HILIC separation and quantitation of water‐soluble vitamins using diol column

Hydrophilic interaction liquid‐chromatography (HILIC) in conjunction with diode array detection has been applied for the separation of selected‐water‐soluble vitamins using an end‐capped HILIC‐diol column. Vitamins with significant biological importance, such as thiamine (B₁), riboflavin (B₂), nicotinic acid (B₃), nicotinamide (B₃), pyridoxine (B₆), folic acid (B₉), cyanocobalamin (B₁₂) and ascorbic acid (vitamin C) were simultaneously separated. Chromatographic conditions including type and percentage of organic modifier in the mobile phase, pH, type and concentration of buffer salt and flow rate were investigated. ACN was shown to offer superior separation for the compounds tested as compared to methanol, isopropanol and THF. Isocratic separation and analysis were achieved for six vitamins (B₁, B₂, nicotinic acid/nicotinamide, B₆ and C) at ACN–H₂O 90:10, containing ammonium acetate 10 mM, triethylamine 20 mM, pH 5.0, using a flow rate of 0.8 mL/min, while a gradient was necessary to resolve a mixture of all eight water‐soluble vitamins. The HILIC method was validated and successfully applied to the analysis of a pharmaceutical formulation and an energy drink negating the need for time consuming clean‐up steps.     

Ion‐pair cloud‐point extraction: A new method for the determination of water‐soluble vitamins in plasma and urine

A novel, simple, and effective ion‐pair cloud‐point extraction coupled with a gradient high‐performance liquid chromatography method was developed for determination of thiamine (vitamin B₁), niacinamide (vitamin B₃), pyridoxine (vitamin B₆), and riboflavin (vitamin B₂) in plasma and urine samples. The extraction and separation of vitamins were achieved based on an ion‐pair formation approach between these ionizable analytes and 1‐heptanesulfonic acid sodium salt as an ion‐pairing agent. Influential variables on the ion‐pair cloud‐point extraction efficiency, such as the ion‐pairing agent concentration, ionic strength, pH, volume of Triton X‐100, extraction temperature, and incubation time have been fully evaluated and optimized. Water‐soluble vitamins were successfully extracted by 1‐heptanesulfonic acid sodium salt (0.2% w/v) as ion‐pairing agent with Triton X‐100 (4% w/v) as surfactant phase at 50°C for 10 min. The calibration curves showed good linearity (r² > 0.9916) and precision in the concentration ranges of 1‐50 μg/mL for thiamine and niacinamide, 5–100 μg/mL for pyridoxine, and 0.5–20 μg/mL for riboflavin. The recoveries were in the range of 78.0–88.0% with relative standard deviations ranging from 6.2 to 8.2%.     

Spectroscopic Properties of 4‐Pyridoxic Acid as a Function of pH and Solvent

The photophysical properties and acid/base equilibria of 4‐pyridoxic acid (=3‐hydroxy‐5‐(hydroxymethyl)‐2‐methylpyridine‐4‐carboxylic acid), the final product of the catabolism of vitamin B₆, have been studied in aqueous solutions. The ground state of 4‐pyridoxic acid exhibits the different protonated forms A – D in the range of H₀=?6 to pH 11.5. HMQC‐ and HMBC‐NMR Studies allowed the pH‐dependent assignment of the different C‐atoms, and the evaluation of the deprotonation sequence. The 3‐OH group in the ground state has a ‘pK_a’ of H₀=?0.64, which is much lower than that found for other vitamin B₆ related compounds. The pK_a value of the 4‐COOH group is 5.4. Fluorescence studies showed that the same species exist at the lowest excited singlet state, but in different pH ranges. The 3‐OH group is four pH units more acidic in the lowest excited singlet state than in the ground state. Excitation spectra and emission decays in the pH range of 8 to 11.5 indicate that the pyridine N‐atom is more basic in the excited singlet state than in the ground state. The emission spectra are red‐shifted in protic solvents, in agreement with an intramolecular H‐bond between the ionized 3‐OH group and the nonionized 4‐COOH group.     

