Method For Determination Of Vitamin Research Articles

Using and Validation of a new Spectrophotometric method for determination of Vitamin C in Pharmaceutical samples

A new simple and rapid spectrophotometric method for determination the accurate amount of vitamin C containedin pharmaceutical samples has been developed. The ferric chloride is reduced by vitamin C and then reacts with potassium ferricyanide to create a dark blue solution of Prussian blue. The resulted color was observant at the absorption wavelength of 697nm. Optimum reaction conditions of Ferric chloride, Potassium Ferricyanide concentrations, reaction time, temperature and pH where investigated. Influence of potential interfering substances were also examined.Analyzes of commercial pharmaceutical samples showed good correlation with the valuesgiven by the manufacturers and the results obtained by a titration method with Iodine. The apparent molar absorptivity value is found to be 3.25 × 103. The results of analysis have been validated statistically, with good correlation coefficient (0.999). The proposed method has better LOD and LOQ values than many methods used to determine vitamin C in pharmaceutical forms.

Development and Optimization of Electrochemical Method for Determination of Vitamin C

The focus of this work was to develop a simple electrochemical method for the determination of vitamin C (VitC) by using a specially constructed microelectrode made from pyrolytic graphite sheet (PGS). A procedure for quantifying VitC in a real sample was established. VitC shows a single quasi-reversible reaction. The method was optimized, and analytical determination was performed by using cyclic voltammetry and square wave voltammetry for electroanalytical purposes. The obtained results show a linear response of the PGS electrode in a wide concentrations range. For the lower concentration range, 0.18–7.04 µg L−1, the sensitivity is 11.7 µAcm−2/mgL−1, while for the higher concentration range, 10.6–70.4 µg L−1, the sensitivity is 134 µAcm−2/mgL−1, preserving the linearity of 0.998 and 0.999. The second objective was to determine the effect of the addition of five different types of “green” biowaste on plant growth, VitC content, and antioxidant activity in arugula (Eruca sativa L.) using the developed method. After three weeks of cultivation, small differences in growth and large differences in certain nutritional characteristics were observed. The addition of black coffee makes the soil slightly alkaline and causes a significant increase in VitC content and antioxidant activity.

Novel method for determination of Vitamins E and C by Cloud-point extraction

A new, simple, fast and accurate spectrophotometric method has been found for the determination of vitamin E and C in their complexes by cloud point method. The Beer-Lambert Law was complied with for a range of concentrations ranging from 5-50 µg.ml-1 for vitamin E and 5-40 µg.ml-1 for vitamin C, and the value of the correlation coefficient R2: 9987 and 9997, and the value of the molar absorption coefficient Ԑ: 24421.25 and 10760.93, Sandel's sensitivity was 0.0176 μg.cm-2 and 0.0164 μg.cm-2 for vitamins E and C, respectively, while the detection limit was 0.0008 and 0.0009, and the quantitative limit was 0.0026 and 0.0030 for vitamins E and C, respectively. The method has been successfully applied for the determination of vitamin E and C in their pure forms and pharmaceutical preparations.

DETERMINATION OF VITAMIN C (ASCORBIC ACID) FROM Annona squamossa AND Valvet tamarind FRUITS

This research was designed to compare two methods for determination of vitamin C (Ascorbicacid) content in Annona squamosa peel and pulp as well as in Velvet tamarind pulp. Vitamin Ccontent of fresh fruit parts were determined by titrimetric and spectrophotometric methods usingpotassium permanganate as chromogenic reagent. Absorbance was measuredspectrophotometrically at 530nm. The titration method was carried out using iodometric backtitration. The results obtained from the study revealed that there was significant differencebetween the two methods and the higher vitamin C content was recorded in Annona squamosa.Both samples could therefore be used to supplement vitamin C daily intake requirements.Keywords: Annona squamossa, Valvettamarind, Vitamin C, Ascorbic acid, Spectrophotometer.

