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.     

Differential pulse polarographic determination of pyridoxine in multivitamins tablets

Pyridoxine (vitamin B₆) is easily oxidized to pyridoxal by active manganese dioxide. Pulse polarograms recorded from alkaline media (pH 12–13) containing pyridoxal are very well-defined. The current is diffusion-controlled and the peak current is proportional to the concentration of pyriodoxine. This provides a simple determination of pyridoxine in multivitamin tablets. There is no interference from other vitamins; nicotinamide can be determined simultaneously from the same polarogram. The method is not applicable to tablets containing the coloids Methocel 4000 or Kollidon, which are strongly adsorbed on the electrode and inhibit the electroreduction of pyridoxal.     

Liquid chromatography of vitamin B6 with electrochemical detection using a carbon fibre electrode

A flow-through electrochemical detector with a carbon fibre electrode was fabricated. A chromatographic method for the determination of vitamin B₆ using this detector is described, and the results are compared with those obtained by detection with a thin-layer glassy carbon electrode or spectrophotometry. The detection limit of vitamin B₆ is 2.5 ng for pyridoxine, 1 ng for pyridoxal and 1 ng for pyridoxamine. The technique was applied to the determination of vitamin B₆ in tablets.     

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.     

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 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.     

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.     

An immediate luminescence enhancement method for determination of vitamin B1 using long-wavelength emitting water-soluble CdTe nanorods

A method for determination of vitamin B₁ has been developed that is based on the enhancement effect of vitamin B₁ on the luminescence of water-soluble CdTe nanorods modified with thioglycolic acid and cysteine. The effect of variables including the size of the nanorods on the enhancement of luminescence have been investigated. A preliminary mechanistic study showed that the passivating action of vitamin B₁ on the surface of the CdTe nanorods is likely to be responsible for the enhancement. Interferences by shortwave fluorescence are effectively eliminated because measurements are performed in the near-infrared. Due to the near-infrared measurement character, the fluorescence interference of vitamin B₂ can be effectively eliminated. Under the optimum conditions, the extent of luminescence enhancement is proportional to the concentration of vitamin B₁ in the range from 0.1 to 3.0 μmol L^?1 and the detection limit is 0.03 μmol L^?1. The relative standard deviation for 2.0 μmol L^?1 vitamin B₁ is 1.3% (n?=?6). The method is highly sensitive and selective, avoids the sample treatment needed in other procedures, and can be applied to the determination of vitamin B₁ in real samples with satisfactory results.     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.

     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.     

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...     

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.     

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.     

A solid-phase enzyme-linked assay for vitamin B12

A new solid-phase enzyme-linked competitive binding assay for vitamin B₁₂ (cyanocobalamin) is described. The assay is based on the competition between analyte B₁₂ molecules and a glucose-6-phosphate dehydrogenase-vitamin B₁₂ conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solid-phase is inversely related to the concentration of B₁₂ in the sample. Under optimized conditions, the method can detect B₁₂ in the range of 3×10^–10–1×10^–8 M (using 100l sample) with high selectivity over other biological molecules.     

Determination of Vitamin B6 Compounds in Foods Using Liquid Chromatography with Post-Column Derivatization Fluorescence Detection

The liquid chromatographic determination of six vitamin B₆-related compounds, the three B₆ vitamers, their corresponding phosphorylated esters, and an excretion product, is optimized using the reversed-phase technique with a stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl. The isocratic mobile phase consisted of a phosphate buffer, and fluorescence detection involved a post-column derivatization reaction using sodium hydrogensulphite to enhance the fluorescence of the phosphate ester. Peaks were identified by the retention characteristics and fluorescence spectra. Detection limits ranged from 1–25 ng mL^–1. Two extraction procedures using acid hydrolysis and enzymatic hydrolysis were compared. The method was applied to the determination of B₆ vitamin derivatives in different types of food including beef liver, egg yolk, baby food cereals and honey. The natural free vitamers appeared in honey and baby food cereals, while the phosphorylated esters were found in the foods of animal origin. An assay using two certified reference materials gave results within the certified range.     

Study on the inclusion interactions of β-cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application

The inclusion interactions of β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) with dyes were developed by spectrofluorimetry, and the inclusion constants of inclusion complexes were determined by direct fluorescence technique. The main factors (the host molecule, the guest molecule, and the pH) for the inclusion interaction were discussed in detail. At the same time, the inclusion interaction of HP-β-CD and vitamin B₆ (VB₆) was investigated with the competitive fluorescence inclusion method and the inclusion constant of HP-β-CD and vitamin B₆ (VB₆) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change of fluorescence intensity (ΔF) and the concentration of VB₆, a competitive fluorescence inclusion method was used to the determination of VB₆. The method has been successfully applied to the analysis of VB₆ in synthetic samples, tablets and injections with satisfactory results.     

High-performance liquid chromatography determination of potential content of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) in resins, oils, dry concentrates and multivitamin formulations.

The determination of potential vitamin D concentrations in raw materials and in multivitamin formulations by high-performance liquid chromatography is reported. Simple experimental conditions allow the rapid separation of the vitamins from their respective pro- and pre-vitamins, from their irradiation side-products and from some of their overirradiation products. Vitamin extraction is performed directly from dry concentrates, tablets and capsules, without saponification; all procedures are carried out at room temperature so as to preserve the ratio of vitamin D and pre-vitamin D present in the prepatation. The relative standard deviation is less than 1.5%.     

Semiquantitative determination of ten vitamins by the nichrome wire ring chamber

Summary A method for microidentification and semiquantitative determination of vitamins A, B₁, B₂, B₆, C, D₂, D₃, E, K₃, andp-aminobenzoic acid from a single drop of solution using the all glass nichrome wire ring chamber is described. The accuracy is± 5% and the method is selective and rapid and other vitamins do not interfere. The application of the method is demonstrated by the determination of vitamins from commercially available multivitamin tablets and injections.

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Zusammenfassung Der Mikronachweis und die semiquantitative Bestimmung der Vitamine A, B₁, B₂, B₆, C, D₂, D₃, E, K₃ und p-Aminobenzoesäure in einem Tropfen Lösung mit Hilfe der aus Glas gefertigten, mit einem Nichromdraht versehenen Ringkammer wurde beschrieben. Die Genauigkeit beträgt ± 5%das Verfahren ist selektiv, andere Vitamine stören nicht. Es wurde an handelsüblichen Multivitamintabletten und -Injektionslösungen erprobt.

     

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.     

Determination of nicotinamide (NA) using its polarographic catalytic wave in the presence of KIO3

Nicotinamide (NA) yields a polarographic catalytic wave with a peak potential –1.38 V (vs. SCE) in 0.1 mol/L HAc-NaAc (pH 4.7)/4 × 10^–3 mol/L KIO₃ buffer solution. The sensitivity of the catalytic wave increased in one order of magnitude as compared to that of the responding reduction wave without KIO₃. Based on this observation, a new method for the determination of NA was recommended. The second order derivative peak current was proportional to the NA concentration in the range of 5 × 10^–8 – 6 × 10^–7 mol/L. 0.11-fold vitamin B₁, 0.13-fold B₂, 0.14-fold B₆ and 8-fold nicotinic acid amounts do not interfere the determination of 1 × 10^–6 mol/L NA. The proposed method was used to determine the NA content in multivitamin tablets, with good agreement to the declared amount. Received: 25 October 1999 / Revised: 28 March 2000 / /Accepted: 31 March 2000