Simultaneous determination of some antidepressant drugs and vitamin B12 in pharmaceutical products and urine sample using HPLC method.

Three sensitive, accurate and precise HPLC methods were devolved for the simultaneous determination of vilazodone HCl (VILHC), agomelatine (AGO) or duloxetine HCl (DULHC) and vitamin B12 (cyanocoblamine B12) in bulk, pharmaceutical dosage form and in urine samples. Both similar methods (I and II) were carried out using 0.04 M phosphate buffer (pH 7.0), acetonitrile and methanol in the ratio (30:30:40, v/v) as a mobile phase. Thermo BDS hypersil-C18 column, with 5 µm particle size and 250 × 4.5 mm dimensions, at flow rate 1.0 mL min-1 and UV detection at 277 nm at ambient temperature 25 °C were used. The retention times were 5.12 and 2.54 min for VILHC and vitamin B12, 4.98 and 2.53 min for AGO and vitamin B12, respectively, with linearity range from 0.001 to 200 µg mL-1. However, for the separation of DULHC and B12, UV detection at 230 nm and Agilent Eclipse XDA-C8 (150 × 4.5 mm, 5 µm) column, were used (method III). The retention time of DULHC and vitamin B12 was found to be 4.53 and 1.35 min, respectively, with linearity range from 0.0005 to 200 µg mL-1. The proposed methods were validated as per the ICH guideline with very low LOD and LOQ. The values of %RSD for precision was less than 2% and the value of % recovery were found to be 99.20-100.9% and 99.23-100.67% for determination of the drugs in pure and pharmaceutical formulations, respectively, for all methods confirming that the methods are precise, accurate and selective for separation and determination of VILHC, AGO or DULHC from B12 in tablets and in urine samples without any interference from each other or from common excipients.

Efficient vitamin B12-imprinted boronate affinity magnetic nanoparticles for the specific capture of vitamin B12.

Vitamin B12 (VB12) has an important function in human physiology. However, analysis of VB12 at natural levels in foods or biological samples is difficult because of its very low concentration level and the presence of high-abundance components which can interfere with the measuring system. Thus, it is essential to develop efficient and selective enrichment approaches for VB12. Molecularly imprinted polymers (MIPs) have important applications from separation and sensing to catalysis. However, there is no report on the preparation of MIPs for VB12. Here, we use boronate affinity-based oriented surface imprinting to prepare MIPs for VB12. A VB12 template was first covalently immobilized onto the surface of boronic acid functionalized magnetic nanoparticles. Subsequently, a thin imprinting coating of poly(2-anilinoethanol) was formed to cover the substrate surface via in-water polymerization. After removing the template, 3D cavities complementary to the molecular size and shape of the template were formed in the imprinting layer. The imprinting coating was highly hydrophilic and presented limited residual boronic acid, thus non-specific binding was avoided. The prepared MIPs exhibited several highly favorable features, including excellent specificity, high binding strength and low binding pH. The prepared MIPs were successfully applied to the analysis of VB12 in human milk.

Improved Extraction Repeatability and Spectral Reproducibility for Liquid Extraction Surface Analysis-Mass Spectrometry Using Superhydrophobic-Superhydrophilic Patterning.

A major problem limiting reproducible use of liquid extraction surface analysis (LESA) array sampling of dried surface-deposited liquid samples is the unwanted spread of extraction solvent beyond the dried sample limits, resulting in unreliable data. Here, we explore the use of the Droplet Microarray (DMA), which consists of an array of superhydrophilic spots bordered by a superhydrophobic material giving the potential to confine both the sample spot and the LESA extraction solvent in a defined area. We investigated the DMA method in comparison with a standard glass substrate using LESA analysis of a mixture of biologically relevant compounds with a wide mass range and different physicochemical properties. The optimized DMA method was subsequently applied to urine samples from a human intervention study. Relative standard deviations for the signal intensities were all reduced at least 3-fold when performing LESA-MS on the DMA surface compared with a standard glass surface. Principal component analysis revealed more tight clusters indicating improved spectral reproducibility for a human urine sample extracted from the DMA compared to glass. Lastly, in urine samples from an intervention study, more significant ions (145) were identified when using LESA-MS spectra of control and test urine extracted from the DMA. We demonstrate that DMA provides a surface-assisted LESA-MS method delivering significant improvement of the surface extraction repeatability leading to the acquisition of more robust and higher quality data. The DMA shows potential to be used for LESA-MS for controlled and reproducible surface extraction and for acquisition of high quality, qualitative data in a high-throughput manner.