UV Spectroscopic Method for Determination of Vitamin C (Ascorbic Acid) Content in Different Fruits in South Gujarat Region

In the present study, determination of ascorbic acid content is determined in different fruits by using UV spectroscopy. In this method bromine water is added which oxidizes the ascorbic acid into dehydroascorbic acid. 2,4 dinitrophenyl hydrazine gives coupling reaction at 37°C temperature for 3 hours. After 3 hours solution is treated with 85% H2SO4 which gives coloured complex and the absorbance was measured at 491nm. The content of vitamin C was found maximum in Black currant and minimum in Grapes

Candidate reference method for determination of vitamin D from dried blood spot samples.

Background The current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies. Methods Blood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators. Results 25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5-376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86). Conclusions We have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.

A Fast and Simple Method for Determination of Vitamin E in Infant Formula by Dispersive Liquid-Liquid Microextraction Combined with HPLC-UV

Developing a simple and fast method for quantification of vitamins in complex matrixes is one of the important issues in quality control of foods such as milk. In this study, after introducing a new method for quantification of vitamin E in infant formula, its content was compared to the label claims. In this study, dispersive liquid-liquid microextraction (DLLME) procedure with acetonitrile and chloroform as dispersive and extraction solvents, respectively, was used for isolation and clean-up of vitamin E from infant formula samples without the need for saponification. Then, reversed-phase high-performance liquid chromatography (RP-HPLC) composed of a C18 column as stationary phase and the mixture of acetonitrile, methanol, and water (91:8:1%) as mobile phase using a standard addition method was employed for quantification of vitamin E (α-tocopheryl acetate) with UV detection at 296 nm. After method validation under the optimum conditions, the method provided a linear range with a determination coefficient (R2) of 0.99 and acceptable accuracy and precision. Results showed that the developed method is an appropriate method for quality control of infant formulas. The advantage of DLLME compared with saponification process and liquid-liquid extraction is the decrease of organic solvent consumption and proposing of a simple and fast method for analysis of vitamin E in infant formula. Application of the developed method for analysis of vitamin E in infant formulas on the market showed there was a considerable difference between labeled and obtained content in most of the samples.

Process for Preparation of Vitamin C and Method for Determination of Vitamin C in Tablets

Procedure for calcium ascorbate (VITAMIN-C) includes such technological steps as reaction of ascorbic acid on calcium carbonate in water. In recent years, the determination of vitamin C has become an important subject in the field of biochemistry and commercial foods.

酢酸エチル・ヘキサン抽出-HPLCによる食品中のビタミンD定量法

酢酸エチル・ヘキサン抽出-HPLCによる食品中のビタミンD定量法

Determination of vitamin E in margarines and edible oils using square wave anodic stripping voltammetry with a glassy carbon paste electrode.

A new electroanalytical method for determination of vitamin E in the form of the total content of tocopherols present in margarines and edible oils has been developed. The method is based on extraction of these biologically active compounds into silicone oil, acting as lipophilic binder of glassy carbon paste electrode, with subsequent electrochemical detection by square wave anodic stripping voltammetry (SWASV) in 0.1M HNO3. The values of vitamin E contents were expressed as mass equivalent of α-tocopherol known as the most active form of this lipophilic vitamin. The linear ranges for α-tocopherol determination were 5×10-7-4×10-5 and 5×10-8-1×10-5molL-1 with the detection limits of 1×10-7 and 3.3×10-9molL-1 for 5 and 15min accumulation, respectively. The results have shown that SWASV with extraction step is very sensitive method for the determination of vitamin E, being comparable to reversed-phase high performance liquid chromatography chosen as reference method.