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.     

Determination of Water Soluble Vitamins in Laboratory Animal Feeds by Micellar Electrokinetic Chromatography

Seven water-soluble vitamins: thiamine hydrochloride (B₁), riboflavin hydrochloride (B₂), pyridoxine hydrochloride (B₆), calcium pantotenate (B₅), ascorbic acid (C), folic acid (B₉), and biotin (H, B₇) were separated using the micellar electrokinetic chromatographic (MEKC) method in a single run with the electrolyte solution consisting of 100 mM H₃BO₃, 5 mM Na₂B₄O₇, and 30 mM sodium dodecylsulphate (SDS). The separation was achieved within 8 min. and the detection was performed at 200 nm. The calibration graphs plotted with six concentrations of each vitamin were linear with the determination coefficient r > 0.999. The method was applied for quantification of vitamins B₁, B₂, B₅, B₆, B₉, C, and H in novel feed for laboratory animals. The sample preparation involved solid-phase extraction of the vitamins, used prior to the MEKC analysis. The attained precision level was good with the recoveries between 98.4% and 105.6%. The simplicity of the procedure should make it highly useful for feed quality control in husbandry and the animal feed industry.     

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.     

Design of a Vitamin B1‐Selective Electrode Based on an Ion‐Pair and Its Application to Pharmaceutical Analysis

Vitamin B₁‐selective electrodes with PVC membrane were developed that contain ion associates of vitamin B₁ with an inorganic anion, BiI₄^?, and an organic anion, brilliant yellow, as electrode‐active substances. The linearity ranges of the electrode function are 1.0×10^?5–1.0×10^?2 and 1.0×10^?4–1.0×10^?2 M, the electrode function slopes are 33.0±1.0 and 33.1±1.1 mV decade^?1, the detection limits are 5.5×10^?6 and 8.3×10^?5 M for BiI₄^? and brilliant yellow respectively. The working range of pH is 5–12. The efficiency of the use of electrodes for the vitamin B₁ content control in multivitamin pharmaceutical preparations was shown by direct potentiometry and potentiometric titration methods.     

Analysis of Levodopa in the Presence of Vitamin B6 Using Carbon Paste Electrode Modified with 1‐Butyl‐3 methylimidazolium Hexafluorophosphate and CuO Nanoparticles

A carbon paste electrode modified with 1‐butyl‐3‐methylimidazolium hexafluorophosphate ionic liquid (BMIPF6) and CuO nanoparticles (CuO/NPs) (CPE/BMIPF₆/CuO/NPs) was fabricated and used for square wave voltammetric analysis of levodopa in the presence of vitamin B₆. The elemental analysis, SEM and XRD methods were used for characterization of synthesized CuO nanoparticle. CPE/BMIPF₆/CuO/NPs exhibited high electrical conductivity toward the electro‐oxidation of levodopa at a pH=7.0 as best experimental condition. Using CPE/BMIPF₆/CuO/NPs the levodopa and vitamin B₆ peaks are separated and oxidized at potentials of 0.565 V and 0.835 V, respectively; hence levodopa can be detected in the presence of vitamin B₆. The electrochemical response shows a linear relationship from concentration of levodopa and vitamin B₆ in the ranges of 0.06‐1000 μM and 0.1‐700.0 μM, respectively. Finally, CPE/BMIPF₆/CuO/NPs were applied as high performance tool for determination of levodopa and vitamin B₆ in real samples.     

