Chemiluminescent Determination of Vitamin B12 Using Charge Coupled Device (CCD)

A method was developed for the determination of vitamin B₁₂ based on the enhancement of cobalt (II) on the chemiluminescence (CL) reaction between luminol and percarbonate (powerful source of hydrogen peroxide). The release of cobalt (II) from the vitamin B₁₂ was reached by a simple and fast microwave digestion (20 s microwave digestion time and a mix of nitric acid and hydrogen peroxide). A charge coupled device (CCD) photodetector, directly connected to the cell, coupled with a simple continuous flow system was used to obtain the full spectral characteristics of cobalt (II) catalyzed luminol-percarbonate reaction.

The optima experimental conditions were established: 8.0 m mol L^?1 luminol in a 0.075 mol L^?1 carbonate buffer (pH 10.0) and 0.15 mol L^?1 sodium percarbonate, in addition to others experimental parameters as 0.33 mL s^?1 flow rate and 2 s integration time, were the experimental conditions which proportionate the optimum CL emission intensity. The emission data were best fitted with a second-order calibration graph over the cobalt (II) concentration range from 4.00 to 300 µ g L^?1 (r² = 0.9990), with a detection limit of 0.42 µ g L^?1. The proposed method was successfully applied to the determination of vitamin B₁₂ in pharmaceuticals.     

A New Spectrophotometric Method for Determination of Vitamin B12 as Cobalts

Abstract

A spectrophotometric method for determination of Vitamin B₁₂ (Cyanocobalamin) as cobalt in pharmaceutical preparations is described. The sample is first decomposed by concentrated sulfuric and nitric acids and the dry residue is solubilized in distilled water. The cobalt present is then spectrophotometrically determined at Λ= 317 nm as Co-HMPA-SCN complex. The complex is stable for a long time in the range of pH 3–10. The presence of many other metal cations does not interfere.     

Iodination of cisplatin adduct of Vitamin B12 [{B12}-CN-{cis-PtCl(NH3)2}]

The cisplatin adduct of vitamin B₁₂, [{B₁₂}-CN-{cis-PtCl(NH₃)₂}]⁺ (1), reacts with iodide in aqueous solution to form [{B₁₂}-CN-{trans-PtI₂(NH₃)}] (3) in good yield. Mono-substitution of chloride was not observed since a subsequent replacement of one NH₃ by a second iodide is very fast as compared to the Cl⁻ → I⁻ exchange. The same reaction conditions allowed to introduce radioiodide ¹³¹I. Vitamin B₁₂ can therefore be labeled with radionuclides via binding to the Pt(II) center.     

Vitamin B12-mediated electrochemical cyclopropanation of styrene

Vitamin B₁₂ (a cobalt corrin complex) mediated the reduction of methylene chloride in the presence of styrene to give cyclopropylbenzene in quantitative yields in DMF containing tetrabutylammonium bromide (TBAB) and acetic acid with current efficiency 45% and turnover rate 4-fold larger than a typical vitamin B₁₂-mediated 6-membered ring cyclization. A pathway involving formation and electroreductive cleavage of a chloromethylene-Co^III intermediate to yield a chloromethylene radical that adds to styrene is suggested. The reaction is highly sensitive to solvent composition, and water in the solvent acts as a proton donor to facilitate production of 1-chloro-3-phenylpropane in a competitive pathway.

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Hydrophobic Vitamin B12. X. Steric Effect in Electrochemical Carbon-Skeleton Rearrangement Catalyzed by Hydrophobic Vitamin B12 in Nonaqueous Media