Different chromatographic methods for the simultaneous determination of vitamin E and vinpocetine in their combined dosage form and in the presence of the alkaline-induced degradation product of vinpocetine

Accurate, selective, and sensitive thin-layer chromatography (TLC)—densitometry and reversed-phase high-performance liquid chromatography (RP-HPLC) methods have been developed and validated for the simultaneous determination of vitamin E (VIT E) and vinpocetine (VINP) in the presence of the alkaline-induced degradation product of vinpocetine (DEG). The proposed TLC—densitometric method depends on the separation and quantitation of VIT E, VINP, and VINP alkaline-induced degradation product on TLC silica gel 60 F254 plates, using methanol—chloroform—ethyl acetate—glacial acetic acid—ammonia solution (6:2:2:0.5:0.1, by volume) as the developing system followed by densitometric measurement at 235 nm. The studied components were well resolved from each other with significantly different RF values of 0.81, 0.62, and 0.41 for VIT E, VINP, and DEG, respectively. On the other hand, the developed RP-HPLC method was based on the separation of the studied components using 0.05 M KH2PO4 (adjusted to pH = 3) and methanol in gradient elution mode on C8 column at a flow rate of 1.5 mL min−1 and ultraviolet (UV) detection at 235 nm. The studied components were well resolved from each other with significantly different Rt values of 10.90, 2.89, and 1.90 min for VIT E, VINP, and DEG, respectively. The developed methods were validated according to the International Conference on Harmonization (ICH) guidelines demonstrating good accuracy and precision. The results were statistically compared with those obtained by the reported method, and no significant difference was found. The developed methods are the first developed stability-indicating assay methods (SIAMs) for the analysis of the studied binary mixture.

Stability Indicating Spectrophotometric Methods for Determination of Vitamin E and Vinpocetine in Their Combined Dosage Form

Stability indicating spectrophotometric methods have been developed and validated for determination of Vitamin E (VIT E) and Vinpocetine (VINP) in presence of alkaline induced degradation product of Vinpocetine (DEG) without prior separation. In method (A), two wavelengths were selected for each drug in such a way that the difference in absorbance was zero for the other drugs. Absorbance difference between 217.4 and 234.4 nm were used to determine VIT E while difference between 250.4 and 314.0 nm were selected for VINP determination and the difference between 258.6 and 312.0 nm were selected to determine DEG. In method (B), area under the curve method (AUC) was used for measuring VINP concentration by direct measuring AUC in the range of 330-350 nm, at which no interference from other components. While for determination of VIT E and DEG, the ternary mixture was divided by standard spectrum of 6 μg/mL VINP, in the obtained ratio spectra VIT E and DEG has been directly determined at 210.0 and 230.0 nm, respectively. In method (C), VIT E and DEG were determined by second derivative of ratio spectra (2DD) method by measuring their peak amplitudes at 215.2 and 237.2 nm, respectively, using 6 μg/mL standard spectrum of VINP as a divisor, while the absorbance at isoabsorptive point (λiso=264.4 nm) was used to determine the total concentration of VINP and DEG (where no contribution from VIT E) in the mixture and then by subtraction, VINP concentration could be obtained. The developed methods were validated according to ICH guidelines revealing good accuracy and precision. The results obtained by the proposed methods were statistically compared with those obtained by the reported HPLC methods and no significant difference was obtained. These methods are the first developed SIAM ones for analysis of the studied components.

Development and validation of a high performance liquid chromatographic method for simultaneous determination of vitamins A and D3 in fluid milk products.

An HPLC method for simultaneous determination of vitamins A and D3 in fluid milk was developed and validated. Saponification and extraction conditions were studied for optimum recovery and simplicity. An RP HPLC system equipped with a C18 column and diode array detector was used for quantitation. The method was subjected to a single-laboratory validation using skim, 2% fat, and whole milk samples at concentrations of 50, 100, and 200% of the recommended fortification levels for vitamins A and D3 for Grade "A" fluid milk. The method quantitation limits for vitamins A and D3 were 0.0072 and 0.0026 μg/mL, respectively. Average recoveries between 94 and 110% and SD values ranging from 2.7 to 6.9% were obtained for both vitamins A and D3. The accuracy of the method was evaluated using a National Institute of Standards and Technology standard reference material (1849a) and proficiency test samples.