Sequential determination of fat- and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry

A rapid method was developed to determine both types of vitamins in Rhodiola imbricata root for the accurate quantification of free vitamin forms. Rapid resolution liquid chromatography/tandem mass spectrometry (RRLC–MS/MS) with electrospray ionization (ESI) source operating in multiple reactions monitoring (MRM) mode was optimized for the sequential analysis of nine water-soluble vitamins (B₁, B₂, two B₃ vitamins, B₅, B₆, B₇, B₉, and B₁₂) and six fat-soluble vitamins (A, E, D₂, D₃, K₁, and K₂). Both types of vitamins were separated by ion-suppression reversed-phase liquid chromatography with gradient elution within 30 min and detected in positive ion mode. Deviations in the intra- and inter-day precision were always below 0.6% and 0.3% for recoveries and retention time. Intra- and inter-day relative standard deviation (RSD) values of retention time for water- and fat-soluble vitamin were ranged between 0.02–0.20% and 0.01–0.15%, respectively. The mean recoveries were ranged between 88.95 and 107.07%. Sensitivity and specificity of this method allowed the limits of detection (LOD) and limits of quantitation (LOQ) of the analytes at ppb levels. The linear range was achieved for fat- and water-soluble vitamins at 100–1000 ppb and 10–100 ppb. Vitamin B-complex and vitamin E were detected as the principle vitamins in the root of this adaptogen which would be of great interest to develop novel foods from the Indian trans-Himalaya.     

TiO2‐assisted Photocatalytic Degradation of Vitamin B12 in Aqueous Solution:Influencing Factors,Kinetics, and Reaction Investigations

The photodegradation (λ=365 nm) of the biomolecule vitamin B₁₂, catalyzed by the photocatalyst TiO₂ nanoparticles (NPs), has been investigated in aqueous suspension. The photodegradation process of vitamin B₁₂ has been monitored by means of electronic absorption (Abs), Fourier‐transform infrared (FT‐IR), and resonance Raman (RR) spectroscopies, respectively. The results show that only under UV illumination in the presence of TiO₂ is there effective degradation, and the photocatalytic degradation of vitamin B₁₂ is strongly influenced by the amount of TiO₂ NPs, the pH, and the initial concentration of vitamin B₁₂. The photocatalytic reaction kinetics of vitamin B₁₂ conforms to a Langmuir‐Hinshelwood isotherm model. Changes involving the three structural units of the carbon‐metal bond C–Co, the organic corrin macrocycle combined with the benzimidazole nucleotide, and the inorganic CN in the vitamin B₁₂ molecule during the photocatalytic degradation are also discussed.     

Determination of aflatoxins in cereal flours by solid-phase microextraction coupled with liquid chromatography and post-column photochemical derivatization-fluorescence detection

A new method for the determination of aflatoxins B₁, B₂, G₁, and G₂ (AFB₁, AFB₂, AFG₁, AFG₂) in cereal flours based on solid-phase microextraction (SPME) coupled with high performance liquid chromatography with post-column photochemical derivatization and fluorescence detection (SPME–HPLC–PD–FD) has been developed. Aflatoxins were extracted from cereal flour samples by a methanol:phosphate buffer (pH 5.8, I = 0.1) (80:20, v/v) solution, followed by a SPME step. Different SPME and HPLC–PD–FD parameters (fiber polarity, temperature, pH, ionic strength, adsorption and desorption time, mobile phase) have been investigated and optimized. This method, which was assessed for the analysis of different cereal flours, showed interesting results in terms of LOD (from 0.035 to 0.2 ng g⁻¹), LOQ (from 0.1 to 0.63 ng g⁻¹, respectively), within and inter-day repeatability (2.27% and 5.38%, respectively) linear ranges (up to 20 ng g⁻¹ for AFB₁ and AFG₁ and 6 ng g⁻¹ for AFB₂ and AFG₂), and total raw extraction efficiency (in the range 55–59% at concentrations in the range 0.3–1 ng g⁻¹ and 49–52% at concentrations in the range 1–10 ng g⁻¹). The results were also compared with the purification step carried out by conventional immunoaffinity columns.     