Abstract

A steric effect in the carbon-skeleton rearrangement catalyzed by heptamethyl cobyrinate perchlorate, [Cob(II)7C₁ester]C10₄, was investigated under electrochemical conditions. The controlled-potential electrolyses of alkyl halides having two carboxylic ester groups of different bulkiness on the same carbon atom, such as 2,2-bis(ethoxycarbonyl)-l-bromopropane, l-bromo-2-tert-butoxycarbonyl-2-ethoxy-carbonylpropane, l-bromo-2-cyclohexyloxycarbonyl-2-ethoxycarbonylpropane, and l-bromo-2-ethoxy-carbonyl-2-phenoxycarbonylpropane, were carried out in N,N-dimethylformamide, as catalyzed by [Cob(II)7C₁ester]CIO₄, to give the corresponding ester-migrated products in the dark at—1.5 V vs SCE in the presence of acetic acid and at—2.0 V vs SCE without acetic acid. As regards a correlation between bulkiness of an ester group and a migration aptitude, a smaller ester group tends to migrate to the adjacent carbon atom more readily than a larger one. The origin of such a steric effect is discussed with attention to the rate-determining step.     

Investigation on the Inclusion Behavior of ApoCopC with Vitamin B6

In neutral phosphate buffer solution of pH 7.0, the interaction between apoCopC and Vitamin B₆ has been investigated in detail by means of fluorescence spectroscopy. According to the change of Vitamin B₆ fluorescence spectra and fluorescence polarization, we can conclude that a novel supramolecular system is generated. ApoCopC can form a 1:5 host-guest inclusion supramolecular complex with Vitamin B₆, and the formation constant has been calculated to be (2.24 ± 0.08) × 10⁴ M^? 1. It suggests the strong inclusion ability of apoCopC to the guest molecules. In addition, the mechanism of the apoCopC protein fluorescence quenching by Vitamin B₆ was also discussed. And based on the Stern-Volmer equation, the apparent quenching constant was estimated to be (2.75 ± 0.05) × 10⁴ M^? 1.     

维生素B12与12-钨磷酸相互作用的双波长共振瑞利散射光谱及其分析应用

建立了一种双波长共振瑞利散射光谱测定维生素B12的新方法. 在pH=1.0的HCl介质中, 维生素B12(VB12)与12-钨磷酸(TP)形成摩尔比为3:1的离子缔合物, 导致双波长共振瑞利散射(DWO-RRS)、 二级散射(SOS)和倍频散射(FDS)光谱显著增强, 其最大散射波长分别位于330和370 nm(RRS), 608 nm(SOS)和386 nm(FDS). 在一定范围内, 3种散射增强(ΔIRRS, ΔISOS和ΔIFDS)均与VB12的浓度成线性关系. 该方法具有较高的灵敏度, RRS, SOS和FDS法对VB12的检出限(3σ)在2.0~7.6 ng/mL之间. 研究了反应条件和共存物质的影响, 结果表明, 该方法具有良好的选择性. 据此, 提出了简便、 快速、 准确且高灵敏测定痕量VB12的光散射新方法, 适用于片剂和尿样中VB12的测定. 还对反应机理和散射光谱增强的原因进行了讨论.     

Use of packed-fiber solid-phase extraction for sample clean-up and preconcentration of vitamin B_(12) before determination

A rapid and simple preconcentration step applying packed-fiber solid-phase extraction columns has been investigated to vitamin B_(12).The extraction performance of the new method was investigated preliminarily on vitamin functional drink.The analysis used a reversed-phase C_(18) column,with a photo-diode array detector at 220 nm.The samples were preconcentrated with packed-fiber solid-phase extraction columns.Good linearity was observed in vitamin functional drink.The repeatability of extraction performa...     

HPLC-ESI-MS analysis of Vitamin B12 in food products and in multivitamins-multimineral tablets

