Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, -tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C₁₈ AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C₁₈ (150×3.9 mm, 4 μm) analytical column, using CH₃OH–0.05 M CH₃COONH₄ as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C₁₈ sorbent, on the above column. The mobile phase employed was MeOH–CH₃CN (95:5, v/v) working at a flow-rate of 2 ml min⁻¹ in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B₁₂) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.     

Simultaneous determination of vitamin E homologs in chicken meat by liquid chromatography with fluorescence detection

A simple and reliable method for the simultaneous determination of all eight homologs of Vitamin E in chicken meat is described. All analytes, including the internal standard (alpha-tocopherol acetate), were eluted within 35 min and detected using their native fluorescence (295 nm excitation and 330 nm emission). Chromatography using hexane based eluent on a normal phase silica column included an initial column conditioning step to prevent irreversible adsorption of tocopherols and tocotrienols on silica. Lowest detectable levels of alpha-tocopherol, gamma-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol were 0.73, 0.86, 1.0, 1.2, 1.7 and 1.3 ng, respectively.     

Rapid determination by reversed-phase high-performance liquid chromatography of Vitamins A and E in infant formulas

A rapid, sensitive method has been developed for the simultaneous determination of retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate. We compare two experimental procedures for simultaneous direct solvent extraction of these vitamins without previous saponification. Method I: the fat milk sample was extracted with ethanol-hexane and injected directly into the chromatographic column. Method II: the power milk sample was extracted with ethanol-hexane and also injected directly into the column. Under optimum conditions the limits of detection for retinol acetate, delta-, gamma-, alpha-tocopherol and alpha-tocopherol acetate were 0.33, 21.2, 32.9, 32.5 and 3.2 ng and the limits of quantification were 0.42, 25.3, 37.9, 36.8 and 6.3 ng, respectively. The precision results showed that the relative standard deviations of repeatability and reproducibility were between 0.74 and 5.7%.     

Detection of flavins by liquid chromatography using an electrochemical detector with two electrodes in series

A liquid chromatographic method for the selective detection of riboflavin (vitamin B₂) and flavin adenine dinucleotide (FAD) utilizing a thin-layer amperometric detector with two electrodes in series is described. The upstream electrode was held at ?0.4 V (vs. SCE) with the downstream electrode at +0.1 V (vs. SCE). The linear ranges are 40 ng?10 μg for riboflavin and 200 ng?8 μg for FAD and the limits of detection are 8 and 40 ng, respectively. The interference of thirteen different vitamins on the spectrophotometric or electrochemical detection of FAD and vitamin B₂ was studied and no interference was found using the two-electrode detector. The effects of light on riboflavin and FAD is discussed. The method is convenient, rapid and economic, and has high selectivity.     

Biosynthesis of Vitamin B1 (Thiamin): An Instance of Biochemical Diversity

The elucidation of the biosynthetic pathway to thiamin (Vitamin B₁) and its pyrophosphate ester, the important coenzyme “cocarboxylase”, has challenged researchers for many years and continues to do so. The problem of the origin of thiamin can be separated into three parts: the independent pathways to the pyrimidine moiety 4-amino-5-hy-droxymethyl-2-methylpyrimidine and to the thiazole moiety 5-(2-hydroxyethyl)-4-methylthiazole, and the route from these subunits to the vitamin. The steps in the latter process were fully established some twenty years ago, and it was shown that the route in aerobic bacteria and yeast differs to some extent from that in enteric bacteria. The pathways to the subunits, on the other hand, are still not clarified. Significant differences exist in the routes whereby each of the two subunits, the pyrimidine moiety and the thiazole moiety, originate in bacteria and yeast. One difficulty that delayed progress was that the incorporation patterns of labeled precursors, which were observed by different research groups in different microorganisms, could not be reconciled on the basis of a single pathway to each of the two subunits. It is now accepted that in each case different pathways exist in enteric bacteria and yeast, and that the biosynthesis of Vitamin B₁ represents an instance of biochemical diversity. A second factor that added to the difficulties is the minute amount of thiamin synthesized in microbiological cultures (about 15 μg per L culture). This limited the investigations until very recently either to the use of radioactive tracers or to the use of stable isotopes in conjunction with mass spectrometric analysis. It is widely recognized that both methods are associated with pitfalls in the interpretation of results. High-field ¹³C NMR, the most powerful modern method available for the determination of incorporation patterns, has only very recently been successfully employed in investigations of thiamin biosynthesis. As a result of the conceptual and experimental problems, even the primary precursors of each of the two relatively simple heterocyclic subunits of thiamin are still not completely established. A search for committed intermediates, the study of the enzymes, and identification of the genes that are involved are the matter of current research.     

Simultaneous analysis of vitamins and caffeine in energy drinks by surfactant-mediated matrix-assisted laser desorption/ionization

As a new approach to rapid small-molecule analysis, surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF-MS) was successfully used in the analysis of caffeine and the vitamins riboflavin, nicotinamide, and pyridoxine in various energy drinks. Of five common MALDI matrices tested (α-cyano-4-hydroxycinnamic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, dithranol, and 2′,4′,6′-trihydroxyacetophenone), α-cyano-4-hydroxycinnamic acid was found to be most suitable for analysis of high-sugar-containing energy drinks. Cetyltrimethylammonium bromide (CTAB) surfactant was used as a matrix-ion suppressor, at a matrix:surfactant mole ratio of approximately 500:1. The resulting mass spectra show very few matrix-related ions, while analyte signals were clearly observed. For comparative purposes the same analytes were identified and quantified in energy drinks by LC–ESI–MS with UV detection. Quantitatively the calibration curves of all four analytes showed a marked improvement when the surfactant-mediated method was used compared with traditional MALDI–TOF-MS; correlation coefficients of 0.989 (nicotinamide), 0.991 (pyridoxine), 0.983(caffeine) and 0.987 (riboflavin) were obtained. It was found that in quantitation of the energy drink analytes the surfactant-mediated MALDI–TOF-MS results were comparable with those from LC analysis. In reproducibility experiments RSD values ranged from 9.7 to 18.1%. The work has demonstrated that this mass spectrometric approach can be used as a rapid screening technique for fortified drinks.     

Improving the Yield of α-Tocopherol from Natural Sources—Chemistry versus Molecular Biology

The conversion of naturally occurring pools of tocopherols in plants into α-tocopherol can be accomplished with a molecular biological and with a chemical approach (see scheme). Therefore, the nutritional value of natural vitamin E sources is enhanced.     

荔枝中水溶性维生素的毛细管电泳快速分离分析

本研究通过双模对接高压电源实现了毛细管电泳中0～40 kV及以上的超高电压。通过考察电压、缓冲溶液浓度、pH等对分离的影响,确定了优化的实验条件。结果表明,在40 kV的超高电压下,缓冲液为25 mmol/L硼砂-H3BO3溶液(pH 8.8),8种水溶性维生素(VB1、VB2、VB6、VC、D-泛酸钙、D-生物素和烟酸、叶酸)在2.2 min内获得了较好的基线分离。同时对荔枝中的水溶性维生素进行定量分析得到了令人满意的结果。     

Selectivity differences of water‐soluble vitamins separated on hydrophilic interaction stationary phases

In this study, the retention behavior and selectivity differences of water‐soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.