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.     

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...     

Determination of vitamin B<Subscript>12</Subscript> using a chemiluminescence flow system

A method to determine vitamin B₁₂ by measuring the chemiluminescence (CL) intensities using a flow injection (FI) system has been proposed. It is based on the catalytic effect of cobalt(II) in vitamin B₁₂ on the CL reaction between luminol and hydrogen peroxide in a basic medium. The increment of the CL intensity is proportional to the concentration of vitamin B₁₂ in the range 8.68–86.9 ng/mL (r ² = 0.9984) with a detection limit (3σ) of 0.89 ng/mL. The CL response is obtained in 10 s at a flow rate of 3.0 mL/min with a relative standard deviation (RSD) of less than 2.5% (n = 6). The method has been successfully applied to the determination of vitamin B₁₂ in pharmaceutical injections. The text was submitted by the authors in English.     

Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: A novel approach

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B₁₂ in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B₁₂ antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B₁₂ and vitamin B₁₂–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B₁₂ concentration. After systematic optimization, the limit of detection was 1 ng mL⁻¹. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B₁₂ was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B₁₂ in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.     

Ultrasensitive Determination of Vitamin B12 Using Disposable Graphite Screen‐Printed Electrodes and Anodic Adsorptive Voltammetry

A facile, rapid and ultra‐sensitive method for the determination of vitamin B₁₂ (cyanocobalamin) at the sub‐nanomolar concentration range by using low‐cost, disposable graphite screen‐printed electrodes is described. The method is based on the cathodic preconcentration of square planar vitamin B_12s, as occurred due to the electro reduction of Co(III) center in vitamin B_12a to Co(I), at ?1.3 V versus Ag/AgCl/3 M KCl for 40 s. Then, an anodic square wave scan was applied and the height of the peak appeared at ca. ?0.73 V versus Ag/AgCl/3 M KCl, due to the oxidation of Co(I) to Co(II) in the adsorbed molecule, was related to the concentration of the vitamin B₁₂ in the sample. EDTA was found to serve as a key‐component of the electrolyte by eliminating the background signal caused by metal cations impurities contained in the electrolyte (0.1 M phosphate buffer in 0.1 M KCl, pH 3). It also blocks trace metals contained in real samples, thus eliminating their interference effect. The method was optimized to various working parameters and under the selected conditions the calibration curve was linear over the range 1×10^?10–8×10^?9 mol L^?1 vitamin B₁₂ (R²=0.994), while the limit of detection for a signal‐to‐noise ratio of 3 (7×10^?11 mol L^?1 vitamin B₁₂) is the lowest value of any reported in the literature for the electrochemical determination of vitamin B₁₂. The sensors were successfully applied to the determination of vitamin B₁₂ in pharmaceutical products.     

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.     

Effects of γ-irradiation and cooking on vitamins B6 and B12 in grass prawns (Penaeus monodon)

The effects of irradiation doses, irradiation temperature and a combined treatment of irradiation and cooking on the vitamin B₆ and B₁₂ contents of grass prawns have been studied. Grass prawns were irradiated at refrigerated (4°C) or frozen (-20°C) temperatures with different doses. A domestic cooking procedure was followed after irradiation. The changes in vitamins B₆ and B₁₂ of both raw and cooked grass prawns were evaluated. Results showed no significant changes of vitamin B₆ and B₁₂ in grass prawns with a radiation dose up to 7 kGy at either 4°C or -20°C. Irradiation at 4°C caused more destruction of vitamin B₁₂ but not vitamin B₆ than did irradiation at -20°C in grass prawns. There was significant destruction of both vitamins B₆ and B₁₂ in unirradiated samples during cooking. The introduction of the irradiation process before cooking had no effect on either vitamin. These results indicate that the loss of vitamins B₆ and B₁₂ in the combined treatments was caused mainly by thermal destruction.     

Features of laser desorption/ionization mass spectrometry fragmentation of vitamin B<Subscript>12</Subscript>

A comparative analysis of the laser desorption/ionization of vitamin B₁₂ by matrix-assisted laser desorption/ionization (MALDI) and desorption/ionization on porous silicon (DIOS) was carried out. The mass spectra obtained were interpreted and the pathways for ion formation and decomposition were established. The MALDI fragmentation of the positive vitamin B₁₂ ions is more extensive than the DIOS fragmentation. The most extensive fragmentation was found using the MALDI method for negative vitamin B₁₂ ions, which are lacking when using the DIOS method. __________ Translated from Teoreticheskaya i éksperimental’naya Khimiya, Vol. 43, No. 4, pp. 251–256, July–August, 2007.     

Determination of B-group vitamins in Turkish honey using ultra-performance liquid chromatography with electrospray ionization coupled to tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (UPLC–ESI–MS/MS) for analysis of B-group vitamins in honey has been presented. Aim of this study is the characterization of different types of Turkish honeys according to B-group vitamins. Vitamins were determined in 17 different types of Turkish honey samples by UPLC–ESI–MS/MS. Heather honey samples were distinguished among the studied honeys with the richest vitamin content with 286.10?mg/kg, and it is followed by sunflower honey and thyme honey with the total vitamin contents of 206.01 and 163.27?mg/kg, respectively. The presence of vitamin B₁ (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (nicotinamide, B₃N and nicotinic acid, B₃H), vitamin B₆ (pyridoxine), and vitamin B₉ (folic acid) was detected in all the honey samples analyzed. Moreover, vitamin B₅ (pantothenic acid) was observed in most of them. Vitamin B₁₂ (cyanocobalamin) and vitamin B₇ (biotine) were not detected in the studied honey samples. Turkish honey samples showed efficacious vitamin content for the consumers.     

