Indirect determination of iron in a flow-injection system with amperometric detection

The change in peak current resulting from the reaction of Fe(II) with nitroso-R salt in a flow-injection system is used to quantify Fe(II) with either single- or dual-electrode amperometric detectors. The current change varies linearly with Fe(II) concentration from 0 to 200 mg 1^?1. The relative standard deviation was about 5% with the single-electrode detector and about 10% with the dual-electrode detector. The method is evaluated for the determination of iron in dietary supplements.     

Chelation controlled regiospecific O-substitution of myo-inositol orthoesters: convenient access to orthogonally protected myo-inositol derivatives

A general method for the completely regioselective protection of the three secondary hydroxyl groups of orthoester derivatives of myo-inositol, utilizing the subtle differences in reactivity exhibited by its alkali metal alkoxides due to differences in their ability to form chelates, is described. This method provides convenient access to orthogonally protected myo-inositol derivatives. A comparison of the methylation of racemic 4-O-trityl-myo-inositol 1,3,5-orthoformate in the presence of sodium or lithium ions showed that stabilization of the C4-alkoxide by chelation with lithium overrides steric hindrance offered by the C6-axial substituent in deciding the regioselectivity during the nucleophilic O-substitution.     

A flow-injection system for the amperometric determination of xylose and xylulose with co-immobilized enzymes and a modified electrode

A flow-injection system is described for the detection of xylose and xylulose. The detection is based on three successive reactions taking place in an enzyme reactor containing xylose isomerase, mutarotase and glucose dehydrogenase co-immobilized on controlled-pore glass. The final product, NADH, is electrocatalytically oxidized at 0 mV vs. SCE at a chemically modified electrode. Straight calibration graphs for the two pentoses were obtained between 50 μM and 2 mM. The maximal sample throughput was 30 h^?1.     

Synthesis of caged myo-inositol 1,3,4,5-tetrakisphosphate

The total synthesis of an enantiomerically pure Ins(1,3,4,5)P₄ derivative equipped with a photosensitive nitroveratryl group at the 3-O-phosphate is reported.     

Rapid measurement of fish freshness indices by an amperometric flow-injection system with a 16-way switching valve and immobilized enzyme reactors

A novel flow-injection system is proposed for the rapid measurement of the fish freshness indices K₁ and K₂: K₁=[([HxR+[Hx])×100/([IMP]+[HxR]+[Hx])] and K₂=[[Hx]×100/([HxR]+[Hx])], where [IMP], [HxR] and [Hx] are inosine-5′-monophosphate, inosine and hypoxanthine concentrations, respectively. For the estimation of index K₁, 5′-nucleotidase immobilized reactor and nucleoside phosphorylase (NP)/xanthine oxidase (XO) coimmobilized reactor were incorporated in series in the flow-injection line made up by a 16-way switching valve with two sample loops. For the estimation of index K₂, NP and XO immobilized reactors were also incorporated in the similar flow-line. Two sample portions passed through the flow-line with different residence times so that two peaks were obtained. The first and second peaks obtained in the K₁-determining system corresponded to the total of HxR and Hx and the total of Hx, HxR and IMP, respectively. Similarly, the first and second peaks obtained in the K₂-determining system corresponded to Hx and the total of Hx and HxR, respectively. Therefore, the indices K₁ and K₂ can be estimated by

where i₁ and i₂ present the peak current of the first and second peaks, respectively, and f the ratio of the peak currents of the first and second peaks for a Hx standard solution. A sea bream was selected as a model fish and it was stored at 4 °C after death. A good correlation was found between the index K₁ and the storage time over a period of 400 h, with a correlation coefficient of 0.992, but no correlation between the index K₂ and the storage time. The measurements could be performed at a rate of 22 samples per hour with satisfactory precision (0.6–1.2% R.S.D.), without calibration for each species, accurate weighing of fish meat and any interferences in fish meat.     

Voltammetric/amperometric detection for flow-injection systems

A dual-electrode voltammetric/amperometric detector for flow-injection systems is described. The detector provides qualitative voltammetric information without the charging currents associated with scanning the potential. Selectivity is enhanced relative to direct voltammetric detection at a single electrode because only chemically reversible redox couples are detected. A preliminary evaluation with hexacyanoferrate(III) and ascorbic acid is presented.     

