Ether Elution Research Articles

Optimized Formation of Benzyl Isothiocyanate by Endogenous Enzyme and its Extraction from <i>Carica Papaya</i> Seed

Purpose : To use endogenous myrosinase in Carica papaya seed to convert benzyl glucosinolate (BG) to benzyl isothiocyanate (BITC) and then extract it for further studies. Methods : Process variables including seed powder particle size, sample-to-solvent ratio, pH of buffer solution, enzymolysis temperature, enzymolysis time were investigated using single-factor experiments and response surface methodology coupled with Box-Behnken design for further optimization of conversion conditions. The formed benzyl isothiocyanate was extracted by steam distillation method and purified by thin-layer chromatography (TLC). Relevant process variables were also studied. Finally, the purified benzyl isothiocyanate was analyzed by gas chromatography-mass spectrometer (GC-MS) and compared to BITC standard. Results : Optimum enzymolysis conditions were seed powder particle size, 90 - 120 µm; sample-tosolvent ratio, 1:20; pH of buffer solution, 4.8; enzymolysis temperature, 40 0C; and enzymolysis time, 27 min. Enzymolysis liquid was steam-distilled for 2 h, extracted twice using dichloromethane (DCM) (using 3 times the volume of distillate each time) as extractant. Finally, 1:20 was adopted as the optimal dichloromethane-petroleum ether (DCM-PE) elution proportion to wash out the unnecessary compounds. Conclusion : The result showed that benzyl isothiocyanate was formed under the optimized conditions and was no doubt the basic component of the extraction. Keywords : Carica papaya, Benzyl isothiocyanate, Endogenous myrosinase, Enzymolysis, Extraction

Confirmatory analysis of stanozolol metabolites in bovine, pig and sheep urines using an optimized clean-up and liquid chromatography–tandem mass spectrometry

This paper describes a new liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3′-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC–MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core–shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples.

Method development for the determination of 52 pesticides in tobacco by liquid chromatography–tandem mass spectrometry

A method using reversed phase liquid chromatography–electrospray ionization–tandem mass spectrometry was developed for the determination of 52 pesticides in tobacco. The influence of mobile phase additives was investigated to improve sensitivity and accuracy of the method and to reduce matrix effects. The tobacco extracts were purified via a Chem Elut partition cartridge by consecutive elution with pentane followed by dichloromethane. The two fractions were further purified by Florisil solid-phase extraction with acetone or diethyl ether elution. An additional dispersive solid-phase extraction step with primary–secondary amine led to decreased recoveries of several pesticides due to degradation or binding to the sorbent. The method was validated for the tobacco types Burley, Oriental and Virginia. The recovery rates of almost all pesticides ranged between 70 and 120%. The limits of quantification were below or near the 10 ng/g level. Few but significant differences between the tobacco types could be found regarding recovery and sensitivity.

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group O-test red cell panels at 37 degrees C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.

Vacuum Ether Elution of Red Blood Cell-Bound Antibodies – A Fast Modification of the Rubin’s Ether Elution Technique

<i>Background:</i> The complete evaporation of the ether from the eluates derived from Rubin’s ether elution technique takes at least 1 h in a 37 °C water bath, preferably followed by overnight incubation in the refrigerator. A fast modification of this technique was developed. <i>Materials and Methods</i>: The ether was evaporated by water aspiration. Administration of vacuum evaporation takes only 10 min for the complete elimination of the ether. Determination of the eluted antibodies was performed by indirect antiglobulin test (tube technique). We compared the eluates of 103 patients who had positive direct anti-globulin tests. Agglutination intensity was scored from 0 to 4. <i>Results</i>: 84 eluates were negative in both techniques. 15 eluates showed a positive reaction with corresponding agglutination scores in both methods. 4 samples showed different results: Two of them became positive only with one method, each. In one case, the probe obtained by vacuum ether elution (VE) reacted one degree weaker than the probe derived from conventional ether elution (CE). In the fourth case, only 1 of 2 antigen-positive cells had a positive reaction with the VE probe in contrast to that of the CE. <i>Conclusion:</i> The results obtained with both methods did not differ significantly. Ether evaporation by administration of vacuum seems to be a fast and easy modification which can be carried out with a simple equipment and in a short time.

