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%.     

Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion,peptide separation,and protein identification using electrospray ionization mass spectrometry

Separation and determination of water- and fat-soluble vitamins by micellar (MEKC) and microemulsion electrokinetic chromatography (MEEKC) are compared. MEKC is only useful in the quantitative analysis of water-soluble vitamins when sodium dodecylsulfate (SDS) is used as the surfactant. However, the separation of mixtures containing water- and fat-soluble vitamins is only achieved by MEEKC using a microemulsion prepared by mixing SDS as the surfactant, butanol as the co-surfactant, octane as the non-polar modifier and propanol as the second co-surfactant. The injection time and the solvent used for the dilution of samples have a significant effect on the analysis of lypophilic compounds. The most reproducible results in the analysis of fat-soluble vitamins are obtained by using the same microemulsion electrolyte as the solvent for samples and an injection time of 10 s.     

Routine clinical determination of carotene, vitamin E, vitamin A, 25-hydroxy vitamin D3 and trans-vitamin K1 in human serum by straight phase HPLC

A universal extraction procedure is described for fat-soluble vitamins in human serum. Methods are presented for routine quantitative analysis by isocratic straight phase HPLC with UV-detection of (alpha + beta)-carotene, vitamin E (alpha-tocopherol) and vitamin A (all-trans-retinol) in one single run, and of vitamin K1 (trans-phylloquinone) and 25-hydroxy vitamin D3 after sample clean-up using disposable reversed-phase cartridges. The limits of detection, precisions and selectivities of the developed assays are shown to be satisfactory after more than three years' experience. The routine clinical determination of fat-soluble vitamins can be performed in less than 5 mL of serum. Analyses of external quality control and randomly taken outpatient samples are shown to be of great value in assessing laboratory performance.     

Determination of vitamins A and E in milk samples by fluorescence in micellar media

The fat-soluble vitamins A and E in milk samples were determined by fluorescence at room temperature in an aqueous media of micellar solutions. Different types of surfactants were studied; the cationic hexadecyltrimethylammonium bromide (CTAB), the anionic sodium dodecylsulfate (SDS) and the non-ionic polyoxyethylene(23)laurylether (Brij 35). The detection limits ranged between 50 and 90 ng.L-1 for both vitamins in CTAB and Brij 35. The method has been applied to the determination of vitamins A and E in milk samples.     

Rapid determination of water- and fat-soluble vitamins with microemulsion electrokinetic chromatography

A rapid, reliable and reproducible method based on microemulsion electrokinetic chromatography (MEEKC) for simultaneous determination of 13 kinds of water- and fat-soluble vitamins has been developed in this work. A novel microemulsion system consisting of 1.2% (w/w) sodium lauryl sulphate (SDS), 21% (v/v) 1-butanol, 18% (v/v) acetonitrile, 0.8% (w/w) n-hexane, 20mM borax buffer (pH 8.7) was applied to improve selectivity and efficiency, as well as shorten analysis time. The composition of microemulsion used as the MEEKC running buffer was investigated thoroughly to obtain stable separation medium, as well as the optimum determination conditions. Acetonitrile as the organic solvent modifier, pH of the running buffer and 1-butanol as the co-surfactant played the most important roles for the separation of the fat-soluble vitamins, water-soluble vitamins and stabilization of system, respectively. The 13 water- and fat-soluble vitamins were baseline separated within 30 min. The system was applied to determine water- and fat-soluble vitamins in commercial multivitamin pharmaceutical formulation, good accuracy and precision were obtained with recoveries between 97% and 105%, relative standard derivations (RSDs) less than 1.8% except vitamin C, and acceptable quantitative results corresponding to label claim.     

