Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction (original) (raw)
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Journal of Separation Science, 2015
A review of chromatographic methods for the determination of water-and fat-soluble vitamins in biological fluids Vitamins are an essential element of nutrition and thus contribute to human health. Vitamins catalyze many biochemical reactions and their lack or excess can cause health problems. Therefore, monitoring vitamin concentrations in plasma or other biological fluids may be useful in the diagnosis of various disorders as well as in the treatment process. Several chromatographic methods have been developed for the determination of these compounds in biological samples, including high-performance liquid chromatography with UV and fluorescence detection. Recently, high-performance liquid chromatography with tandem mass spectrometry methods have been widely used for the determination of vitamins in complex matrices because of their high sensitivity and selectivity. This method requires preconditioning of samples for analysis, including protein precipitation and/or various extraction techniques. The choice of method may depend on the desired cost, convenience, turnaround time, specificity, and accuracy of the information to be obtained. This article reviews the recently reported chromatographic methods used for determination of vitamins in biological fluids. Relevant papers published mostly during the last 5 years were identified by an extensive PubMed search using appropriate keywords. Particular attention was given to the preparation steps and extraction techniques. This report may be helpful in the selection of procedures that are appropriate for certain types of biological materials and analytes.
Journal of chromatography. A, 2017
Fat-soluble vitamins and antioxidants are of relevance in health and disease. Current methods to extract these compounds from biological fluids mainly need use of multi-steps and multi organic solvents. They are time-consuming and difficult to apply to treat simultaneously large sample number. We here describe a single-step, one solvent extraction of fat-soluble vitamins and antioxidants from biological fluids, and the chromatographic separation of all-trans-retinoic acid, 25-hydroxycholecalciferol, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8'-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, phylloquinone, lycopene, α-carotene, β-carotene and coenzyme Q. Extraction is obtained by adding one volume of biological fluid to two acetonitrile volumes, vortexing for 60s and incubating for 60min at 37°C under agitation. HPLC separation occurs in 30min using Hypersil C18, 100×4.6mm, 5μm particle size column, gradient from 70% methanol+30% HO to 100% acetonitr...
Journal of Al-Nahrain University Science, 2013
A new stationary phase was prepared by the reaction of a crystal violet solution with silica gel. The capacity of the new prepared resin was calculated and the found average was 6.6meq./g. The resulted resin was highly rigid with high stability and used as stationary phase for HPLC column. Some water-soluble vitamin C, B2, B6, and B12 were examined by this column with isocratic eluention distilled water and methanol (2:98, (v/v)) as a mobile phase with flow rate of 0.5 ml/min and UV detection of 230 nm. The chromatographic performance of the packed column was characterized. The number of plate numbers (N), height equivalent of a theoretical plates (H), capacity factors (K), selectivity factors (α), peaks asymmetry and Resolution (Rs) were measured by analyzing different analytes on the new columns using different mobile phase compositions and flow rates.
Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2004
In the present study, a simple and rapid reversed-phase HPLC procedure has been developed for the simultaneous determination of eight fat-soluble vitamins (retinol, menadione, menaquinone, δ-tocopherol, cholecalciferol, α-tocopherol, α-tocopherol acetate and phylloquinone) in biological fluids: blood serum and urine. The analytical column, Phenomenex Luna C18 (150mm×4.6mm) 3μm, was operating at ambient temperature. Mobile phase consisted of a mixture of CH3OH–CH3CN
HILIC separation and quantitation of water-soluble vitamins using diol column
Journal of Separation Science, 2009
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 (B 1 ), riboflavin (B 2 ), nicotinic acid (B 3 ), nicotinamide (B 3 ), pyridoxine (B 6 ), folic acid (B 9 ), cyanocobalamin (B 12 ) 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 (B 1 , B 2 , nicotinic acid/ nicotinamide, B 6 and C) at ACN -H 2 O 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.
Journal of Pharmaceutical and Biomedical Analysis, 1997
A method was developed for the quantitative analysis of six water-soluble vitamins (thiamine, nicotinamide, riboflavine, pyridoxine, ascorbic acid and pantothenic acid) in a pharmaceutical formulation, using free solution capillary zone electrophoresis (CZE) in uncoated fused silica capillaries and UV detection. The influence of different parameters, such as the nature of the buffer anionic component and buffer concentration on the CZE separation of vitamins was investigated using four vitamins of the B group as model compounds. A good compromise between resolution, analysis time and analyte stability was obtained by use of a 50 mM borax buffer of pH 8.5. This CZE method was found to be very useful for the separation of more complex samples, a mixture of ten water-soluble vitamins being completely resolved in about 10 min. However, cyanocobalamine could not be separated t¥om nicotinamide in this CZE system, the two compounds being in uncharged form at the pH used. These two compounds could easily be resolved by micellar electrokinetic chromatography (MEKC), the anionic surfactant dodecylsulfate being added to the running buffer at 25 mM concentration. In the pharmaceutical formulation, some excipients were found to be adsorbed to the capillary surface, giving rise to a progressive decrease of the electroosmotic flow and consequently to a simultaneous increase of analyte migration times. A capillary wash with sodium hydroxide had to be made between successive runs in order to minimize these effects. Good results with respect to linearity, precision and accuracy were obtained in the concentration range studied for the six vitamins, using nicotinic acid as internal standard. © 1997 Elsevier Science B.V.
Journal of Chromatography A, 2000
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, a-tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C AR 18 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 C (15033.9 mm, 4 mm) analytical 18 column, using CH OH-0.05 M CH COONH as mobile phase The chromatographic analysis of the fat-soluble vitamins 3 3 4 was carried out after their sequential elution with methanol and chloroform from C sorbent, on the above column. The 18 21 mobile phase employed was MeOH-CH CN (95:5, v / v) working at a flow-rate of 2 ml min in isocratic mode. The 3 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 B) and 285 nm for the water-soluble and fat-soluble vitamins present in real 12 samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.
ChemInform, 2005
High-performance liquid chromatographic methods are the most often used methods for the determination of water-soluble vitamins (WSV) and fat-soluble vitamins (FSV). General approaches in quantification, occurring forms of vitamins, influences, which can affect stability of vitamins, necessary precautions in sample handling, pre-run sample stabilization, extractions procedures, and HPLC quantifications are mentioned and compared. This paper provides basic guidance for using HPLC in analysis of WSV and FSV. Finally, some methods for the quantification of WSV and FSV in pharmaceutical preparations, food supplements, and biological samples are reviewed.
Journal of Chromatography A, 1984
The advent of multi-vitamin analysis by high-performance liquid chromatography (HPLC) can possibly be attributed to the use of ion-exchange principles*. Although the future of these techniques can not be entirely disregarded, reversedphase chromatography has largely superceded ion-exchange and many of the underlying principles and practicalities are well known 2. The introduction of ion-pairing reagents has, perhaps more than anything else, allowed a greater versatility and scope in controlling vitamin assays3. Separations of B-group vitamins have often been used by manufacturers of stationary phases to illustrate the efficiency of their products.