The stability of water‐ and fat‐soluble vitamin in dentifrices according to pH level and storage type

The purpose of this study is to evaluate the vitamin stabilities in dentifrices by analyzing various vitamins according to the level and storage temperature. The stabilities of water‐ and fat‐soluble vitamins were investigated in buffer solution at different pH values (4, 7, 8, 10 and 11) for 14 days and in dentifrices at different pH (7 and 10) for 5 months at two temperature conditions (room and refrigeration temperature) by analyzing the remaining amounts using HPLC methods. In the buffer solution, the stability of vitamins B₁, B₆ and C was increased as the pH values increased. Vitamins E and K showed poor stability at pH 4, and vitamin B₃ showed poor stability at pH 11. In dentifrices, the storage temperature highly influenced vitamin stability, especially vitamins C and E, but the stabilities of vitamins B₁ and C according to pH values did not correspond to the buffer solution tests. Vitamin B group was relatively stable in dentifrices, but vitamin C completely disappeared after 5 months. Vitamin K showed the least initial preservation rates. Vitamins were not detected in commercial dentifrices for adults and detected amounts were less than the advertised contents in dentifrices for children. Copyright © 2015 John Wiley & Sons, Ltd.     

Voltammetric determination of vitamin B<Subscript>6</Subscript> (pyridoxine) at a graphite screen-printed electrode modified with graphene oxide/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>@SiO<Subscript>2</Subscript> nanocomposite

The preparation and study of electrochemical properties of a graphite screen-printed electrode (SPE) modified with the GO/Fe₃O₄@SiO₂ (GO is graphene oxide) nanocomposites are described. The morphologies of the GO/Fe₃O₄@SiO₂ nanocomposites were examined by scanning electron microscopy. The electrochemical oxidation of vitamin B₆ (pyridoxine) on SPE modified with the GO/Fe₃O₄@SiO₂ nanocomposite was investigated by cyclic voltammetry, differential pulse voltammetry, and chronoamperometry. Under optimum conditions (pH 7.0), the vitamin B₆ oxidation at the surface of the modified SPE occurs at a potential about 190 mV less positive than that at the unmodified SPE. A linear voltammetric response for vitamin B₆ was obtained in the concentration range 1.0?10 ⁶—9.0?10 ⁴ mol L^–1 with a detection limit of 5.2?10 ⁷ mol L^–1 using differential pulse voltammetry. The developed sensor was also successfully applied for determination of trace level of vitamin B₆ in both the standard vitamin B₆ sample and biological samples (urine).     

The kinetics of photostabilization of cyanocobalamin in liposomal preparations

Cyanocobalamin (B₁₂) is a photosensitive vitamin, and its photodegradation to hydroxocobalamin (B_12b) in liposomes has been investigated. The values of apparent first-order rate constants (k_obs) for the photodegradation of B₁₂ in liposomes (nine preparations) are in the range of (0.52-2.24) × 10^–3 min^–1, compared to 3.21 × 10^–3 min^–1 for B₁₂ in aqueous solution (pH 5.0). The entrapment efficiency of B₁₂ in liposomes is 26.4-38.8%. The values of k_obs show a linear relation with phosphatidylcholine (PC) content in liposomes, indicating the influence of PC in inhibiting the rate of photolysis of B₁₂. The value of the bimolecular rate constant for photochemical interaction of B₁₂ and PC is 0.32 M^–1 min^–1, indicating the stabilizing effect of PC on the photolysis of B₁₂. The ratio of B₁₂ stabilization in liposomal preparations is in the range 2-6 compared to that of the unentrapped vitamin The stabilization of B₁₂ is mediated by a photoinduced charge-transfer B₁₂-PC complex that leads to the reduction of B₁₂ to B_12r, which is then oxidized to B_12b that has low susceptibility to photolysis. The extent of stabilization of B₁₂ probably depends on the degree of interaction between the two compounds under the reaction conditions, indicated by the loss of B₁₂ fluorescence.     

