Multi-lumen capillary based trypsin micro-reactor for the rapid digestion of proteins.

In this work we evaluated a novel microreactor prepared using a surface modified, high surface-to-volume ratio multi-lumen fused silica capillary (MLC). The MLC investigated contained 126 parallel channels, each of 4 µm internal diameter. The MLC, along with conventional fused silica capillaries of 25 µm and 50 µm internal diameter, were treated by (3-aminopropyl)triethoxysilane (APTES) and then modified with gold nanoparticles, of ∼20 nm in diameter, to ultimately provide immobilisation sites for the proteolytic enzyme, trypsin. The modified capillaries and MLCs were characterised and profiled using non-invasive scanning capacitively coupled contactless conductivity detection (sC4D). The sC4D profiles confirmed a significantly higher amount of enzyme was immobilised to the MLC when compared to the fused silica capillaries, attributable to the increased surface to volume ratio. The MLC was used for dynamic protein digestion, where peptide fragments were collected and subjected to off-line chromatographic evaluation. The digestion was achieved with the MLC reactor, using a residence time of just 1.26 min, following which the HPLC peak associated with the intact protein decreased by >70%. The MLC reactors behaved similarly to the classical in vitro or in-solution approach, but provided a reduction in digestion time, and fewer peaks associated with trypsin auto-digestion, which is common using in-solution digestion. The digestion of cytochrome C using both the MLC-IMER and the in-solution approach, resulted in a sequence coverage of ∼80%. The preparation of the MLC microreactor was reproducible with <2.5% RSD between reactors (n = 3) as determined by sC4D.

Determination of trace labile copper in environmental waters by magnetic nanoparticle solid phase extraction and high-performance chelation ion chromatography.

Cobalt magnetic nanoparticles surface functionalised with iminodiacetic acid were evaluated as a nano-particulate solid phase extraction absorbent for copper ions (Cu(2+)) from environmental water samples. Using an external magnetic field, the collector nanoparticles could be separated from the aqueous phase, and adsorbed ions simply decomplexed using dilute HNO3. Effects of pH, buffer concentration, sample and sorbent volume, extraction equilibrium time, and interfering ion concentration on extraction efficiency were investigated. Optimal conditions were then applied to the extraction of Cu(2+) ions from natural water samples, prior to their quantitation using high-performance chelation ion chromatography. The limits of detection (LOD) of the combined extraction and chromatographic method were ~0.1 ng ml(-1), based upon a 100-fold preconcentration factor (chromatographic performance; LOD=9.2 ng ml(-1) Cu(2+)), analytical linear range from 20 to 5000 ng mL(-1), and relative standard deviations=4.9% (c=1000 ng ml(-1), n=7). Accuracy and precision of the combined approach was verified using a certified reference standard estuarine water sample (SLEW-2) and comparison of sample determinations with sector field inductively coupled plasma mass spectrometry. Recoveries from the addition of Cu(2+) to impacted estuarine and rain water samples were 103.5% and 108.5%, respectively. Coastal seawater samples, both with and without prior UV irradiation and dissolved organic matter removal were also investigated using the new methodology. The effect of DOM concentration on copper availability was demonstrated.

3D printed metal columns for capillary liquid chromatography.

Coiled planar capillary chromatography columns (0.9 mm I.D. × 60 cm L) were 3D printed in stainless steel (316L), and titanium (Ti-6Al-4V) alloys (external dimensions of ~5 × 30 × 58 mm), and either slurry packed with various sized reversed-phase octadecylsilica particles, or filled with an in situ prepared methacrylate based monolith. Coiled printed columns were coupled directly with 30 × 30 mm Peltier thermoelectric direct contact heater/cooler modules. Preliminary results show the potential of using such 3D printed columns in future portable chromatographic devices.

Practical method for evaluation of linearity and effective pathlength of on-capillary photometric detectors in capillary electrophoresis.

The optical characteristics of on-capillary photometric detectors for capillary electrophoresis were evaluated and five commercial detectors were compared. Plots of sensitivity (absorbance/concentration) versus absorbance obtained with a suitable testing solution yield both the linear range and the effective path length of the detector. The detector linearity is a crucial parameter when using absorbing electrolytes, such as for indirect photometric detection, and especially for highly absorbing electrolyte probe ions. The upper limits of the linear ranges (determined as 5% decline in sensitivity) for five commercial detectors ranged from 0.175 to 1.2 AU. The effective pathlength reflects the quality of the optical design of the detector and is equal to the capillary internal diameter only for a light beam passing exactly through the capillary centre, but becomes progressively shorter for imperfect optical designs. The determined effective pathlength for the five investigated detectors ranged from 49.7 to 64.6 microm for a 75 microm I.D. capillary.

Fast separation of UV absorbing anions using ion-interaction chromatography.