Methods for the determination of Vitamin B₁₂ remain limited due to their low sensitivity and poor selectivity. In the present work, a simple and sensitive HPLC-ESI-MS method for determining Vitamin B₁₂ in food products and in multivitamin-multimineral tablets was developed. Vitamin B₁₂ was extracted from food products with 50 mM sodium acetate buffer (pH 4.0) in the presence of sodium cyanide. Total Vitamin B₁₂ content in food product can be obtained by efficient enzymatic hydrolysis to release the bound Vitamin B₁₂. Vitamin B₁₂ was quantified with ginsenoside Re as internal standard (I.S.) after their separations on a C₁₈ column with a gradient of mobile phase made of water and acetonitrile. MS with SIR mode at m/z 930.8 was used for Vitamin B₁₂ quantification. The calibration graphs plotted with five concentrations of Vitamin B₁₂ was linear with a regression coefficient r² = 0.9994. The intra-assay R.S.D. and the inter-assay R.S.D. were 2.6% (n = 5) and 3.5% (n = 6), respectively. The recoveries evaluated at spiking three different concentrations on fortified products were above 93%. The method has been applied successfully in the determination of Vitamin B₁₂ in fortified food products and multivitamin-multimineral tablets.     

Adenosyl-176-norcobinamide - A likely biosynthetic precursor to natural 176-norvitamin B12 derivatives

The “complete” corrinoid 176-norpseudovitamin B₁₂ was recently isolated as the cyano-Co(III)-form of the corrinoid cofactor of tetrachlorethene reductive dehalogenase of the anaerobe Sulfurospirillum (formerly Dehalospirillum) multivorans. 176-Norpseudovitamin B₁₂ represents the first example of (the cyano-Co(III)-form of) a naturally occurring “complete” B₁₂ cofactor lacking a characteristic peripheral methyl group of the cobamide ligand. Its discovery has generated interest in 176-nor-B₁₂ derivatives, “complete” corrinoids lacking the methyl group attached to carbon 176. Here, we report the preparation of Co_β-5′-adenosyl-176-norcobinamide by in situ alkylation of Co(I)-176-norcobinamide, obtained from electrochemical reduction of Co_α,Co_β-dicyano-176-norcobinamide. Since Co_β-5′-adenosylcobinamide is a biosynthetic intermediate of the complete cobamides, Co_β-5′-adenosyl-176-norcobinamide is a “rational” biosynthetic precursor for natural 176-nor-B₁₂ derivatives. The spectroscopic data for adenosyl-176-norcobinamide establish the suggested structure of the title compound and give further evidence for the extensive flexibility and conformational dynamics of the organometallic 5′-deoxy-5′-adenosyl ligand.     

the Mechanism of the Oxidation of Cob(I)alamins

Abstract

Spectroelectrochemical investigations of the reoxidation sequence of the reduced cob(I)alamin to the oxidized cob(III)alamin show that two different cob(II)alamin intermediates are formed during the processes which appear to correlate to base-on and base-off cob(II)-alamin species.     

Enhanced application of square wave voltammetry with glassy carbon electrode coupled to multivariate calibration tools for the determination of B(6) and B(12) vitamins in pharmaceutical preparations

Vitamins B₆ (VB₆) and B₁₂ (VB₁₂) were simultaneously determined in pharmaceutical preparations by using square wave voltametry (SWV) together with artificial neural networks (ANNs). Supporting electrolyte solution, pH and voltametric technique were optimised. The calibration set was built with several artificial samples containing both active ingredients and excipients. Deviations from linearity were observed for both analytes. It is probably due to interactions among the electro active components and competition by the electrode surface, fact that supports the use of ANNs. Recoveries when analysing a nine sample validation set, of 100.2 and 96.4 were calculated for VB₆ and VB₁₂, respectively. Commercial samples were analysed with reasonably good results considering the complexity of the mixture studied.     

Sub-picogram determination of Vitamin B12 in pharmaceuticals and human serum using flow injection with chemiluminescence detection

A sensitive chemiluminescence (CL) method, based on the enhancive effect of cobalt(II) on the CL reaction between luminol and dissolved oxygen in a flow injection (FI) system, was proposed for determination of Vitamin B₁₂. The increment of the CL intensity was proportional to the concentration of Vitamin B₁₂, giving a calibration graph linear over the concentration from 2.0×10⁻¹⁰ to 1.2×10⁻⁶ g l⁻¹ (r²=0.9992) with the detection limit of 5.0×10⁻¹¹ g l⁻¹ (3σ). At a flow rate of 2.0 ml min⁻¹, a complete determination of Vitamin B₁₂, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (R.S.D.) of less than 5.0%. The proposed method was applied successfully to the determination of Vitamin B₁₂ in pharmaceuticals, human serum, egg yolk and fish tissue.     