Silk fibroin as a platform for dual sensing of vitamin B12 using photoluminescence and electrical techniques.

The amalgamation of natural origin materials and technologies for label-free and real time detection is pertinent in analytical field. In this study, Bombyx mori silk fibroin protein (BMSF) has been utilized for the dual sensing of vitamin B12 via fluorescence and electrical impedance techniques. The processing of BMSF is done to generate an aqueous solution of BMSF along with the development of micro patterned thin films via soft lithography technique. The BMSF aqueous solution exhibit auto fluorescence property and thereby utilized for label free detection of vitamin B12 with an estimated limit of detection (LOD) of about 0.003 × 10-6 g/uL. This is followed by impedimetric detection of vitamin B12 using the micro patterned BMSF thin films. A LOD of 17.8 ppm and 0.25 ppm are achieved in aqueous solution and human blood serum, respectively. Taken together, this work demonstrates a potential label free dual sensing mode for sensitive detection of micronutrient vitamin B12.

Isolation and analysis of vitamin B12 from plant samples.

Based on increased demands of strict vegetarians, an investigation of vitamin B12 content in plant sources, was carried out. The vitamin B12 concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B12 was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B12, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004µg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n=6). Significant amounts of vitamin B12 in plants were detected in Hippophae rhamnoides (37µg/100g dry weight), in Elymus (26µg/100g dry weight) and in Inula helenium (11µg/100g dry weight).

Cobalamin production by Lactobacillus coryniformis: biochemical identification of the synthetized corrinoid and genomic analysis of the biosynthetic cluster.

BACKGROUND: Despite the fact that most vitamins are present in a variety of foods, malnutrition, unbalanced diets or insufficient intake of foods are still the cause of vitamin deficiencies in humans in some countries. Vitamin B12 (Cobalamin) is a complex compound that is only naturally produced by bacteria and archea. It has been reported that certain strains belonging to lactic acid bacteria group are capable of synthesized water-soluble vitamins such as those included in the B-group, as vitamin B12. In this context, the goal of the present paper was to evaluate and characterize the production of vitamin B12 in Lactobacillus coryniformis CRL 1001, a heterofermentative strain isolated from silage. RESULTS: Cell extract of L. coryniformis CRL 1001, isolated from silage, is able to correct the coenzyme B12 requirement of Salmonella enterica serovar Typhimurium AR 2680 in minimal medium. The chemical characterization of the corrinoid-like molecule isolated from CRL 1001 cell extract using HPLC and mass spectrometry is reported. The majority of the corrinoid produced by this strain has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. Genomic studies revealed the presence of the complete machinery of the anaerobic biosynthesis pathway of coenzyme B12. The detected genes encode all proteins for the corrin ring biosynthesis and for the binding of upper (ß) and lower (α) ligands in one continuous stretch of the chromosome. CONCLUSIONS: The results here described show for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce pseudocobalamin containing adenine instead of 5,6-dimethlbenzimidazole in the Coα-ligand. Genomic analysis allowed the identification and characterization of the complete de novo biosynthetic pathway of the corrinoid produced by the CRL 1001 strain.

Evaluation of oxidized buckypaper as material for the solid phase extraction of cobalamins from milk: Its efficacy as individual and support sorbent of a hydrophilic-lipophilic balance copolymer.