Determination of vitamins A and E in milk samples by fluorescence in micellar media

The fat-soluble vitamins A and E in milk samples were determined by fluorescence at room temperature in an aqueous media of micellar solutions. Different types of surfactants were studied; the cationic hexadecyltrimethylammonium bromide (CTAB), the anionic sodium dodecylsulfate (SDS) and the non-ionic polyoxyethylene(23)laurylether (Brij 35). The detection limits ranged between 50 and 90 ng · L^–1 for both vitamins in CTAB and Brij 35. The method has been applied to the determination of vitamins A and E in milk samples. Received: 30 June 2000 / Revised: 13 September 2000 / Accepted: 16 September 2000     

Application of a polarity parameter model to the separation of fat-soluble vitamins by reversed-phase HPLC

The retention behavior of a series of fat-soluble vitamins has been established on the basis of a polarity retention model: log k = (log k)(0) + p (P(m) (N) - P(s) (N)), with p being the polarity of the solute, P(m) (N) the mobile phase polarity, and (log k)(0) and P(m) (N) two parameters for the characterization of the stationary phase. To estimate the p-values of solutes, two approaches have been considered. The first one is based on the application of a QSPR model, derived from the molecular structure of solutes and their log P(o/w), while in the second one, the p-values are obtained from several experimental measurements. The quality of prediction of both approaches has also been evaluated, with the second one giving more accurate results for the most lipophilic vitamins. This model allows establishing the best conditions to separate and determine simultaneously some fat-soluble vitamins in dairy foods.     

Validation of a screening method for the simultaneous identification of fat-soluble and water-soluble vitamins (A,E, B<Subscript>1</Subscript>, B<Subscript>2</Subscript> and B<Subscript>6</Subscript>) in an aqueous micellar medium of hexadecyltrimethylammonium chloride

Simultaneous determination of the fat-soluble vitamins A and E and the water-soluble vitamins B₁, B₂ and B₆ has been carried using a screening method from fluorescence contour graphs. These graphs show different colour zones in relation to the fluorescence intensity measured for the pair of excitation/emission wavelengths. The identification of the corresponding excitation/emission wavelength zones allows the detection of different vitamins in an aqueous medium regardless of the fat or water solubility of each vitamin, owing to the presence of a surfactant which forms micelles in water at the used concentration (over the critical micelle concentration). The micelles dissolve very water insoluble compounds, such as fat-soluble vitamins, inside the aggregates. This approach avoids the use of organic solvents in determining these vitamins and offers the possibility of analysing fat- and water-soluble vitamins simultaneously. The method has been validated in terms of detection limit, cut-off limit, sensitivity, number of false positives, number of false negatives and uncertainty range. The detection limit is about g L^–1. The screening method was applied to different samples such as pharmaceuticals, juices and isotonic drinks.     

超高效合相色谱法快速测定保健食品中10种脂溶性维生素

脂溶性维生素作为保健食品重要的功效指标,现有的标准方法存在测定组分少、样品前处理过程复杂、对人员操作能力要求较高等问题,因此建立一种快速、简便、准确,且能同时检测多种常见脂溶性维生素的方法具有重要的现实意义。该研究采用超高效合相色谱(UPC²)建立了同时测定保健食品中维生素A棕榈酸酯、维生素A醋酸酯、维生素E醋酸酯、维生素K₁、α-生育酚、β-生育酚、γ-生育酚、δ-生育酚、维生素D₂、维生素D₃等10种常用脂溶性维生素的方法。样品经含75%二甲基亚砜(DMSO)的水溶液在45 ℃水浴超声15 min破乳,再加入正己烷振摇提取90 min, 3000 r/min离心10 min,取上清液过滤后进行分析。使用LC-Simulator软件对色谱条件进行模拟及优化,选用ACQUITY UPC² Viridis HSS C18 SB色谱柱进行分离,CO₂和乙腈-甲醇(85∶15, v/v)为流动相,梯度洗脱,流速1.9 mL/min,柱温30 ℃,选取10种脂溶性维生素各自的最大吸收波长检测,外标法进行定量。结果表明,10种脂溶性维生素在各自范围内线性关系良好,相关系数(r)均大于0.9997,检出限(LOD)和定量限(LOQ)分别为片剂:0.2~30 μg/g和0.8~75 μg/g,胶囊:0.4~60 μg/g和2~150 μg/g,样品平均加标回收率在96.5%~113.9%之间,RSD均小于4%,精密度、稳定性、重复性测定结果的RSD值也均小于2%。经比较,该方法测定结果与现有的国家食品安全标准基本一致,但该方法简单、快速、灵敏、准确,可满足保健食品中10种脂溶性维生素的检测要求,为保健食品中脂溶性维生素的快速同时检测奠定基础。     

