Melamine sponges incorporated azo-linked porous organic polymer as adsorbent for extraction and determination of six B vitamins using pipette tip micro solid-phase extraction.

A novel azo-linked porous organic polymer (AL-POP) was synthesized from caffeic acid and benzidine via an azo-coupling reaction and characterized by FTIR, SEM-EDS, BET, TGA, XRD and zeta potential analysis. AL-POPs were incorporated into melamine sponges and used for pipette tip micro solid-phase extraction (PT-MSPE) of six types of B vitamins (including thiamine, riboflavin, nicotinamide, pyridoxine, folic acid, and cyanocobalamin). After extraction, the samples were analyzed using high performance liquid chromatography-diode array detection (HPLC-DAD) system. The effect of AL-POP composition on the extraction efficiency (EE) of vitamins was investigated and benzidine to caffeic acid mol ratio of 1.5, 3.35 mmol of NaNO2, and reaction time of 8 h were selected as optimum conditions. The efficiency of the extraction process was improved by optimizing various parameters such as the amount of sorbent, pH and ionic strength of the sample, sample volume, number of sorption and desorption cycles, type of wash solvent, and type and volume of eluent solvent. Linearity (R2≥0.9987), Limit of detection (LOD) (11.88-18.97 ng/mL), limit of quantification (LOQ) (39.62-63.23 ng/mL), and enrichment factor (EF) (1.27-4.31) were obtained using calibration curves plotted under optimum conditions. Recovery values of these six B vitamins in the spiked multivitamin syrup samples varied from 80.01% to 108.35%, with a relative standard deviation (RSD) below 5.44%. Eventually, the optimized method was successfully used to extract and quantify the B vitamins in multivitamin syrup and non-alcoholic beer.

Bio-metal-organic frameworks for molecular recognition and sorbent extraction of hydrophilic vitamins followed by their determination using HPLC-UV.

A bio-metal-organic framework (bio-MOF) derived from the amino acid L-serine has been prepared in bulk form and evaluated as sorbent for the molecular recognition and extraction of B-vitamins. The functional pores of bio-MOF exhibit high amounts of hydroxyl groups jointly directing other supramolecular host-guest interactions thus providing the recognition of B-vitamins in fruit juices and energy drinks. Single-crystal X-ray diffraction studies reveal the specific B-vitamin binding sites and the existence of multiple hydrogen bonds between these target molecules and the framework. It offered unique snapshots to accomplish an efficient capture of these solutes in complex aqueous matrices. Four B-vitamins (thiamin, nicotinic acid, nicotinamide, and pyridoxine) were investigated. They were eluted from the sorbent with phosphate buffer at pH 7 and analyzed by HPLC with UV detection. The sorbent was compared with commercial C18 cartridges. Following the procedure, acceptable reproducibility (RSD values < 14%) was achieved, and the detection limits were in the range 0.4 to 1.4 ng mL-1. The method was applied to the analysis of energy drink and juice samples and the recoveries were between 75 and 123% in spiked beverage samples. Graphical abstractA bio-MOF as SPE sorbent was prepared and applied to the extraction of B-vitamins in fruit juices and energy drinks.

Fast separation of water-soluble vitamins by hydrophilic interaction liquid chromatography based on submicrometer flow-through silica microspheres.

In the present study, submicrometer flow-through silica microspheres (Sub-FTSiO2) was for the first time obtained via a suspension polymerization method coupled with sol-gel transition and phase separation. The Sub-FTSiO2 was characteristic of rich mesopores, penetrable macropores and small particle size, which would be beneficial to fast mass transfer, low column backpressure and high column efficiency. It was directly used as the hydrophilic interaction liquid chromatographic (HILIC) stationary phase, and the fast separation of seven water-soluble vitamins in 2.2 min was realized. The proposed method was successfully applied to the determination of water-soluble vitamins in two functional beverages on the market. The prepared Sub-FTSiO2 was well demonstrated for fast HILIC, and would be potential as the stationary phase matrix for fast liquid chromatography in diverse separation modes.