Ion-interaction chromatography on a short (30 x 4.6 mm) 3 microm ODS column has been investigated with the aim of developing fast chromatographic separations of selected inorganic anions. Tetrabutylammonium chloride (TBA-Cl) was used as the ion-interaction reagent in mobile phases that also contained up to 20% methanol. Separations of simple test mixtures of up to eight UV absorbing anions illustrated how excellent efficiencies (>50,000 plates/m) could be obtained under optimized conditions. The use of an optimised mobile phase containing 20 mM TBA-Cl and 20% methanol resulted in the baseline separation of five important anions (iodate, bromate, nitrite, bromide and nitrate) in a separation window of just 28 s, with a shortest total analysis time of 50 s. The method was briefly applied to the rapid analysis of nitrite and nitrate in both a drinking water and a river water sample with a view to future on-line monitoring.

Sensitive and selective ion chromatographic method for the determination of trace beryllium in water samples.

A selective and sensitive ion chromatographic method has been developed for the determination of beryllium in a number of water samples at low-microg/l concentrations. The separation was performed on a 250x4.0 mm I.D. iminodiacetic acid functionalised silica gel column. Chromatographed Be(II) was detected using visible detection at 590 nm following post-column reaction with chrome azurol S (CAS). The optimum separation and derivatisation conditions were studied in detail. The optimum eluent conditions were found to be 0.4 M KNO3, adjusted to pH 2.5 using HNO3, with optimum post-column detection being achieved using a solution containing 0.26 mM CAS, 2% Triton X-100, 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0. Under the above conditions, the concentration detection limit for Be(II) was found to be 3 microg/l in a standard solution and 4 microg/l in a typical tap water sample, using a 250 microl injection. The method was linear over the investigated range of 10 microg/l to 10 mg/l and highly reproducible. The method was successfully applied to a number of water samples of varying matrix complexity, including simulated seawater, and also to a natural freshwater certified reference material NIST 1640.

Determination of trace alkaline earth metals in brines using chelation ion chromatography with an iminodiacetic acid bonded silica column.

The chromatographic behaviour of alkaline earth metals on iminodiacetic acid bonded silica was studied. It was found that the ionic strength of the eluent greatly affected both retention time and selectivity by controlling the extent to which either simple ion exchange or surface complexation was responsible for retention. With a 0.1 M KNO3 eluent, the retention order was Mg(II), Sr(II), Ca(II) and Ba(II), indicating a strong contribution to retention from ion exchange. However, when using a 1.5 M KNO3 eluent, Ba(II) was found to elute first, indicating complexation to be more dominant under these conditions (pH 4.2). The effect of the ionic strength of the sample was also studied and it was found that by matching the eluent cation with that of the sample matrix, efficient separations of alkaline earth metals in 1.0 M NaCl and KCl brines could be obtained without matrix system peaks. Using post-column reaction with o-cresolphthalein complexone, trace levels of Ca(II) and Mg(II) were determined in medicinal NaCl saline solution and laboratory-grade KCl.

Determination of trace cadmium in environmental water samples using ion-interaction reversed-phase liquid chromatography with fluorescence detection.

An ion-interaction reversed-phase liquid chromatographic method has been developed for the determination of cadmium at low microg/l concentrations in environmental water samples. Cadmium and other matrix metals were separated through on-column complexation with 8-hydroxyquinoline sulphonate, using an octadecylsilica column and a mobile phase containing 15% acetonitrile, 10-13 mM tetrabutylammonium hydroxide, 5 mM 8-hydroxyquinoline 5-sulphonic acid and 10 mM acetic acid-acetate buffer (pH 4.8-5.4). Under the above conditions Cd(II) could be easily resolved from excess concentrations of matrix metals and could be detected at concentrations as low as 2 microg/l using fluorescence detection at 500 nm (based upon a 100-microl injection). The method showed a slightly curved detector response over the range of interest [up to 1 mg/l Cd(II)] and was successfully applied to the determination of trace Cd(II) in water samples containing large excesses of Mg(II) and Zn(II) and other matrix metals.

Retention modelling of electrostatic and adsorption effects of aliphatic and aromatic carboxylic acids in ion-exclusion chromatography.

The retention mechanism of aliphatic and aromatic carboxylic acids in ion-exclusion chromatography has been investigated with consideration of simultaneous electrostatic repulsion effects and hydrophobic adsorption effects. A mathematical relationship between the retention factor of the analyte and the mobile-phase composition (sulfuric acid concentration and percentage of methanol), the type of analyte (pKa and hydrophobicity) and some physical characteristics of the stationary phase has been derived. Thirteen carboxylic acids (comprising mono- and divalent, aliphatic and aromatic acids) were chosen and used to acquire retention data on three different cation-exchange stationary phases (in which the sulfonate functional groups are bound to polystyrene-divinylbenzene, polymethacrylate or silica) using 14 mobile-phase compositions of varying pH and percent methanol. These retention data were used to derive the parameters necessary to solve the retention model using non-linear regression. In this way, a quantitative measure of the effects of adsorption phenomena on analyte retention were obtained. The model was then used to optimise the separation of nine carboxylic acids.