A formal synthesis of valiolamine from myo-inositol

An efficient formal synthesis of racemic valiolamine starting from readily available myo-inositol is reported. In all the synthetic steps only one regioisomer is formed, which circumvents laborious purification of products. Regioselective benzylation of myo-inositol orthoformate, super-hydride mediated deoxygenation of a cyclitol derivative and stereoselective addition of dichloromethyllithium to an inosose are the key reactions in the synthesis.     

Determination of l-malate in wine by flow-injection with co-immobilized malate dehydrogenase/oxaloacetate decarboxylase

A method for determination of l-malate in wine is described. Malate dehydrogenase and oxaloacetate decarboxylase were immobilized on poly (vinyl alcohol) beads and incorporated in a flow-injection system with fluorescence detection. Sample solution (50 mul) was injected into the carrier stream [4mM NAD(+) in glycine buffer (pH 10.0)]. The fluorescence based on the NADH formed could be directly related to the amount of malate. The calibration graph was linear over the range 0.4-300muM. The detection limit was 0.2muM. Sampling throughout was 25 samples/hr.     

Determination of l-alanine in a flow-injection system with an immobilized enzyme reactor

A flow-injection system for the determination of l-alanine is described. Alanine dehydrogenase is immobilized on poly(vinyl alcohol) beads and used in a packed-bed enzyme reactor. The system responds linearly to injected samples (50 μl) in the concentration range 0.5–500 μM. The maximum throughput was 40 samples per hour. The immobilized enzyme reactor was stable for at least 6 weeks. Its usefulness for assay of l-alanine in serum and beverages is described.     

Simultaneous determination of sulfite and phosphate in wine by means of immobilized enzyme reactions and amperometric detection in a flow-injection system

A flow-injection system is proposed for the simultaneous determination of sulfite and phosphate in wine. A sulfite oxidase immobilized reactor and purine nucleoside phosphorylase-xanthine oxidase co-immobilized reactor are incorporated at fixed positions (parallel configuration) in the flow line, which is based on the splitting of the flow after sample injection and subsequent confluence. A poly(1,2-diaminobenzene)-coated platinum electrode is used as an amperometric detector to detect selectively hydrogen peroxide generated enzymatically in the enzyme reactors, without any interference from oxidizable species and proteins present in wine. Because each channel has a different residence time, two peaks are obtained. The first peak corresponds to sulfite and the second peak to phosphate. The peak current is linearly related to the concentrations of sulfite between 1 × 10⁻⁵ and 2 × 10⁻³M and phosphate between 2 × 10⁻⁵ and 5 × 10⁻³M. The simultaneous determination of sulfite and phosphate in wine can be performed at a rate of 30 samples/hr with satisfactory precision (less than 1.2% RSD) and no pretreatment except for the sample dilution.     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Efficient syntheses of optically pure chiro- and allo-inositol derivatives, azidocyclitols and aminocyclitols from myo-inositol

Efficient routes for the syntheses of optically pure and hitherto unknown l-chiro- and d-allo-inositol derivatives, azido- and aminocyclitols of l-chiro-configuration, diazido- and diaminocyclitols of d-allo-configuration from economically viable myo-inositol are described. These routes provide access to synthetically flexible 1,2:4,5-di-O-isopropylidene-chiro-inositol and 1,6:3,4-di-O-isopropylidene-allo-inositol, which are otherwise difficult to synthesize directly from their parent inositols. A one pot methodology that allows rapid access to both chiro- and allo-inositol derivatives has also been developed. Investigations on the glycosidase inhibitory properties of these novel azido- and amino-inositols unraveled the potentials of these classes of compounds as novel class of glycosidase inhibitors. Both d and l forms of these cyclitols could be synthesized from myo-inositol in gram scales and hence by exploiting the difference in reactivities of cis- and trans-ketals, a variety of protected derivatives, which are useful for the synthesis of unnatural phosphoinositols and natural products, can be synthesized.     