Simultaneous microemulsion liquid chromatographic analysis of fat-soluble vitamins in pharmaceutical formulations: Optimization using genetic algorithm

An environmentally benign and simple method has been proposed for separation and determination of fat-soluble vitamins using isocratic microemulsion liquid chromatography. Optimization of parameters affecting the separation selectivity and efficiency including surfactant concentration, percent of cosurfactant (1-butanol), and percent of organic oily solvent (diethyl ether), temperature and pH were performed simultaneously using genetic algorithm method. A new software package, MLR-GA, was developed for this purpose. The results indicated that 73.6 mM sodium dodecyl sulfate, 13.64% (v/v) 1-butanol, 0.48% (v/v) diethyl ether, column temperature of 32.5 °C and 0.02 M phosphate buffer of pH 6.99 are the best conditions for separation of fat-soluble vitamins. At the optimized conditions, the calibration plots for the vitamins were obtained and detection limits (1.06–3.69 μg mL⁻¹), accuracy (recoveries > 94.3), precision (RSD < 3.96) and linearity (0.01–10 mg mL⁻¹) were estimated. Finally, the amount of vitamins in multivitamin syrup and a sample of fish oil capsule were determined. The results showed a good agreement with those reported by manufactures.     

In Process Citation

The separation, identification and determination of the fat- and water-soluble vitamins are realized by partition chromatography with a reversed-phase system made by bonding a C(18) group to silica. The water-soluble vitamins are directly separated with the mobile phase 1% acetic acid/acetonitrile (89:11 v/v) and are revealed by an ultraviolet detector, except for pantothenic acid. The separation efficiency and precision of determination of the fat-soluble vitamins depend on the operational conditions. The composition of the excipients and all the constituents of pharmaceuticals (aqueous and oil solutions, injections, dispersions, emulsions) determine the choice of the extraction solvents and the preparation of the solution to be injected; the polarity of the mobile phase (acetonitrile/water 95:5 v/v) can be changed, and the choice depends on the components to be separated. The experimental conditions are specified and some examples are given of application of HPLC to determination of water-soluble vitamins (B1, B2, C, PP, B6) and fat-soluble vitamins (non-saponifiable oils, vitamin A and its esters, cholecalciferol, ergocalciferol, and tocopherol and its acetate) in multivitamin formulations (solutions, suspensions, syrups, fatty excipients etc.).     

Determination of vitamins in mixtures of various composition by spectrophotometry with self-modeling curve resolution

A simple and reliable method for the analysis of mixtures of water- and fat-soluble vitamins in the UV spectral region has been developed using the chemometrical algorithm of self-modeling curve resolution for the decomposition of the spectra of mixtures. The influence of various factors on the result of spectra decomposition has been investigated. The proposed method has been applied to the analysis of model vitamin mixtures as well as multivitamin preparations and energetic drinks.     

高效液相色谱法同时测定秋葵干蔬中的脂溶性维生素A、E、K3

建立了同时测定秋葵干蔬中脂溶性维生素A、E、K3的反相高效液相色谱法.样品皂化后,采用C18色谱柱(150 mm×4.6 mm,5μm i.d.)进行分离,以甲醇为流动相,流速1.0 mL/min,二极管阵列检测器(DAD)在325、290、244 nm波长下同时检测,外标法定量.结果表明,脂溶性维生素A、E、K3在0...     

A liquid chromatography method for determination of selected amino acids, coenzymes, growth regulators, and vitamins from Cicer arietinum (L.) and Solanum lycopersicum (L.)

A bottleneck in crosstalk and QC research has been the quantification of diverse chemotypes in small amounts of tissue. An LC-UV method for estimating 28 selected metabolites of the regulatory network underlying growth, development, maintenance, vital functions, defense reactions, and food quality is reported. The method was based on binary gradient resolutions of the analytes in an RP C18 column. The mobile phase comprised solvent A [water+0.1% trifluoroacetic acid (TFA)] and B (acetonitrile + 0.085% TFA at a flow rate of 1 ml/min. Twenty-three metabolites (selected amino acids, coenzymes, growth regulators, phenolic antioxidant, and water-soluble vitamins) were detected at 254 nm, and four fat-soluble vitamins at 280 nm. Jasmonic acid was quantified at 210 nm. The RSDs of peak area and retention time for each metabolite were <5.8%. The calibration graphs were linear with R2 values ranging from 0.98 to 0.99. The LODs (microg/mL) were about 0.01-1.0 for 23 metabolites quantified at 254 nm, 0.1-0.2 for fat-soluble vitamins, and 0.1 for jasmonic acid. The recoveries ranged from 80 to 105%, with RSDs of 2.8 to 11.2%. The method has been satisfactorily applied for determination of 28 metabolites from Cicer arietinum (L.) and Solanum lycopersicum (L.). It could be an alternative and competitive method of choice that can cheaply and easily perform routine analysis for food quality and targeted metabolomics of chickpea and tomato in response to stressors.     

