GC-MS analysis is labor-intensive and consumes large amounts of toxic solvents. My study provides an alternative automated SPE-LC-APPI-MS/MS analysis with minimal sample preparation and a lower solvent consumption. The system can inject, extract, clean, separate and detect 28 PAHs and 15 families of alkylated PAHs in 28 minutes. The methodology was tested with environmental samples from Miami. Airborne Particulate Matter is a mixture of particles of chemical and biological origin. Assessment of its elemental composition is critical for the protection of sensitive ecosystems and public health. The APM collected from Port Everglades between 2005 and 2010 was analyzed by ICP-MS after acid digestion of filters. The most abundant elements were Fe and Al, followed by Cu, V and Zn. Enrichment factors show that hazardous elements (Cd, Pb, As, Co, Ni and Cr) are introduced by anthropogenic activities. Data suggest that the major sources of APM were an electricity plant, road dust, industrial emissions and marine vessels.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Micropartículas de quitosana estruturadas com aerosil®: estabilidade, adsorção, encapsulação e liberação de substâncias ativas

Biodegradable microspheres used as controlled release systems are important in pharmaceutics. Chitosan biopolymer represents an attractive biomaterial alternative because of its physicochemical and biological characteristics. Chitosan microspheres are expected to become promising carrier systems for drug and vaccine delivery, especially for non-invasive ways oral, mucosal and transdermal routes. Controlling the swelling rate and swelling capacity of the hydrogel and improving the fragile nature of microspheres under acidic conditions are the key challenges that need to be overcomed in order to enable the exploration of the full pharmaceutical potential use of these microparticles. Many studies have focused on the modification of chitosan microsphere structures with cross-linkers, various polymers blends and new organic-inorganic hybrid systems in order to obtain improved properties. In this work, microspheres made of chitosan and nanosized hydrophobic silica (Aerosil R972) were produced by a method consisting of two steps. First, a preparation of a macroscopically homogeneous chitosan-hydrophobic silica dispersion was prepared followed by spray drying. FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM) were used to characterize the microspheres. Also, the were conducted acid stability, moisture sorption capacity, release properties and biological assays. The chitosan-hydrophobic silica composite microspheres showed improved thermal degradation, lower water affinity, better acid stability and ability to retard rifampicin and propranolol hydrochloride (drug models) release under simulated physiological conditions. In vitro biocompatibility studies indicated low cytotoxicity and low capacity to activate cell production of the pro-inflammatory mediator nitric oxide. The results show here encourage further studies on the use of the new chitosan-hydrophobic silica composite microspheres as drug carrier systems via oral or nasal routes.

Síntese, caracterização e estudo da associação em meio aquoso de copolímeros responsivos de carboximetilcelulose-g-Jeffamine®

This thesis presents the synthesis, characterization and study of the associative behaviour in aqueous media of new responsive graft copolymers, based on carboxymethylcellulose as the water-soluble backbone and Jeffamine® M-2070 e Jeffamine® M-600 (commercial polyetheramines) as the thermoresponsive grafts with high cloud point temperatures in water. The synthesis was performed on aqueous medium, by using 1-ethyl-3- (3-(dimethylamino)-propyl)carbodiimide hydrochloride and N-hydroxysuccinimide as activators of the reaction between carboxylategroupsfrom carboxymethylcellulose and amino groups from polyetheramines. The grafting reaction was confirmed by infrared spectroscopy and the grafting percentage by 1H NMR. The molar mass of the polyetheramines was determined by 1H NMR, whereas the molar mass of CMC and graft copolymers was determined by static light scattering. The salt effect on the association behaviour of the copolymers was evaluated in different aqueous media (Milli-Q water, 0.5M NaCl, 0.5M K2CO3 and synthetic sea water), at different temperatures, through UV-vis, rheology and dynamic light scattering. None of the copolymers solutions, at 5 g/L, turned turbid in Milli-Q water when heated from 25 to 95 °C, probably because of the increase in hydrophibicity promoted by CMC backbone. However, they became turbid in the presence of salts, due to the salting out effect, where the lowest cloud point was observed in 0.5M K2CO3, which was attributed to the highest ionic strength in water, combined to the ability of CO3 2- to decrease polymer-solvents interactions. The hydrodynamic radius and apparent viscosity of the copolymers in aqueous medium changed as a function of salts dissolved in the medium, temperature and copolymer composition. Thermothickening behaviour was observed in 0.5M K2CO3 when the temperature was raised from 25 to 60°C. This performance can be attributed to intermolecular associations as a physical network, since the temperature is above the cloud point of the copolymers in this solvent.