This work describes a new analytical method for the determination of four cobalamins (adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) in cow's milk. The extraction procedure is fast and based on dilution/protein precipitation of a milk sample with 50mM sodium acetate buffer (pH 4.6), followed by solid phase extraction (SPE) of the filtered supernatant. Relative recoveries higher than 60% have been obtained for all the cobalamins by combining two different types of sorbents in the same SPE cartridge: two disks of buckypaper (BP), a nanoporous felt composed of oxidized multiwalled carbon nanotubes (MWCNTs), separated by a Teflon frit from OASIS HLB (500mg), a hydrophilic-lipophilic balance copolymer. Before its use as sorbent, BP was characterized in terms of porosity, permeability, surface area, specific adsorption capacity and tested for a potential reuse after adequate chemical regeneration. The analysis of the extracts was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) on an analytical C18 column in less than 10min. After validation, the method was applied to the determination of the natural content of the four B12 homologues in cow's milk samples, providing data lacking in the literature.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry.

Here we present a novel coupling of thin-layer chromatography (TLC) to diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). DLTV is a new technique of aerosol generation which uses a diode laser to induce pyrolysis of a substrate. In this case the cellulose stationary phase on aluminum-backed TLC sheets overprinted with black ink to absorb laser light. The experimental arrangement relies on economic instrumentation: an 808-nm 1.2-W continuous-wave infrared diode laser attached to a syringe pump serving as the movable stage. Using a glass tubular cell, the entire length of a TLC separation channel is scanned. The 8-cm long lanes were scanned in ∼35 s. The TLC - DLTV ICP MS coupling is demonstrated on the separation of four cobalamins (hydroxo-; adenosyl-; cyano-; and methylcobalamin) with limits of detection ∼2 pg and repeatability ∼15% for each individual species.

A "three-in-one" sample preparation method for simultaneous determination of B-group water-soluble vitamins in infant formula using VitaFast(®) kits.

VitaFast(®) test kits designed for the microbiological assay in microtiter plate format can be applied to quantitative determination of B-group water-soluble vitamins such as vitamin B12, folic acid and biotin, et al. Compared to traditional microbiological methods, VitaFast(®) kits significantly reduce sample processing time and provide greater reliability, higher productivity and better accuracy. Recently, simultaneous determination of vitamin B12, folic acid and biotin in one sample is urgently required when evaluating the quality of infant formulae in our practical work. However, the present sample preparation protocols which are developed for individual test systems, are incompatible with simultaneous determination of several analytes. To solve this problem, a novel "three-in-one" sample preparation method is herein developed for simultaneous determination of B-group water-soluble vitamins using VitaFast(®) kits. The performance of this novel "three-in-one" sample preparation method was systematically evaluated through comparing with individual sample preparation protocols. The experimental results of the assays which employed "three-in-one" sample preparation method were in good agreement with those obtained from conventional VitaFast(®) extraction methods, indicating that the proposed "three-in-one" sample preparation method is applicable to the present three VitaFast(®) vitamin test systems, thus offering a promising alternative for the three independent sample preparation methods. The proposed new sample preparation method will significantly improve the efficiency of infant formulae inspection.

Effect of electric field on the partitioning behavior of solutes in entropic interaction chromatography.

In this study, a novel column design with a round cross-section was proposed to be suitable for a transverse electric field (EF). Additionally, two beads for entropic interaction chromatography (EIC) were prepared by grafting glycidyl methacrylate onto Toyopearl HW-65F (T65F) beads. Solute partitioning was then investigated to elucidate the role of graft polymerization with and without an EF. In a T65F column, solute partitioning was attributed to the distinct pore structure in the beads and was governed by pore flow. Under EF, partition coefficients (Kp) for solutes decreased with increasing EF strength. In the two EIC columns, a decrease of Kp was also observed without an EF while the fractionation windows were extended. It was more pronounced in the EIC column with a high grafting density (T65F-H). This was explained by the decrease in the effective pore size of solutes caused by the steric hindrance of polymer chains. Under an EF, the solutes showed different partitioning behaviours in the T65F-H column. With increasing EF strength, Kp for vitamin B12 and myoglobin was decreased. In contrast, Kp for large solutes increased as a result of concentration polarization on the bead surface. Both behaviors were related to the modulation of graft polymerization to residual charge on the matrix and the pore size of the solutes.