Determination of niobium(V) and tantalum(V) as 4-(2-pyridylazo)resorcinol-citrate ternary complexes in geological materials by ion-interaction reversed-phase high-performance liquid chromatography.

A method for the simultaneous separation and determination of Nb(V) and Ta(V) as ternary complexes formed with 4-(2-pyridylazo)resorcinol (PAR) and citrate was developed using ion-interaction reversed-phase high-performance liquid chromatography on a C18 column. Method parameters, such as pre-column complex formation conditions and composition of the complexes were investigated using spectrophotometry and HPLC. Under the optimum conditions, the Nb(V) and Ta(V) complexes were eluted within 12 min with a mobile phase of methanol-water (32:68, v/v) containing 5 mM acetate, 5 mM TBABr and 5 mM citrate buffer at pH 6.5, with detection at 540 nm. A typical separation efficiency was 33,000 and 20,000 theoretical plates per metre for Nb(V) and Ta(V), respectively. The relative standard deviation of retention times for the Nb(V) and Ta(V) complexes were 0.16% and 0.17% and for peak areas were 0.28% and 1.36%, respectively. The detection limits (signal-to-noise ratio = 3) for Nb(V) and Ta(V) were 0.4 ppb and 1.4 ppb, respectively. Results obtained for standard reference rock samples agreed well with certified values and results obtained by inductively coupled plasma MS.

Effect of RG 12525, a new leukotriene antagonist, on pulmonary function of asthmatic adults.

RG 12525 is a new oral leukotriene D4 (LTD4) antagonist with proven activity in animal and human models of leukotriene-induced bronchoconstriction. In this randomized, double-blind, parallel-group, placebo-controlled study the bronchodilator effects of single oral doses of RG 12525 of 25 and 200 mg or placebo were evaluated in 62 adult asthmatic patients. All patients had previously demonstrated 20% reversibility of FEV1 with an inhaled beta-agonist. They had an unmedicated FEV1 less than 80% of predicted value at the time of the study. Bronchodilator activity was assessed by spirometry before and at multiple time points after dosing for eight hours. A single 200-mg dose of RG 12525 of mg resulted in statistically significantly greater increases in mean maximum change above baseline for FEV1 and FEF25-75% than placebo. The peak effect was observed four to five hours after dosing. The RG 12525 dose of 25 mg dose induced better bronchodilation than placebo but the differences were not significant. Adverse clinical experiences or laboratory abnormalities were not noted.

Fluticasone propionate given once daily is as effective for seasonal allergic rhinitis as beclomethasone dipropionate given twice daily.

Fluticasone propionate was compared with beclomethasone dipropionate for the treatment of allergic rhinitis in a multicenter, double-blind, randomized, placebo-controlled study during the mountain cedar (Juniperus ashei) pollination season in central Texas. Adults (n = 313) with moderate to severe symptoms were treated with fluticasone propionate aqueous nasal spray 200 micrograms once a day or beclomethasone dipropionate aqueous nasal spray 168 micrograms twice a day or placebo for 2 weeks. Fluticasone propionate administered once daily and beclomethasone dipropionate administered twice daily were equally effective as assessed by clinician- and patient-rated scores for nasal obstruction, rhinorrhea, sneezing, and nasal itching throughout the treatment and follow-up periods. Both regimens were more effective than placebo. Adverse events were related to topical administration and were similar in frequency and nature in all three treatment groups. Fluticasone propionate and beclomethasone dipropionate displayed a similar safety profile that did not differ from placebo. We conclude that fluticasone propionate aqueous nasal spray administered as 200 micrograms once daily in the morning is as safe and effective as beclomethasone dipropionate aqueous nasal spray administered as 168 micrograms twice daily for seasonal allergic rhinitis.

A dose-ranging study of fluticasone propionate aqueous nasal spray for seasonal allergic rhinitis assessed by symptoms, rhinomanometry, and nasal cytology.