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).

Characterization and Quantitation of Vitamin B12 Compounds in Various Chlorella Supplements.

Vitamin B12 was determined and characterized in 19 dried Chlorella health supplements. Vitamin contents of dried Chlorella cells varied from <0.1 µg to approximately 415 µg per 100 g of dry weight. Subsequent liquid chromatography/electrospray ionization-tandem mass spectrometry analyses showed the presence of inactive corrinoid compounds, a cobalt-free corrinoid, and 5-methoxybenzimidazolyl cyanocobamide (factor IIIm) in four and three high vitamin B12-containing Chlorella tablets, respectively. In four Chlorella tablet types with high and moderate vitamin B12 contents, the coenzyme forms of vitamin B12 5'-deoxyadenosylcobalamin (approximately 32%) and methylcobalamin (approximately 8%) were considerably present, whereas the unnaturally occurring corrinoid cyanocobalamin was present at the lowest concentrations. The species Chlorella sorokiniana (formerly Chlorella pyrenoidosa) is commonly used in dietary supplements and did not show an absolute requirement of vitamin B12 for growth despite vitamin B12 uptake from the medium being observed. In further experiments, vitamin B12-dependent methylmalonyl-CoA mutase and methionine synthase activities were detected in cell homogenates. In particular, methionine synthase activity was significantly increased following the addition of vitamin B12 to the medium. These results suggest that vitamin B12 contents of Chlorella tablets reflect the presence of vitamin B12-generating organic ingredients in the medium or the concomitant growth of vitamin B12-synthesizing bacteria under open culture conditions.

Methods of biological fluids sample preparation - biogenic amines, methylxanthines, water-soluble vitamins.

In recent years demands on the amount of information that can be obtained from the analysis of a single sample have increased. For time and economic reasons it is necessary to examine at the same time larger number of compounds, and compounds from different groups. This can best be seen in such areas as clinical analysis. In many diseases, the best results for patients are obtained when treatment fits the individual characteristics of the patient. Dosage monitoring is important at the beginning of therapy and in the full process of treatment. In the treatment of many diseases biogenic amines (dopamine, serotonin) and methylxanthines (theophylline, theobromine, caffeine) play an important role. They are used as drugs separately or in combination with others to support and strengthen the action of other drugs - for example, the combination of caffeine and paracetamol. Vitamin supplementation may be also an integral part of the treatment process. Specification of complete sample preparation parameters for extraction of the above compounds from biological matrices has been reviewed. Particular attention was given to the preparation stage and extraction methods. This review provides universal guidance on establishing a common procedures across laboratories to facilitate the preparation and analysis of all discussed compounds.

Comparison of different extraction methods to determine free and bound forms of B-group vitamins in quinoa.

The distribution of free and bound forms of B-group vitamins (B1, B2, B3, B5, and B6) was quantified in quinoa seeds using LC-MS-TOF combined with a stable isotope dilution assay. The effectiveness of liberating thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine from the food matrix and cofactors was evaluated for a variety of extraction conditions, including the addition of enzymes. Phosphatase and protease inhibitors, as well as ultrafiltration, were evaluated for their ability to suppress vitamer liberation via enzymes endogenous to quinoa. Cold extraction, together with a mixture of phosphatase and protease inhibitors, is identified as the most efficient treatment to prevent the conversion of cofactors into simple vitamers. Overnight incubation at 37 °C both with and without additional hydrolytic enzyme preparations containing phosphatase and ß-glucosidase activity was almost equally effective in releasing the bound forms of the vitamers. This indicates that the endogenous enzymes within quinoa seeds have high activity. ß-Glucosidase should be used when the total pyridoxine content is to be determined, and thermal treatment followed by enzymatic treatment with phosphatase activity is recommended to liberate the bound forms of pyridoxal prior to quantification.

Comparison of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-TOF and stable isotope dilution assay.