Analysis of bio-derived platform chemicals in ionic liquid media by desorption/ionization mass spectrometry

In this work, desorption/ionization mass spectrometry was employed for the analysis of sugars and small platform chemicals that are common intermediates in biomass transformation reactions. Specifically, matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometric techniques were employed as alternatives to traditional chromatographic methods. Ionic liquid matrices (ILMs) were designed based on traditional solid MALDI matrices (2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA)) and 1,3-dialkylimidazolium ionic liquids ([BMIM]Cl, [EMIM]Cl, and [EMIM]OAc) that have been employed as reaction media for biomass transformation reactions such as the conversion of carbohydrates to valuable platform chemicals. Although two new ILMs were synthesized ([EMIM][DHB] and [EMIM][CHCA] from [EMIM]OAc), chloride-containing ILs did not react with matrices and resulted in mixtures of IL and matrix in solution. Compared to the parent solid matrices, much less matrix interference was observed in the low mass region of the mass spectrum (< 500 Da) using each of the IL-matrices. Furthermore, the formation of a true ILM (i.e. a new ion pair) does not appear to be necessary for analyte ionization. MALDI sample preparation techniques were optimized based on the compatibility with analyte, IL and matrix. ILMs and IL-matrix mixtures of DHB allowed for qualitative analysis of glucose, fructose, sucrose and N-acetyl-D-glucosamine. Analogous CHCA-containing ILMs did not result in appreciable analyte signals under similar conditions. Small platform compounds such as 5-hydroxymethylfurfural (HMF) and levulinic acid were not detected by direct analysis using MALDI-MS. Furthermore, sugar analyte signals were only detected at relatively high matrix:IL:analyte ratios (1:1:1) due to significant matrix and analyte suppression by the IL ions. Therefore, chemical modification of analytes with glycidyltrimethylammonium chloride (GTMA) was employed to extend this method to quantitative applications. Derivatization was accomplished in aqueous IL solutions with fair reaction efficiencies (36.9 – 48.4 % glucose conversion). Calibration curves of derivatized glucose-GTMA yielded good linearity in all solvent systems tested, with decreased % RSDs of analyte ion signals in IL solutions as compared to purely aqueous systems (1.2 – 7.2 % and 4.2 – 8.7 %, respectively). Derivatization resulted in a substantial increase in sensitivity for MALDI-MS analyses: glucose was reliably detected at IL:analyte ratios of 100:1 (as compared to 1:1 prior to derivatization). Screening of all test analytes resulted in appreciable analyte signals in MALDI-MS spectra, including both HMF and levulinic acid. Using appropriate internal standards, calibration curves were constructed and this method was employed for monitoring a model dehydration reaction of fructose to HMF in [BMIM]Cl. Calibration curves showed wide dynamic ranges (LOD – 100 ng fructose/μg [BMIM]Cl, LOD – 75 ng HMF/μg [BMIM]Cl) with correlation coefficients of 0.9973 (fructose) and 0.9931 (HMF). LODs were estimated from the calibration data to be 7.2 ng fructose/μg [BMIM]Cl and 7.5 ng HMF/μg [BMIM]Cl, however relatively high S/N ratios at these concentrations indicate that these values are likely overestimated. Application of this method allowed for the rapid acquisition of quantitative data without the need for prior separation of analyte and IL. Finally, small molecule platform chemicals HMF and levulinic acid were qualitatively analyzed by DESI-MS. Both HMF and levulinic acid were easily ionized and the corresponding molecular ions were easily detected in the presence of 10 – 100 times IL, without the need for chemical modification prior to analysis. DESI-MS analysis of ILs in positive and negative ion modes resulted in few ions in the low mass region, showing great potential for the analysis of small molecules in IL media.

Measurement of H2S in vivo and in vitro by the monobromobimane method

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels.

Gas/particle partitioning of carbonyls in the photooxidation of isoprene and 1,3,5-trimethylbenzene

A new denuder-filter sampling technique has been used to investigate the gas/particle partitioning behaviour of the carbonyl products from the photooxidation of isoprene and 1,3,5-trimethylbenzene. A series of experiments was performed in two atmospheric simulation chambers at atmospheric pressure and ambient temperature in the presence of NOx and at a relative humidity of approximately 50%. The denuder and filter were both coated with the derivatizing agent O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA) to enable the efficient collection of gas- and particle-phase carbonyls respectively. The tubes and filters were extracted and carbonyls identified as their oxime derivatives by GC-MS. The carbonyl products identified in the experiments accounted for around 5% and 10% of the mass of secondary organic aerosol formed from the photooxidation of isoprene and 1,3,5-trimethylbenzene respectively. Experimental gas/particle partitioning coefficients were determined for a wide range of carbonyl products formed from the photooxidation of isoprene and 1,3,5-trimethylbenzene and compared with the theoretical values based on standard absorptive partitioning theory. Photooxidation products with a single carbonyl moiety were not observed in the particle phase, but dicarbonyls, and in particular, glyoxal and methylglyoxal, exhibited gas/particle partitioning coefficients several orders of magnitude higher than expected theoretically. These findings support the importance of heterogeneous and particle-phase chemical reactions for SOA formation and growth during the atmospheric degradation of anthropogenic and biogenic hydrocarbons.