A new liquid membrane electrode for determination of vitamin B1 in multivitamin preparations

Summary A new liquid membrane electrode was developed which is highly selective and sensitive for vitamin B₁. It is based on the thiamine-picrolonate ion-pair complex as an electroactive material in nitrobenzene solvent. The electrode has a remarkable selectivity for vitamin B₁ in the presence of vitamins B₂, B₆, B₁₂ and nicotinamide. The results obtained for the determination of vitamin B₁ in various multivitamin preparations at levels as low as 1 g/ml show an average recovery of 98% (mean standard deviation 1.8%) and are favourably compared with those obtained by the official methods.

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Neue Flüssig-Membranelektrode zur Bestimmung von Vitamin B₁ in Multivitaminpräparaten

Zusammenfassung Eine neue, für Vitamin B₁ hochselektive Flüssig-Membranelektrode wurde entwickelt. Sie beruht auf dem Thiamin-Picrolonat-Ionenpaarkomplex als elektroaktives Material im Lösungsmittel Nitrobenzol. Die Elektrode besitzt eine beachtliche Selektivität für Vitamin B₁ in Gegenwart der Vitamine B₂, B₆, B₁₂ und Nicotinamid. Bei Bestimmungen in verschiedenen Multivitaminpräparaten mit Konzentrationen bis herab zu 1 g/ml erhielt man eine durchschnittliche Wiederfindung von 98% (mittlere Standardabweichung 1,8%). Die erhaltenen Resultate zeigen gute Übereinstimmung mit den nach amtlichen Methoden erhaltenen Werten.

     

Interactions and Encapsulation of Vitamins C,B3, and B6 with Dendrimers in Water

Titrations of commercial diaminobutane (DAB) and polyamidoamine (PAMAM) dendrimers by vitamins C (ascorbic acid, AA), B₃ (nicotinic acid), and B₆ (pyridoxine) were monitored by ¹H NMR spectroscopy using the chemical shifts of both dendrimer and vitamin protons and analyzed by comparison with the titration of propylamine. Quaternarizations of the terminal primary amino groups and intradendritic tertiary amino groups, which are nearly quantitative with vitamin C, were characterized by more or less sharp variations (Δδ) of the ¹H chemical shift (δ) at the equivalence points. The peripheral primary amino groups of the DAB dendrimers were quaternarized first, but not selectively, whereas a sharp chemical‐shift variation was recorded for the inner methylene protons near the tertiary amines, thereby indicating encapsulation, when all the dendritic amines were quaternarized. With DAB‐G5‐64‐NH₂, some excess acid is required to protonate the inner amino groups, presumably because of basicity decrease due to excess charge repulsion. On the other hand, this selectivity was not observed with PAMAM dendrimers. The special case of the titration of the dendrimers by vitamin B₆ indicates only dominant supramolecular hydrogen‐bonding interactions and no quaternarization, with core amino groups being privileged, which indicates the strong tendency to encapsulate vitamins. With vitamin B₃, a carboxylic acid, titration of DAB‐G3‐16‐NH₂ shows that only six peripheral amino groups are protonated on average, even with excess vitamin B₃, because protonation is all the more difficult due to increased charge repulsion, as positive charges accumulate around the dendrimer. Inner amino groups interact with this vitamin, however, thus indicating encapsulation presumably with supramolecular hydrogen bonding without much charge transfer.     

Fluorescence of New o‐Carborane Compounds with Different Fluorophores: Can it be Tuned?