Analysis of pharmaceuticals and other solutes of biochemical importance by supercritical fluid chromatography

The feasibility of using supercritical fluid chromatography (SFC) for analysis of polar and/or ionic analytes of interest to the pharmaceutical industry is described. Specifically, the analyses of cyclopsporin (a cyclic undecapeptide), several ionophores, and a group of fat-soluble vitamins are illustrated. The separation of group of fat-soluble vitamins is evaluated on two bonded stationary phases, DB-5 (95% dimethyl-5%-diphenylpolysiloxane) and DB-WAX (Carbowax 20M type, “bonded”). Lastly, a new restrictor technology known as a converging-diverging restrictor is described.     

Simultaneous determination of fat- and water-soluble vitamins by microemulsion electrokinetic chromatography

A new procedure was developed for the simultaneous determination of water- and fat-soluble vitamins by microemulsion electrokinetic chromatography. Using microemulsions based on sodium dodecyl sulfate, Brij 35, 1-butanol, and heptane, 10 vitamins were separated (4 water-soluble and 6 fat-soluble) within 35 min. The efficiency of the separation was 800000 theoretical plates (effective capillary length, 40 cm). To enhance the selectivity, 2-propanol was used as a hydrophobizing addition into the microemulsion. The procedure was used for analyzing a kaolin-based vitamin premix. The use of microemulsion as an agent for extracting vitamins from the support considerably simplifies the sample preparation.     

Characterization of Column Packings in Normal-Phase Liquid Chromatography. I. Retention Behavior of Fat-Soluble Vitamins in Silica Gel-Binary Solvent Systems

Abstract

To characterize packing columns in high performance liquid chromatography, the retention indices of ten fat-soluble vitamins were systematically measured using binary solvents each containing ethyl acetate, tetrahydrofuran and 2-propanol in n-hexane for silica gel chromatography. A linear relationship between the logarithm of the capacity ratio and that of the concentration of the polar solvents was confirmed. The retention sequence of the solutes was determined as follows: retinol > ergocalciferol = cholecalciferol > δ- > γ- > β- > α - tocopherol > menadione > phylloquinone. The retention behavior of retinal was similar to that of tocopherol derivatives, but varied depending on the polar solvent used. Such a retention sequence of fat-soluble vitamins may be explained on the basis of hydrogen bonding interactions between the active functional group on the solute molecules and silanol groups on the silica gel surface. Based on the adsorption selectivity given by the phase systems used, the resolution of each class of vitamins but not that of vitamin D homologues was successfully carried out.     

Determination of fat-soluble vitamins and carotenoids in standard reference material 3280 multivitamin/multielement tablets by liquid chromatography with absorbance detection

The concentrations of selected fat-soluble vitamins and carotenoids in Standard Reference Material (SRM) 3280 Multivitamin/Multielement Tablets have been determined by two independent LC methods, with measurements performed by the National Institute of Standards and Technology (NIST). This SRM has been prepared as part of a collaborative effort between NIST and the National Institutes of Health Office of Dietary Supplements. The SRM is also intended to support the Dietary Supplement Ingredient Database that is being established by the U.S. Department of Agriculture. The methods used at NIST to determine the concentration levels of vitamins A and E, and beta-carotene in the SRM used RPLC with absorbance detection. The relative precision of these methods ranged from 2 to 8% for the analytes measured. SRM 3280 is primarily intended for use in validating analytical methods for the determination of selected vitamins, carotenoids, and elements in multivitamin/multielement tablets and similar matrixes.