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity.

Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

The discovery of vitamin B(12).

The discovery of vitamin B(12), the elucidation of its role in metabolism, and the effects and treatment of its deficiency occurred in distinct phases over more than 100 years, and it was the subject of two separate Nobel Prizes. The valuable contribution of clinical reports and studies of patients with pernicious anemia throughout the 19th century resulted in enough clinical definition to allow Minot and Murphy to put together the first hallmark study on treatment of the condition, leading them to a Nobel Prize. These researchers were not the first to suggest that an inadequacy of nutrients was the cause of pernicious anemia, but their particular input was a carefully designed intervention in well-characterized pernicious anemia patients, of a special diet containing large amounts of liver. They found consistent improvement in the clinical and blood status of all subjects, most of whom remained on remission indefinitely. After the successful intervention studies, the next advance was made by Castle who discovered that a gastric component, which he called intrinsic factor, was missing in pernicious anemia. Many years later, intrinsic factor was found to be a glycoprotein that formed a complex with vitamin B(12), promoting its absorption through ileal receptors. The vitamin was isolated by two groups simultaneously and was crystallized and characterized in the laboratory of Dorothy Hodgkin, contributing to her Nobel Prize in 1964. Subsequently, the various biochemical roles of vitamin B(12) were elucidated, including its important interaction with folate and their common link with megaloblastic anemia. Many of the early clinical studies recognized that vitamin B(12) deficiency also caused a severe neuropathy leading to paralysis and death, while post mortem analysis demonstrated spinal cord demyelination. Vitamin B(12) is still the subject of intense research and, in particular, its role in preventing these irreversible neurological lesions remains unclear.

Characterization of corrinoid compounds from edible cyanobacterium Nostochopsis sp.

Vitamin B12 content of an edible cyanobacterium, Nostochopsis sp. was determined to be 140.6±16.2 µg/100 g dry weight by a microbiological method. To evaluate whether the Nostochopsis cells contain vitamin B12 or inactive corrinoid compounds, corrinoid compounds were purified from the cells and then identified as pseudovitamin B12 (97.4±11.8 µg/100 g dry weight) and vitamin B12 (43.2±6.0 µg/100 g dry weight) on the basis of silica gel 60 TLC bioautograms and LC/ESI-MS/MS chromatograms. Vitamin B12 content was significantly increased in the Nostochopsis cells (254.8±17.6 µg/100 g dry weight) grown in the vitamin B12-supplemented medium.

Selective extraction and determination of vitamin B12 in urine by ionic liquid-based aqueous two-phase system prior to high-performance liquid chromatography.

A rapid and simple extraction technique based on aqueous two-phase system (ATPS) was developed for separation and enrichment of vitamin B(12) in urine samples. The proposed ATPS-based method involves the application of the hydrophilic ionic liquid (IL) 1-hexyl-3-methylimidazolium chloride and K(2)HPO(4). After the extraction procedure, the vitamin B(12)-enriched IL upper phase was directly injected into the high performance liquid chromatography (HPLC) system for analysis. All variables influencing the IL-based ATPS approach (e.g., the composition of ATPS, pH and temperature values) were evaluated. The average extraction efficiency was 97% under optimum conditions. Only 5.0 mL of sample and a single hydrolysis/deproteinization/extraction step were required, followed by direct injection of the IL-rich upper phase into HPLC system for vitamin B(12) determination. A detection limit of 0.09 µg mL(-1), a relative standard deviation (RSD) of 4.50% (n=10) and a linear range of 0.40-8.00 µg mL(-1) were obtained. The proposed green analytical procedure was satisfactorily applied to the analysis of samples with highly complex matrices, such as urine. Finally, the IL-ATPS technique could be considered as an efficient tool for the water-soluble vitamin B(12) extraction.

