Determination of the carbon-13 content of sugars and pulp from fruit juices by isotope-ratio mass spectrometry (internal reference method). A European interlaboratory comparison

An interlaboratory comparison by ¹³C-isotope ratio mass spectrometry was organised by working group No. 1 of the European Commission of Standardisation (CEN/TC 174) in order to define the repeatability (r) and the reproducibility (R) of the ¹³C determination in sugars and pulp, isolated from the same fruit juice. Six unlabelled juices were analysed in 19 laboratories in the European Union, Australia and the USA. Three samples were authentic juices (orange, grapefruit, pineapple) and the remaining samples were the same juices with an addition of sugar at 15 g l⁻¹ (orange, pineapple) or 11.8 g l⁻¹ (grapefruit). The sugar was prepared by mixing equal amounts of beet and cane sucroses. The different laboratories used the same experimental protocol under different conditions (operator, conversion system for CO₂ preparation, mass spectrometer). The results for the sugars (r = 0.27%., R = 0.82%.) were comparable to those from an earlier ring test (r = 0.30%., R = 0.70%.), while the results for the pulp presented higher interlaboratory variability (r = 0.38%., R = 1.89%.) possibly due to experimental difficulties. Nevertheless, the RSD_R for free sugars ranged 0.9–3.2% and for pulp, 0.9–9.3%. On the basis of the Horwitz equation and taking account of the concentration of the isotopes measured, an RSD_R of 7–8% might have been expected. The experimentally determined RSD_R values were less than twice the theoretically expected values, which is a criterion of an acceptable method. The results were therefore considered to be acceptable by specialists in the field of isotopic analysis, and the method applied was found to improve the sensitivity of the ¹³C determination for the detection of sugar addition in fruit juices.     

Flow injection with chemical reaction interface-isotope ratio mass spectrometry: an alternative to off-line combustion for detecting low levels of enriched 13C in mass balance studies

We have evaluated the potential of flow injection chemical reaction interface isotope-ratio mass spectrometry to replace radioactive labeling techniques in material balance studies. A sample is flow injected and transmitted through a desolvation system followed by combustion to form ¹³CO₂ with a microwave-powered chemical reaction interface. We can detect trace amounts of a ¹³C-labeled drug (3′-azido-3′-deoxythymidine, AZT) in urine or feces. Our ability to quantify less than 100 ng/mL of excess ¹³C (∼1 μg/mL of ¹³C-labeled AZT) from a sample equivalent to 10 μL of urine is superior to previous detection limits for ¹³C in urine that use off-line combustion methods. Parallel studies using ¹⁴C-labeled AZT showed that our stable isotope method provides comparable percent excretion data for urine and feces. These results support previous findings that mass balance studies could be carried out with isotope-ratio mass spectrometer, here using doses as low as 1–2 mg/kg.     

Tellurium tetrachloride: an improved method of preparation

An efficient and practical synthesis of tellurium tetrachloride from elemental tellurium and sulfuryl chloride is described.     

Serum and plasma beta-carotene levels measured with an improved method of high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of beta-carotene concentrations in serum and plasma is reported. Using a method modified from a previously published technique, serum and plasma proteins are denatured by exposure to perchloric acid, and beta-carotene is subsequently extracted into an organic matrix consisting of ethyl acetate-tetrahydrofuran (1:1); no evaporation step is required. Separation is achieved using isocratic elution from a reversed-phase C18 column with UV detection at 436 nm. Recovery of beta-carotene from water and plasma was greater than 98.1%; beta-carotene was stable in the extraction matrix for at least 4 h. Three anticoagulants (oxalate, citrate, and EDTA) caused losses of beta-carotene; perchloric acid and tetrahydrofuran could also destroy beta-carotene under certain conditions. Each run required less than 15 min; within-day coefficient of variation for identical samples averaged 2.3%, between-day coefficient of variation was 4.4% and sensitivity was better than 10 ng/ml. Stability of beta-carotene in plasma was also examined. This method permits a simple, rapid, sensitive, precise, and accurate determination of beta-carotene using 0.5 ml of serum or heparinized plasma.     