Two sets of o‐carborane derivatives incorporating fluorene and anthracene fragments as fluorophore groups have been successfully synthesized and characterized, and their photophysical properties studied. The first set, comprising fluorene‐containing carboranes 6 – 9 , was prepared by catalyzed hydrosilylation reactions of ethynylfluorene with appropriate carboranylsilanes. The compound 1‐[(9,9‐dioctyl‐fluorene‐2‐yl)ethynyl]carborane ( 11 ) was synthesized by the reaction of 9,9‐dioctyl‐2‐ethynylfluorene and decaborane (B₁₀H₁₄). Furthermore, reactions of the lithium salt of 11 with 1 equivalent of 4‐(chloromethyl)styrene or 9‐(chloromethyl)anthracene yielded compounds 12 and 13 . Members of the second set of derivatives, comprising anthracene‐containing carboranes, were synthesized by reactions of monolithium or dilithium salts of 1‐Me‐1,2‐C₂B₁₀H₁₁, 1‐Ph‐1,2‐C₂B₁₀H₁₁, and 1,2‐C₂B₁₀H₁₂ with 1 or 2 equivalents of 9‐(chloromethyl)anthracene, respectively, to produce compounds 14 – 16 . In addition, 2 equivalents of the monolithium salts of 1‐Me‐1,2‐C₂B₁₀H₁₁ (Me‐o‐carborane) and 1‐Ph‐1,2‐C₂B₁₀H₁₁ (Ph‐o‐carborane) were reacted with 9,10‐bis(chloromethyl)anthracene to produce compounds 17 and 18 , respectively. Fluorene derivatives 6 – 9 exhibit moderate fluorescence quantum yields (32–44 %), whereas 11 – 13 , in which the fluorophore is bonded to the C_cluster (C_c), show very low emission intensity (6 %) or complete fluorescence quenching. The anthracenyl derivatives containing the Me‐o‐carborane moiety exhibit notably high fluorescence emissions, with ?_F=82 and 94 %, whereas their Ph‐o‐carborane analogues are not fluorescent at all. For these compounds, we have observed a correlation between the C_c?C_c bond length and the fluorescence intensity in CH₂Cl₂ solution, comparable to that observed for previously reported styrene‐containing carboranes. Thus, our hypothesis is that for systems of this type the fluorescence may be tuned and even predicted by changing the substituent on the adjacent C_c.     

A new sample preparation and separation combination for precise,accurate, rapid,and simultaneous determination of vitamins B1, B2, B3, B5, B6, B7, and B9 in infant formula and related nutritionals by LC-MS/MS

An improved method was developed for simultaneous determination of the fortified forms of thiamine (B₁), riboflavin (B₂), nicotinamide and nicotinic acid (B₃), pantothenic acid (B₅), pyridoxine (B₆), biotin (B₇), and folic acid (B₉) in infant formulas and related nutritionals. The method employed a simple, effective, and rapid sample preparation followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). It improved upon previous methodologies by offering facile and rugged sample preparation with improved chromatographic conditions, which culminated in a highly accurate and precise method for water-soluble vitamin determination in a wide range of formulas. The method was validated over six days in ten unique matrices with two analysts and on instruments in two different labs. Intermediate precision averaged 3.4 ± 2.6% relative standard deviation and over-spike recovery averaged 100.2 ± 2.4% (n = 160). Due to refinements in sample preparation, the method had high sample throughput capacity.     

Study on the Interaction of Vitamin B12 with DNA by Spectroscopy and Electrochemical Methods

The Interaction between vitamin B₁₂ (VB₁₂) and fish sperm DNA was investigated in physiological buffer (pH 7.4) using the methylene blue (MB) dye as a spectral probe by spetcrophotometery, viscosity measurements and cyclic voltammetry. The apparent binding constant of vitamin B₁₂ with DNA was found to be 3.2×10⁵ mol⁻¹·L. The voltammetric behavior of vitamin B₁₂ has been investigated at glassy carbon electrode using cyclic voltammetry. Thermodynamic parameters including ΔH⁰, ΔS⁰ and ΔG⁰ for the interaction between VB₁₂ and DNA have determined as −2.3×10⁴, 27.54 and −3.1×10⁴J·mol⁻¹·K⁻¹ respectively. One indication of DNA binding mode with VB₁₂ was the change in viscosity when a small molecule associates with DNA. The diffusion coefficients of VB₁₂ in the absence (D₀)_f and presence of DNA (D₀)_b was calculated as 5.04×10⁻⁶ and 1.13×10⁻⁶ cm²·s⁻¹ respectively. The results indicated that vitamin B₁₂ can bind to DNA and the major binding mode was intercalative binding.