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 B(12) in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B(12) antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B(12) and vitamin B(12)-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 B(12) 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 B(12) 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 B(12) in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride.

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.

Novel in situ product removal technique for simultaneous production of propionic acid and vitamin B12 by expanded bed adsorption bioreactor.

A new type of in situ product removal (ISPR) technique of expanded bed adsorption (EBA) bioreactor was studied to simultaneously produce extracellular propionic acid and intracellular vitamin B12 by Propionibacterium freudenreichii CICC 10019. Resin screening experiments showed that the ZGA330 resin have the best biocompatibility and highest adsorption for propionic acid. Through the EBA bioreactor, propionic acid could be recovered efficiently by semi-continuous recirculation of the unfiltered broth, which eliminated the feedback inhibition of propionic acid. Fed-batch fermentation was carried out using the EBA system, resulting in a propionic acid concentration of 52.5 g L(-1) and vitamin B12 concentration of 43.04 mg L(-1) at 160 h, which correspond to product yields of 0.66 g g(-1) and 0.54 mg g(-1), respectively. The present study suggests that the EBA bioreactor can be utilized for the simple and economical production of propionic acid and vitamin B12 in a single fermentation process.

Magnetic nanoparticle-based solid-phase extraction of vitamin B12 from pharmaceutical formulations.

In the present study, a novel quantitative method, namely magnetic nanoparticle-based solid-phase extraction (MSPE), was applied to extract vitamin B(12) from pharmaceutical formulations. The technique involves the use of Fe(3)O(4) nanoparticles modified by sodium dodecyl sulfate (SDS) as an efficient adsorbent for solid-phase extraction of vitamin B(12). Collection of magnetic nanoparticles (MNPs) from aqueous solution was simply achieved by applying external magnetic field. The analyte was desorbed from MNPs using alkali 1-propanol. The extracted analyte was analyzed by using flow injection inductively coupled plasma-optical emission spectrometry. Factors affecting the extraction efficiency were investigated and optimized. Under the optimum conditions, enhancement factor of 184, linear dynamic range of 2.5-500 µg L(-1) with correlation of determination (R(2) > 0.999), and limit of detection of 1.0 µg L(-1) were obtained for vitamin B(12). The percent relative standard deviation based on five-replicate determination was less than 6.2%. The method was successfully applied for extraction and determination of vitamin B(12) in different types of pharmaceutical samples such as multivitamin tablet, effervescent tablet, and injection sample. The results showed that the proposed method based on SDS-Fe(3)O(4) MSPE was a simple, accurate, and highly efficient approach for analysis of vitamin B(12).

Determination of Vitamin B12 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column.

A quantitative method for the determination of Vitamin B12 in meat products by RP-HPLC and UV detection was developed and compared to the reference method (microbiological assay, MBA). Vitamin B12 was extracted with 50mM sodium acetate buffer in the presence of sodium cyanide. For the quantification of total Vitamin B12, it was necessary to release protein-bound Vitamin B12 by pepsin treatment. Cyanocobalamin was detected as total Vitamin B12 after purification and enrichment on an immunoaffinity column. The calibration with five concentrations of Vitamin B12 was linear with a regression coefficient r2>0.99. The method was validated at three different concentration levels (5-15 ng/g) with salami showing good recovery rates between 80 and 108% and low relative standard deviations between 1.50 and 7.26% (n = 6). The detection limit was found to be 2 ng/g. The Vitamin B12 levels of 50 meat products measured by the developed procedure were similar or significantly lower than those determined by the MBA.