A structural influence on the electrical double-layer characteristics of Al4C3-derived carbon

Several carbon materials were produced by reacting aluminum carbide with chlorine gas at different temperatures (400–900 °C). Chlorination temperature and porosity values showed the inversely related trends whereby the graphitization degree rises with the chlorination temperature. Electrochemical measurements performed in three-electrode test cells with 1.0-M Et₃MeNBF₄ electrolyte revealed that the changes in porosity parameters and the degree of graphitization are in good correlation with specific capacitance values. Capacitance depends on the structure of carbon and varies in studied chlorination range from 109 to 60 F g⁻¹ and from 114 to 64 F g⁻¹ for the negatively and positively charged electrode materials, respectively. An exceptionally low capacitance was observed for the material produced at 700 °C that was explained by the multiwall carbon nanobarrels and the highly ordered curved graphitic flakes, which have low specific surface and possess the relatively low specific surface-related capacitance.     

Determination of the 13C/12C ratio of ethanol derived from fruit juices and maple syrup by isotope ratio mass spectrometry: collaborative study

A collaborative study of the carbon-13 isotope ratio mass spectrometry (13C-IRMS) method based on fermentation ethanol for detecting some sugar additions in fruit juices and maple syrup is reported. This method is complementary to the site-specific natural isotope fractionation by nuclear magnetic resonance (SNIF-NMR) method for detecting added beet sugar in the same products (AOAC Official Methods 995.17 and 2000.19), and uses the same initial steps to recover pure ethanol. The fruit juices or maple syrups are completely fermented with yeast, and the alcohol is distilled with a quantitative yield (>96%). The carbon-13 deviation (delta13C) of ethanol is then determined by IRMS. This parameter becomes less negative when exogenous sugar derived from plants exhibiting a C4 metabolism (e.g., corn or cane) is added to a juice obtained from plants exhibiting a C3 metabolism (most common fruits except pineapple) or to maple syrup. Conversely, the delta13C of ethanol becomes more negative when exogenous sugar derived from C3 plants (e.g., beet, wheat, rice) is added to pineapple products. Twelve laboratories analyzed 2 materials (orange juice and pure cane sugar) in blind duplicate and 4 sugar-adulterated materials (orange juice, maple syrup, pineapple juice, and apple juice) as Youden pairs. The precision of that method for measuring delta13C was similar to that of other methods applied to wine ethanol or extracted sugars in juices. The within-laboratory (Sr) values ranged from 0.06 to 0.16%o (r = 0.17 to 0.46 percent per thousand), and the among-laboratories (SR) values ranged from 0.17 to 0.26 percent per thousand (R = 0.49 to 0.73 percent per thousand). The Study Directors recommend that the method be adopted as First Action by AOAC INTERNATIONAL.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

Nucleophilic addition to C,C-double bonds. VI. Intramolecular addition of primary amines to C,C-double bonds induced by steric compression

The tricyclic olefinic primary amine 1 readily cyclizes to the tetracyclic secondary amine 2 at approximately 200° in protic as well as aprotic solvents although the C, C-double bond is not activated by electron-attracting groups. This unusual intramolecular addition is the consequence of the close proximity of the nucleophile and the double bond. For the synthesis of the sterically highly hindered amine 1 , the tricyclic oxime 4 was reduced with TiCl₃ to the remarkably stable imine 5 and the latter treated with AlH₃. On the other hand, reaction of 4 with AlH₃ yielded the pentacyclic aziridine 6 .     

13C Fourier transform n.m.r.: XIII—reassignment of the 13C spectrum of ergosterol

The complete labeling pattern of ergosterol isolated from Saccharomyces cerevisiae grown with [1-¹³C] acetate is detailed and the results of this study have led to a reassignment of carbons 5, 8, 12, 16, 22 and 23 in the ¹³C n.m.r. spectrum of ergosterol.     

483—Monitoring cell potential of Escherichia coli with use of an electrode sensitive to tetraphenyl phosphonium: Change by addition of various sugars

An electrode sensitive to tetraphenyl phosphonium cation (TPP⁺) was applied to the determination of the membrane potential of E. coli (strain B, IAM 1268). This strain did not require EDTA (ethylenediamine tetraacetic acid)-treatment for accumulation of TPP⁺. As reported previously, some part of the TPP⁺-uptake was due to the binding to cell envelopes. The time course of TPP⁺-uptake by cells on addition of sugars was analysed, and the result indicated that the additional binding of TPP⁺ no longer occured. The change in the membrane potential on addition of various sugars was evaluated.     