The application of LC/MS-TOF method combined with stable isotope dilution assay was studied for determination of thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine in food. Nutritional yeast powder was used as a model food matrix. Acid extraction was compared with various enzymatic treatments in ammonium formate buffer to find a suitable method for the conversion of more complex vitamers into the same forms as the used isotope-labeled internal standards. The enzyme preparations α-amylase, takadiastase, ß-glucosidase, and acid phosphatase were all able to liberate thiamine and riboflavin. The diastatic enzyme preparations α-amylase and takadiastase also expressed proteolytic side activities resulting in the formation of small peptides which interfered with the mass spectra of thiamine and riboflavin. Liberation of nicotinamide and pantothenic acid from NAD(+) and CoA, respectively, could not be achieved with any of the studied enzyme preparations. Hydrochloric acid extraction at 121 °C for 30 min was found to be destructive to pantothenic acid, but increased the liberation of pyridoxal.

Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization.

An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples.

Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography.

An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH(2)PO(4)) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L(-1) to 5 mg L(-1) for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 µg L(-1), and the relative standard deviations (RSD, n=7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.

Purification, identification, and characterization of methylcobalamin from Spirulina platensis.

The present study reports methylcobalamin in Spirulina platensis using high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), microbiological assay, chemiluminescence assay, liquid chromatography-mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS). Extraction of vitamin B12 from S. platensis was carried out without using cyanide. Partial purification was achieved using Amberlite XAD-2 followed by elution with 80% (v/v) methanol. Activated charcoal facilitated removal of impurities in S. platensis extract and in further purification of vitamin B12. The purified fraction was identified to contain methylcobalamin as analyzed by HPLC and TLC. Authenticity of methylcobalamin was further confirmed by LC-MS and MS/MS. Quantitation of methylcobalamin in a test sample of S. platensis biomass was performed using microbiological assay and chemiluminescence assay and was found to be 38.5±2 and 35.7±2 µg/100 g of dry biomass, respectively.

[Improving the efficacy of synergetically facilitated pertraction of p-aminobenzoic acid by increasing the reextraction rate]. / Marirea eficientei pertractiei facilitate sinergetice a acidului p-aminobenzoic prin cresterea vitezei de reextractie din membrana lichida.

The studies on facilitated pertraction of PABA with Amberlite LA-2 and 1-octanol as phase modifier indicated the increase of the process efficiency and, implicitly, of the transport capacity of the liquid membrane by adding KOH in the stripping phase. Thus, the use of KOH led to the diminution of the kinetic resistance of the reextraction process, with positive effects on the acid final mass flow and permeability factor. Compared to the similar pertraction systems containing NaOH in the stripping phase and in direct correlation with the pertraction parameters (pH-gradient between the feed and stripping phases, carrier and alchohol concentrations inside the liquid membrane), the final mass flow can be accelerated for about 1.9 times, and the permeability through liquid membrane can be enhanced for about 1.5 times in presence of KOH.

Esmond E. Snell--the pathfinder of B vitamins and cofactors.

Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

Identification and quantitation of cobalamin and cobalamin analogues in human feces.

BACKGROUND: Cobalamin (vitamin B-12) and cobalamin analogues are present in human feces, but a complete identification has not been established, and the amounts present have not been determined. OBJECTIVES: We aimed to develop a liquid chromatography-mass spectrometry method for cobalamin and cobalamin analogues and to identify and quantitiate the amounts present in human feces. DESIGN: Fecal samples were obtained from 20 human subjects in good general health. The samples were analyzed for the presence and amounts of cobalamin and 12 cobalamin analogues that were synthesized with and without the incorporation of stable isotopes. RESULTS: Cobalamin and 7 cobalamin analogues were identified and quantitated in human feces. The mean for the total amount present in 18 subjects whose daily intake was < or = 25 microg cobalamin from vitamin supplements was 1309 ng cobalamin equivalents/g wet wt of feces. Cobalamin (1.4%) and cobinamide (1.8%) (an incomplete corrinoid) represented a small portion of the total amount. Six cobalamin analogues that contain a base other than the 5,6-dimethylbenzimidizidole in cobalamin were present. The bases and their mean amounts (in %) are 2-methyladenine (60.6%), p-cresol (16.3%), adenine (12.5%), 2-(methylthio)adenine (15.5%), 5-hydroxybenzimidazole (1.8%), and phenol (0.1%). One subject ingested 1 mg cobalamin/d and another ingested 2 mg cobalamin/d, and they appeared to convert most of the cobalamin to cobinamide and the 4 analogues that contain the bases-2-methyladenine, p-cresol, adenine, and 2-(methylthio)adenine. CONCLUSIONS: Cobalamin analogues are present in human feces and account for > 98% of the total of cobalamin plus cobalamin analogues. A major portion of large amounts of ingested cobalamin appears to be converted to cobalamin analogues.