Determination of Vitamin B12 in Multivitamin Tablets by High Performance Liquid Chromatography

Abstract

Two procedures for separation and determination of vitamin B₁₂ in multivitamin tablets by reversed phase high performance liquid chromatography are proposed. Sample preparation is very simple: tablets are dissolved in distilled water, centrifuged and filtered. The sample solution is directly applied in the sample loop injector and chromatograms are obtained with gradient elution using water-methanol and water-acetonitrile as solvents. The peak of vitamin B₁₂ from samples of B-complex tablets is well separated with the two procedures. For multivitamin tablets, however, only the procedure with water and methanol as solvents was good for separation and quantification of vitamin B₁₂. Both procedures were verified by the standard addition method and also compared to a previously developed method using electrothermal atomic absorption spectrometry for vitamin B₁₂ determination.     

Phyllohydroquinone (Vitamin K1 H2) and Phylloquinone (Vitamin K1): Purification and Spectral Analysis

Abstract

Simple procedures for the preparation of pure phyllohydroquinone and phylloquinone are described. Phylloquinone is reduced by sodium dithionite and the reduced vitamin is purified by reverse phase high performance liquid chromatography. The essential spectral properties of the pure vitamins in ethanol and methanol are cited. The data provide a convenient spectral method for the accurate determination of the absolute concentrations of the oxidized and reduced vitamins and the rates of oxidation of the reduced vitamin. Pure, dry phyllohydroquinone may be safely stored at -70[ddot]C under nitrogen for a period of months. The stability of Phyllohydroquinone in methanol at 25.0 ± 0.1[ddot]C is also reported.     

Study on methane fermentation and production of vitamin B12 from alcohol waste slurry

We studied biogas fermentation from alcohol waste fluid to evaluate the anaerobic digestion process and the production of vitamin B₁₂ as a byproduct. Anaerobic digestion using acclimated methanogens was performed using the continuously stirred tank reactor (CSTR) and fixed-bed reactor packed with rock wool as carrier material at 55°C. We also studied the effects of metal ions added to the culture broth on methane and vitamin B₁₂ formation. Vitamin B₁₂ production was 2.92 mg/L in the broth of the fixed-bed reactor, twice that of the CSTR. The optimum concentrations of trace metal ions added to the culture liquid for methane and vitamin B₁₂ production were 1.0 and 8 mL/L for the CSTR and fixed-bed reactor, respectively. Furthermore, an effective method for extracting and purifying vitamin B₁₂ from digested fluid was developed.     

Synthesis and characterization of vitamin B12-hyperbranched polymer

Vitamin B₁₂-hyperbranched polymer was synthesized and characterized by UV-vis and NMR spectroscopy as well as DLS. The shape of hybrid polymer was also directly observed by TEM. Microenvironmental polarity around vitamin B₁₂ derivative on the polymer was evaluated by UV-vis absorption peak with α-band of vitamin B₁₂ chromophore.     

Polarographic Behaviour of Orotic Acid (Vitamin B13). Anodic Wave

Abstract

The electroanalytical anodic behaviour of orotic acid has been studied at several pH values, using DPP and CV techniques. The orotic acid undergoes an anodic wave in both techniques.

The best conditions for the determination of orotic acid with the mentioned techniques was studied.

The determination of Hg(II) by classic polarography, DC, was performed.     

Indirect Determination of Organic Compounds by Atomic Absorption Spectrophotometry: The Study of Vitamin B12 in Pharmaceutical Dosage Forms

Abstract

A method for the determination of vitamin B₁₂ in pharmaceutical dosage forms is presented. The method involves, dissolution of the vitamin B₁₂ and subsequent determination of the complexed cobalt by atomic absorption spectrophotometry. The method is both rapid and sensitive giving applications to quality, control of this type of formulation.