SO3-mediated addition of N-chlorourethanes to the C=C bond

Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 12, pp. 2871–2872, December, 1989.     

An improved method for the extraction of low molecular weight organic acids in variable charge soils.

Due to specific adsorption to variable charge soils, low molecular weight organic acids (LMWOAs) have not been sufficiently extracted, even if common extractants, such as water and 0.1 M sodium hydroxide (NaOH), were employed. In this work, the method for extracting LMWOAs in soils with 0.1 M NaOH was improved for variable charge soils; e.g. 1.0 M potassium fluoride (KF) with pH 4.0 was applied as an extractant jointed with 0.1 M NaOH based on its stronger ability to change the electrochemical properties of variable charge soils by specific adsorption. With the proposed method, the recoveries of oxalic, tartaric, malic, citric and fumaric acids were increased from 83 +/- 4, 93 +/- 1, 22 +/- 2, 63 +/- 5 and 84 +/- 3% to 98 +/- 2, 100 +/- 2, 85 +/- 2, 90 +/- 2 and 89 +/- 2%, respectively, compared with NaOH alone. Simultaneously, the LMWOAs in Agri-Udic Ferrosol with field moisture were measured with a satisfactory result.     

Preparation of 13C4-6-methyl anthranilic ester via a Diels-Alder-type process. an experimental and theoretical study to characterize an unexpected isotope exchange

Buta-2,3-dienoate reacts with vinyl ketenimine to give the corresponding substituted aniline through a Diels-Alder cycloaddition. Besides the expected Diels-Alder adduct 3a, the aniline 3b was also obtained in a ratio of 91:9. The observed (13)C exchange is explained on the basis of a reversible [2 + 2] cycloaddition competing with the [4 + 2] process. This is supported by B3LYP DFT computations, as a stepwise pathway lies very close in energy to the [4 + 2] concerted one. [reaction: see text]     

Cp*(2)TiMe(2): an improved catalyst for the intermolecular addition of n-alkyl- and benzylamines to alkynes

Cp(2)TiMe(2) has been found to be a competent catalyst for the intermolecular addition of sterically less demanding n-alkyl- and benzylamines to internal alkynes. In the presence of 2.0-6.0 mol % of the catalyst, hydroamination reactions between n-propyl-, n-hexyl-, benzyl-, p-methoxybenzyl- or 2-phenylethylamine and diphenylacetylene, 3-hexyne or 4-octyne go to completion within 24 h or less at 114 degrees C (oil bath temperature). After subsequent reduction of the initially formed imines with zinc-modified sodium cyanoborohydride in MeOH at 25 degrees C, the corresponding secondary amines can be isolated in excellent yields (>78%). Hydroamination/reduction sequences employing the unsymmetrically substituted alkyne 1-phenylpropyne give access to mixtures of regioisomeric secondary amines. The observed regioselectivity is low.     

Conjugate addition to chromones: Synthesis of substituted 4-chromanones

Chromones activated by carbonyl substituents at C-3 are transformed into the corresponding 2-methyl 4-chromanones by treating with lithium dimethylcuprate     

Nucleophilic addition to styryl sulphones. part II. Regio- and stereochemistry of the addition of enamines from 4-substituted cyclohexanones

Formation of β- and α-adducts in the reactions of styryl sulphones with enamines from 4-alkylcyclohexanones is reexamined. Steric effects, electrostatic interactions between the nitrogen atom of the enamine and the oxygen atoms of the sulphone group, and medium effects contribute to the regioselectivity of the reaction.     

A highly regio- and chemoselective addition of carbon nucleophiles to pyrimidinones. A new route to C4 elaborated Biginelli compounds

Ethyl 6-methyl-pyrimidine-2-one-5-carboxylates react with C-nucleophiles in a diversity oriented synthetic sequence to afford C4 substituted congeners of medicinally potent Biginelli dihydropyrimidinones, in a highly regioselective manner.