Dosage de traces de vitamines B12

An indirect determination of traces of vitamin B₁₂ is proposed; the vitamin is decomposed and the liberated cobalt is determined colorimetrically with Nitroso-R salt. 5–10 μg of B₁₂ can be determined. The use of tracers and internal standards decreases the error to less than 6%. Before the decomposition of the B₁₂ all traces of ionic cobalt are removed by means of an ion-exchange resin. The method is very sensitive and fairly selective; only homologues of B₁₂ containing cobalt interfere. B₁₂ can be determined in many pharmaceutical products.     

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.     

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.     

RP-HPLC determination of water-soluble vitamins in honey

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B₂, riboflavin; vitamin B₃, nicotinic acid; vitamin B₅, pantothenic acid; vitamin B₉, folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg⁻¹), we have observed a marked dependence of some of their concentrations (i.e. vitamin B₃ and vitamin B₅) and the botanical origin of the honey. This insight might lead to important characterization features for this food item.     

Partial Synthesis of CoαCoβ-Dicyano-176-Norcobinamide

Summary. Recent interest in norvitamin B₁₂-derivatives, homologues of complete vitamin B₁₂-derivatives, lacking the methyl group at carbon 176, stems from the identification of the corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Sulfurospirillum multivorans as 176-nor-pseudovitamin B₁₂. Here we report the partial synthesis of the corrinoid Co_αCo_β-dicyano-176-norcobinamide by condensation of cobyric acid and 2-aminoethanol. In addition, the partial synthesis of crystalline Co_α-aquo-Co_β-cyanocobyric acid by acid catalyzed hydrolysis of vitamin B₁₂ is detailed, improving the method and the isolation procedure worked out earlier by Bernhauer et al. The solution structure of Co_αCo_β-dicyano-176-norcobinamide was studied by spectroscopy and was compared with that of the homologue Co_αCo_β-dicyanocobinamide. The title compound, Co_αCo_β-dicyano-176-norcobinamide, represents the dicyano-form of a potential biosynthetic precursor of the 176-nor-B₁₂-derivatives, such as 176-nor-pseudovitamin B₁₂.     

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.     

Determination of energizers in energy drinks

The method of UV/VIS derivative spectrophotometry for the determination of caffeine and B vitamins in energy drinks after solid phase extraction has been developed. Caffeine has been determined in the mixture with B₂ vitamin with zero-crossing technique from the I derivative spectra (λ = 266.8 nm), and B₃ in mixture with B₆ vitamin from the II derivative spectra (λ = 280.1 nm). B₁₂ vitamin has also been determined in a three-component mixture with vitamins B₃ and B₆. Taurine in drinks has been determined from the basic spectra after derivatization with ninhydrin (λ = 570 nm).     

Quantum chemical study of the effects of interaction of polyhydrated Hg(II) with methylcobalamin — A derivative of vitamin B<Subscript>12</Subscript>

Quantum chemical methods were used to investigate the mechanism of interaction of polyhydrated Hg(II) with methylcobalamin — a derivative of vitamin B₁₂. Migration of the methyl group in the B₁₂·Hg(II) nH₂O cluster, initiating the formation of methylmercury, is considered. The reaction of substitution of methylmercury for the cobalt atom in the corrin fragment of B₁₂ has been studied. The thermodynamic and activation characteristics of these processes have been obtained. The mercury analog of vitamin B₁₂ is shown to exist in two radically different conformations.     

Determination of vitamin B<Subscript>1</Subscript> in seawater and microalgal fermentation media by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of vitamin B₁. Vitamin B₁ was converted into a fluorescent compound by treatment with hydrogen peroxide–horseradish peroxidase and the derivative was subsequently analyzed by HPLC on a Waters Spherisorb ODS2 column (250 mm×4.6 mm ID, 5 μm) with 40:60 methanol–pH 8.5 acetate buffer solution as mobile phase and fluorescence detection at 440 nm (with excitation at 375 nm). The calibration graph was linear from 5.00×10⁻¹⁰ mol L⁻¹ to 5.00×10⁻⁷ mol L⁻¹ for vitamin B₁ with a correlation coefficient of 0.9991 (n=9). The detection limit was 1.0×10⁻¹⁰ mol L⁻¹. The method was successfully used for determination of vitamin B₁ at pg mL⁻¹ levels in microalgal fermentation media and seawater after solid-phase extraction. Recovery was from 89 to 110% and the relative standard deviation was in the range 1.1 to 4.3%.     

Solid-solid synthesis of a hydrophobic vitamin B12 having a benzo-18-crown-6 moiety at the C10 position of the corrin ring

A new hydrophobic vitamin B₁₂ having a benzo-18-crown-6 moiety at the C-10 position of the corrin ring was synthesized by solid-state condensation reaction. The proton NMR titration study in acetonitrile exhibits a potassium ion binding behavior of the hydrophobic vitamin B₁₂ at the benzo-18-crown-6 moiety.     

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.