[Comparative study on separation of p-aminobenzoic and p-hydroxy-benzoic acids by reactive extraction]. / Studiul comparativ al separarii acizilor p-amino- benzoic si p-hidroxibenzoic prin extractie reactiva.

The comparative study on reactive extraction of p-aminobenzoic and p-hydroxybenzoic acid with Amberlite LA-2 and D2EHPA in two solvents with different polarity (n-heptane and dichloromethane) indicated that the nature of the specific substituents, extractant type and solvent polarity control the extraction mechanism. Although the reactive extraction with Amberlite LA-2 of the two acids occurs by means of rather similar interfacial reactions, the extraction with D2EHPA is based on different mechanisms, due to the participation of the two different substituents of aminic and phenolic type. In all cases, the most efficient extractions have been reached for the combination Amberlite LA-2-dichloromethane.

[The extraction and transport of folic acid through liquid membranes]. / Extractia si transportul acidului folic prin membrane lichide.

The study on the extraction and transport of folic acid (vitamin B9) with Amberlite LA-2 underlined the significant influences of the pH-gradient on the aqueous phases, carrier concentration and mixing intensity, in correlation with the extraction mechanism. Thus, for reaching the maximum efficiency of folic acid separation, the pH-value of the initial aqueous phase of 5, the pH-value of the final aqueous solution greater than 11 and the Amberlite LA-2 concentration in solvent layer over 80 g/l are required.

Extraction of water-soluble vitamins from pharmaceutical preparations using AOT (sodium di-2-ethylhexyl sulfosuccinate)/pentane reversed micelles.

Reversed micelles can be used to concentrate water-soluble materials in the water pool. In this study, the extraction of water-soluble vitamins into reversed micelles was attempted, and a flow system was used to determine the time-course of the vitamin extraction. The efficiency of extraction was strongly affected by the extraction temperature and the concentration of reversed micelles, and the selectivity depended on the size of micelles. Water-soluble vitamins could be efficiently and rapidly extracted. The selective extraction of a model mixture of vitamins from pharmaceutical preparations was also attempted. Moreover, the usefulness of the proposed method for the determination of vitamins in various commercial tablets was also demonstrated. Using of this method, the surfactant remains mixed with the extracted compounds, and so we attempted to remove the surfactant from the extract by supercritical fluid extraction. Supercritical carbon dioxide containing 7.5% ethanol as entrainer was found to be the most efficient solvent for removing residual surfactant from the extract.

Micellar electrokinetic capillary chromatography analysis of water-soluble vitamins and multi-vitamin integrators.

Micellar electrokinetic capillary chromatography (MECC) was applied to the determination of water-soluble vitamins (WSV) in pharmaceutical preparations. The analytical procedure employed in the WSV separation by MECC showed a satisfactory average column efficiency (195,000 N) and resolution (more than 100 theoretical peaks separable). The mean reproducibility in the retention time of consecutive and day-to-day runs was 1.1% and 5.1%, respectively. Analyses were performed after extraction of active ingredients by solid phase extraction. Recoveries (92-103%) and relative variation coefficients (less than 5%) confirmed the potential of MECC in the field of pharmaceutical analysis.