Characterization of Elevated Neutrophil-Associated IgG in Various Autoimmune Disorders: Not Anti-Neutrophil Autoantibodies, But Possibly Immune Complexes, Bind to Neutrophils

Neutropenia is frequently observed in a variety of autoimmune disorders. As the mechanism of neutropenia in these disorders, the destruction of neutrophils by anti-neutrophil autoantibodies has been believed since elevated levels of neutrophil-associated IgG (NAIgG) have been described. However, no data exists to characterize the nature of NAIgG and show NAIgG is an anti-neutrophil autoantibodies. We investigated whether the elevated NAIgG in these patients consists of anti-neutrophil autoantibodies. The NAIgGs of 91 patients with autoimmune disorders including 50 patients with idiopathic thrombocytopenic purpura, 13 patients with systemic lupus erythematosus, 11 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease were analyzed. The level of NAIgG was high in 36 of 91 patients. Elution studies were performed to determine whether NAIgG has a nature of autoantibodies. In model experiments, the ether elu-ate from neutrophils sensitized with neutrophil-specific alloantibody (anti-NA2) reacted with donor neutrophils, whereas the eluates from those with model immune complexes (ICs) failed. These data indicated that the ether elution technique is useful to determine whether NAIgG consists of anti-neutrophil autoantibodies. The NAIgG on patient's neutrophils was eluted with ether and the reactivity of the eluate with normal neutrophils was investigated. The eluates from 34 of 36 patients with various autoimmune disorders with elevated NAIgG level failed to react with donor neutrophils. These data indicated that the elevated NAIgG in the majority of these patients did not consist of anti-neutrophil autoantibodies, but possibly of ICs.

A note on individual differences in the urinary excretion of optical enantiomers of styrene metabolites and of styrene-derived mercapturic acids in humans.

Urine samples from 20 male workers in the polyester industry exposed by inhalation to styrene concentrations ranging from 29 to 41 ppm were investigated. Excretion products of styrene metabolism, mandelic acid and mercapturic acids, were purified from the urine over an extraction column packed with Porapak Q, with subsequent ether elution. The optical enantiomers R- and S-mandelic acid were then determined by thin layer chromatography (TLC) using chiral plate material and selective staining with vanadium pentoxide. Quantitative analysis of these compounds was performed using commercial reference substances. Styrene-specific mercapturic acids were analyzed by a modified TLC method, using synthesized reference substances. The concentration of racemic mandelic acid in the individual urine samples ranged from 80 to 1610 mg/l, and the ratio of the R- and S-enantiomers ranged from 0.7 to 2.2. These individual variations are not explained by differences in individual styrene exposure levels, or by differences in the concentration of the urine samples (in relation to creatinine excretion). Styrene-specific mercapturic acids were detected in the urine of only 1 of the 20 workers, at a concentration much lower than expected from previous investigations by others in humans and laboratory animals, in which less specific analytical methods had been used. The results point to marked interindividual differences in metabolism of styrene, probably related to enzyme polymorphisms.

Measurement of plasma testosterone by gas chromatography-negative-ion mass spectrometry using pentafluoropropionic derivatives.

Plasma testosterone was measured by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-MS). The testosterone was extracted from plasma using home-made Extrelut columns and diethyl ether elution. It was quantified as the pentafluoropropionate (PFP) derivative by selected-ion monitoring at m/z 560 (testosterone) and 563 (d3-testosterone), accounting for about 34% of the total ion. The characteristics of the method were: extraction recovery about 95%; linearity over the range 1.7-71.5 nmol l-1 with linear regression equation y = 1.41x + 0.0217, r = 0.999; detection limit 3.5 fmol injected with a signal-to-noise ratio of 7.4; within-day variation, 3% for GC-MS, and 5.8% for the whole process; day-to-day coefficient of variation, 6.6-11%, depending on the concentrations. There was a good correlation between the results obtained by GC-MS and RIA (r = 0.994), but the GC-MS values were significantly lower (p < 0.05) than those obtained by RIA.