Highly selective and sensitive detection of NADP coenzymes using co-immobilized glucose-6-phosphate dehydrogenase/diaphorase reactors as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system

Two enzyme reactors prepared by the co-immobilization of two different glucose-6-phosphate dehydrogenases (G6PDH; from Leuconstoc mescenteroides (LM) and yeast (Y) and diaphorase are employed to enhance the sensitivity of NAD(P) coenzymes as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system. The NAD(P) coenzymes are recycled enzymatically during passage through the reactor in the presence of sufficient glucose-6-phosphate and oxygen in the carrier solution to produce a large amount of hydrogen peroxide, which is detected chemiluminometrically in the subsequent flow line. The G6PDH(LM)/diaphorase co-immobilized reactor is not specific between the NAD and NADP coenzymes, but shows a six fold selectivity towards NADP coenzymes compared to NAD coenzymes; the amplification factors for NAD and NADP coenzymes are 60 and 380, respectively, at a flow rate of 0.3 ml min(-1). In contrast, the G6PDH(Y)/diaphorase co-immobilized reactor is specific for NADP coenzymes with an amplification factor of about 600 (at a flow rate of 0.3 ml min(-1)). The detection limit is 6 fmol for both NADP(+) and NADPH.     

A flow-injection system with amperometric detection for selective determination of antioxidants in foodstuffs and drinks

Amperometric detection in flow-injection systems intended for total determination of antioxidants (flavonoids, oxyaromatic acids, etc.) in various foodstuffs (berries, fruit, vegetables) and drinks (wine, tea, coffee, cognac, beer, etc.) was described and discussed. Advantages of this method were demonstrated. These measurements can underlie building of a bank of data on the content of antioxidants in foodstuffs and drinks.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Determination of parathion in the presence of paraoxon and p-nitrophenol by flow-injection analysis with amperometric detection

The amperometric determination of parathion in the presence of its metabolites paraoxon and p-nitrophenol in a flow-injection system is reported. The method is based on the measurement of the oxidation signal yielded by p-nitrophenol on hydrolysis of parathion in the presence of Pd(II) or Hg(II). The oxidation of the metabolite was preferred to its reduction as it avoids the removal of oxygen, which poses major problems in flow-through configurations.     

Determination of sucrose in the presence of glucose in a flow-injection system with imomobilized multi-enzyme reactors

Sucrose is determined by its reaction in a packed-bed enzyme reactor containing co-immobilized invertase, mutarotase and glucose oxidase. The hydrogen peroxide produced is measured spectrophotometrically after a chromogenic reaction. Glucose in the samples is decomposed in a pre-reactor containing co-immobilized mutarotase, glucose oxidase and catalase. The response is linear over more than three decades. The detection limit is 0.1 μM sucrose for injection volumes of 80 μl. The sample frequency is 80 h^?1 with an r.s.d. of 0.3%. The response for glucose is 0.7% that for sucrose.     

Efficient regioselective protection of myo-inositol via facile protecting group migration

A cis-1,2-cyclohexanediol, 1,4,5,6-tetra-O-benzyl-myo-inositol, was selectively protected at the axial C2-hydroxyl via acid-mediated rearrangement of the corresponding 1,2-orthoacetate, or via the base-induced migration of a protecting group that had previously been easily installed with complete regioselectivity at the adjacent equatorial hydroxyl. Esters 4a-6a were synthesized in high yields (75-82%) while sulfonate 7a and silyl ether 8a were obtained in 85 and 31% yields, respectively. The migration of the esters induced by DBU results in equilibrium between regioisomers favouring the C2 protected isomer, but NaH induced migration of sulfonyl and silyl groups results in complete migration from equatorial to axial hydroxyl groups.     

Cross-correlation in flow-injection analysis with parallel flow streams and amperometric detection

Cross-correlation was implemented for flow-injection analysis by using two parallel flow lines, each with amperometric detectors, and driven by peristaltic pumps. One flow line was used to generate the reference signal for an analog correlator circuit and the other to generate the analyte signal. Cross-correlation was performed by multiplying these signals together at a time delay of zero, followed by low pass filtering. Using dopamine as a test system, improvements in signal-to-noise ratios of about two orders of magnitude were found for the correlation signal over the direct measurement of the electrode current.