Fluticasone propionate is a new glucocorticosteroid with potent topical activity. In a double-blind, randomized, parallel-group study, 423 adult patients with moderate to severe seasonal allergic rhinitis received placebo or fluticasone propionate aqueous nasal spray at doses of 25, 100, or 400 micrograms twice daily (b.i.d.) for 2 weeks. Efficacy was evaluated by nasal symptom scores, nasal airflow, nasal cytology, and global evaluation. All doses of fluticasone propionate were significantly better than placebo in reducing symptoms of seasonal allergic rhinitis. Patients receiving the largest dose of fluticasone propionate (400 micrograms b.i.d.) had a slightly greater reduction (not significant) in symptom scores than patients receiving the smallest dose (25 micrograms b.i.d.). Symptom improvement was evident within 3 days of treatment. Nasal airflow improved in the groups treated with fluticasone propionate, 100 and 400 micrograms b.i.d. Examination of nasal cytograms revealed a striking decrease in both eosinophils and basophils in all three groups receiving active treatment compared with placebo. There were few adverse events and no treatment-related abnormalities in laboratory assays or evaluations of hypothalamo-pituitary-adrenocortical axis function. Comparison of treatment groups indicated that fluticasone propionate aqueous nasal spray was as safe as placebo at the doses studied.

Imported fire ant allergy. Perspectives on diagnosis and treatment.

As the imported fire ant relentlessly expands its territory, more and more people are at risk of the insects' discomforting and sometimes dangerous stings. Thus, all primary care physicians in fire ant-infested areas should be familiar with diagnosis and treatment of fire ant stings and of allergic reactions to the stings. Fire ant venom is unique when compared with other Hymenoptera venoms, as it consists mainly of nonallergenic alkaloids that are of small molecular weight with only a small percentage of allergenic proteins. Fire ant wholebody extract, unlike other Hymenoptera wholebody extracts, retains its allergenic activity and is an adequate material for use in diagnostic testing and immunotherapy. Diagnosis of fire ant allergy can be readily made by the radioallergosorbent test (RAST) or by skin testing using fire ant wholebody extract. Immunotherapy with fire ant whole-body extract in allergic individuals can prevent serious recurrent systemic reactions.

Fire ant venom hypersensitivity. I. Comparison of fire ant venom and whole body extract in the diagnosis of fire ant allergy.

Levels of IgE antibody to fire ant whole body extract (FA-WBE) and fire ant venom (FAV) were evaluated in 60 patients with clinical histories of fire ant sting hypersensitivity and 11 frequently stung individuals without significant clinical reactivity to fire ant. An overlap of levels of IgE antibody to FA-WBE and FAV was seen between groups of patients with increasingly severe clinical reactivity; however, the group mean IgE antibody level of patients without significant clinical reactivity differed markedly from the group means of patients with more severe clinical reactivity (p less than 0.001). In 31 patients a good correlation (r = 0.6935) was found comparing FA-WBE IgE antibody level with FA-WBE skin tests, and in 26 patients a good correlation (r = 0.8564) was obtained comparing FAV IgE antibody levels with FAV skin tests. An excellent Spearman rank correlation coefficient (r = 0.9197) was found on comparison of FAV and FA-WBE IgE antibody levels in 71 individuals. Cross-reactivity of FAV and FA-WBE was demonstrated by the ability of these reagents to competitively inhibit the binding of IgE antibody to solid-phase FA-WBE and FAV in the radioallergosorbent test (RAST). From these results we conclude that FAV and FA-WBE both contain relevant allergens important in fire ant-allergic individuals and that skin tests and RAST are both valid as diagnostic tests for fire ant allergy. Finally, there is a good correlation between RAST and skin tests using FA-WBE or FAV as test reagents, since these reagents are highly cross-reactive as demonstrated by their ability to inhibit each other in the RAST.

Structure and activity of ragweed antigen E. II. Allergenic crossreactivity of the subunits.

Recently we have reported the isolation of the two subunits (alpha and beta) of ragweed antigen E (AgE) in their active form. In the present work we have compared the allergenic activity of the two subunits with native AgE by the radioallergosorbent test (RAST) and by inhibition of the RAST. Also, the amounts of IgE antibodies in 30 ragweed-sensitive sera bound to alpha and beta subunits as well as native AgE were measured by RAST. In all individuals the percentage of RAST binding was greatest to native AgE; however, varying patterns of reactivity to alpha and beta subunits were noted. In RAST inhibition experiments, free beta subunit was equally effective in inhibiting the binding of IgE antibody (from a pooled ragweed-sensitive sera) to immoblized alpha or beta subunit. Similarly, fluid-phase alpha subunit inhibited in a comparable manner the binding of IgE antibody to solid-phase alpha or beta subunits. In further RAST inhibition experiments 7 x 10(-13) moles of fluid-phase native AgE was required for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. Larger molar amounts of fluid phase beta (3.5 x 10(-11) and alpha (3 x 10(-10) were needed for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. AgE allergenicity as measured by RAST could not be totally reconstituted by recombination of equimolar quantities of alpha and beta subunits. Less than 1 x 10(-12) moles of fluid-phase native AgE inhibited the binding of IgE antibody to solid-phase alpha or beta, greater than 50%. Finally, the isolated chains are bioligcally active as demonstrated by their ability to provoke wheal-and-flare skin reactions in subjects allergic to ragweed pollen, and the activities of the chains were approximately equivalent on a weight basis to native AgE.