Elevated platelet‐associated IgG in SLE patients due to anti‐platelet autoantibody: differentiation between autoantibodies and immune complexes by ether elution

The level of platelet-associated IgG (PAIgG) is reported to be elevated in patients with systemic lupus erythematosus (SLE). However, the nature of PAIgG is unclear. We have investigated whether the PAIgG of SLE consists of anti-platelet autoantibodies or immune complexes (IC). The PAIgG values measured by flow cytometry were elevated in 11/25 patients with SLE. 3/6 SLE patients with thrombocytopenia had a high level of PAIgG (the mean fluorescence intensity > 10). We used an ether elution technique to determine whether elevated PAIgG consists of anti-platelet antibodies or IC. Preliminary experiments showed that the eluates prepared from platelets sensitized with anti-HPA-4a antibody reacted with normal platelets, while the eluates prepared from platelets sensitized with heat-aggregated IgG or model IC failed to react with normal platelets. These results indicate that the reactivity of eluates can distinguish between platelet-bound antibody and IC. We applied this technique to analysis of the PAIgG of SLE platelets. The eluates from SLE platelets (the mean fluorescence intensity > 10) reacted with normal platelets, indicating that the PAIgG of SLE platelets has the nature of antiplatelet autoantibodies. Furthermore, we investigated the target antigens which bind PAIgGs of SLE, using the direct immunoprecipitation procedure and modified antigen capture ELISA (MACE). Both methods identified GPIIb/IIIa as the target antigens. We conclude that the ether elution technique can distinguish between anti-platelet antibodies and IC, and that the PAIgGs of SLE with a high PAIgG value and thrombocytopenia have the nature of anti-platelet autoantibodies.

Langkettige Alkylpyridine und Alkylpyrazine als Spurenbestandteile in technischer Ölsäure

AbstractDurch Aufarbeitung von Talg erhaltene technische Ölsäure wurde in einer Folge chromatographischer Verfahren aufgetrennt. Die Fraktion der schwach polaren, mittels Ether eluierbaren Neutralstoffe enthielt unter anderem als Spurenbestandteile α,β‐ungesättigte Cyclopentenone, α‐Alkyl‐ und α‐Methyl‐α‐alkylpyridine, Alkyl‐ und Dialkylpyrazine.

受身凝集法を利用した新赤血球抗体同定法

A new method was developed to identify antibodies bound to red cell antigens using a small volume of sensitized red cells. Red cell (50μl) sensitized with Rh-Hr antibodies were reacted with panel cells (50μl) in test tube under various conditions. After centrifugation, agglutination did not occur with saline, albumin or low ionic strength solution, but did occur with antiglobulin and an enzyme method. However, the agglutination specificity for sensitized cells was observed with the two-stage enzyme method but not with antiglobulin or the one-stage enzyme method. The same results were obtained using umbilical cord cells collected from Rho incompatible pregnant women. The optimum conditions of the reaction between sensitized red cells and panel cells in the two-stage enzyme method were: an incubation time of 15min, centrifugation for 30sec at 1000rpm, and 1.5% sensitized red cells. Compared to the ether elution method, the sensitivity of detecting antibodies with this new method was twice that for anti-D, about 70 times that for anti-E and similar to that for anti-C. These results indicate that the new method is very useful for identifying antibodies bound to red cells when blood sampling from patients is very difficult and the sampling volume is small.

Improvement to aroma and taste by adding roasted tobacco volatiles to cigarettes.

An improvement to the aroma and taste of Burley cigarettes was achieved by adding volatiles from roasted, Japanese flue-cured tobacco. The addition of roasted tobacco volatiles was also found to decrease the offensive odor and taste. Among the fractions obtained from silica gel column chromatography of roasted tobacco volatiles, Fr. 8 (diethyl ether elution) exhibited the best improvement to the organoleptic properties of cigarette smoke. A great part of the improvement was attributable to 4-hydroxy-2, 5-dimethyl-3(2H)-furanone and 3-hydroxy-4, 5-dimethyl-2(5H)-furanone, both found in small amounts in Fr. 8 but possessing a strong impact on the olfactory properties.

Quantitative Evaluation of Elution Methods for Red Cell Antibodies

Two recent techniques using xylene and chloroform for antibody elution from red blood cells (RBC) were compared with the most widely used ether and heat elution by means of a quantitative 125 I-antiglobulin test. Free serum alloantibodies and cell-bound alloantibodies from in vivo sensitization of delayed hemolytic transfusion reactions and of hemolytic disease of the newborn as well as autoantibodies in autoimmune hemolytic disease were studied. With alloantibodies, xylene proved more efficient than chloroform (p<0.025), while both methods were superior to heat or ether elution (p<0.005). Autoantibody elution was equally effective with xylene and chloroform (p>0.1) but again better than heat or ether elution (p<0.02). However, no single method produced the strongest eluate with all antibodies studied. We conclude that xylene and/or chloroform elution are valuable implementations in RBC serology, but, in selected instances, the classical elution methods will retain their importance.

Acid elution of blood group antibodies from intact erythrocytes.

Human IgM and IgG antibodies against blood group antigens (A, B, D, C, c, E, e, Fya, K), autoantibodies and mouse IgM and IgG antibodies against sheep erythrocytes have been eluted from intact human and sheep red cells by glycine-HCl buffer, pH 3.0. The yield of human antibodies was higher with acid than with heat and ether elution, and the contamination of hemoglobin in the eluate was negligible. The acid elution method is very simple and rapid and, therefore, highly suitable for experimental as well as routine immunohematological work.

Acid Elution of Blood Group Antibodies from Intact Erythrocytes

Human IgM and IgG antibodies against blood group antigens (A, B, D, C, c, E, e, Fy^a, K), autoantibodies and mouse IgM and IgG antibodies against sheep erythrocytes have been eluted from intact human and sheep red cells by glycine-HCl buffer, pH 3.0. The yield of human antibodies was higher with acid than with heat and ether elution, and the contamination of hemoglobin in the eluate was negligible. The acid elution method is very simple and rapid and, therefore, highly suitable for experimental as well as routine immunohematological work.

Determination of Polybrominated Biphenyl Residues in Dairy Products

A method for the determination of polybrominated biphenyls (PBBs) in dairy products is described. Fat is extracted from the products by the official AOAC method. The PBB residues are separated from the fatty material by gel permeation chromatography prior to gas-liquid chromatographic (GLC) quantitation. An additional cleanup using petroleum ether elution through a miniature Florisil column is necessary for thin layer chromatographic (TLC) confirmation. Recoveries of PBBs from samples fortified at levels from 0.1 to 0.5 ppm ranged from 94 to 104% with an average of 99%. GLC sensitivity permits the estimation of PBB residue levels as low as 0.007 ppm. Routine TLC confirmation is limited by sensitivity to greater than or equal to 0.2 ppm.

Adsorption Behaviors of Detergents in Lubricating Oil Chromatography

The authors studied the adsorption behaviors of highly alkaline detergents in a lubricating oil chromatography using Silica gel and Alumina gel as the adsorbents. As the typical detergents with high alkalinity, calcium phenate and calcium sulfonate were taken up and the model oil samples having total base numbers of about 30mgKOH/g were prepared by adding these detergents into a base oil. Chromatographic fractionation of the model samples were carried out with Silica gel and Alumina gel having various pore sizes and the results of separation of the detergents and the base oil were studied. It was found that only the detergent was eluted in the first cut of petroleum ether elution when Silica gel have a pore diameter of 23Å. On the other hand, only the base oil was eluted in the chromatography when Silica gel as well as Alumina gel have larger pore size (60∼130Å). Consequenthy, it is possible by analysis, to determine the composition of the base oil. By applying this chromatographic techniques, it is possible to analyse similar highly alkaline detergent oil and identify the nature of detergents and the type of the base oil used. The authors assume such characteristic adsorption behaviors may have relation to the physicochemical properties of these individual adsorbent such as the size of pore.