Comparison of UPLC and HPLC methods for determination of vitamin C

Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehydroascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparations were compared. Both methods are rapid: total time of analysis was 15 and 6min for HPLC and UPLC methods, respectively. The methods were validated in terms of linearity, instrument precision, limits of detection (LOD) and quantification (LOQ), accuracy and recovery. Intra- and inter-day instrument precisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% for UPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively (HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049μg/mL for HPLC and 0.024μg/mL for UPLC while LOQs were 0.149 and 0.073μg/mL for HPLC and UPLC, respectively. These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in pharmaceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and consumes less eluent.

Precision of dehydroascorbic acid quantitation with the use of the subtraction method – Validation of HPLC–DAD method for determination of total vitamin C in food

In food analysis, a method for determination of vitamin C should enable measuring of total content of ascorbic acid (AA) and dehydroascorbic acid (DHAA) because both chemical forms exhibit biological activity. The aim of the work was to confirm applicability of HPLC–DAD method for analysis of total content of vitamin C (TC) and ascorbic acid in various types of food by determination of validation parameters such as: selectivity, precision, accuracy, linearity and limits of detection and quantitation. The results showed that the method applied for determination of TC and AA was selective, linear and precise. Precision of DHAA determination by the subtraction method was also evaluated. It was revealed that the results of DHAA determination obtained by the subtraction method were not precise which resulted directly from the assumption of this method and the principles of uncertainty propagation. The proposed chromatographic method should be recommended for routine determinations of total vitamin C in various food.

Rapid HPLC Method for Determination of Vitamin C, Phenolic Acids, Hydroxycinnamic Acid, and Flavonoids in Seasonal Samples of Emblica officinalis Juice

The work proposes a simple method for simultaneous detection and quantification of vitamin C (ascorbic acid), phenolic acids (gallic acid and ellagic acid), hydroxycinnamic acid (chlorogenic acid), and flavonoids (myricetin, quercetin, and kaempferol) in seasonal samples of emblica juice. The compounds were separated by an intangible curved gradient of 0.1% orthophosphoric acid in water (v/v) and acetonitrile as mobile phase A and B using Zorbax SB RP C-18 column at a wavelength of 254 nm in 18 min. The assay was optimized by varying the mobile phase, gradient type, and detection wavelength. The method was validated in terms of linearity, precision, detection limits, and quantification limits. Good linear response was observed over the range specified for all the analytes, as confirmed by the correlation coefficient which ranged from 0.991 and 0.995. The limit of detection and limit of quantification were found to be in the range of 0.129–0.685 µg mL−1 and 0.43–2.883 µg mL−1, respectively. Complete resolution of the phenolic compounds was achieved without the need of pre-analysis processes. The overall precision values obtained for standards and samples were within the range of 0.01–0.2 and 0.15–0.23, respectively.

Quantitative Determination of Vitamin C in Honey and Bee Pollen Using HPLC-DAD Method

Methods for determination of vitamin C from two used matrices - honey and bee pollen – by liquid chromatography coupled with UV-Vis diode array detector have been established. Separation was performed on a C18 column, using an isocratic 1% (v/v) formic acid in water as mobile phase. Meta-phosphoric acid was used as extraction solvent, each matrix following different sample preparation steps. The proposed methods were applied after their validations on the analysis of 50 honey and 25 bee pollen samples from Romania with different botanical origin and harvest years. Vitamin C concentration found in fresh bee pollen samples was significantly higher than the content in samples from previous years. Vitamin C may be used as important freshness indicator. In honey vitamin C content is not entirely dependent of storage period, but also by botanical origin of the samples. The developed method proved to be sensitive, accurate and without interfering compounds.

A Fast HPLC Method for Determination of Vitamin E Acetate in Dietary Supplements Using Monolithic Column

A simple and fast high-performance liquid chromatography (HPLC) method with rapid separation on monolithic column has been developed for quantitative determination of vitamin E acetate in dietary supplements. Commercial samples of dietary supplements were extracted with 100 % methanol with the help of ultrasound bath. A 20-μL sample volume of the filtered solution was directly injected into the HPLC system. Separation of ballast matrix, vitamin E acetate, and internal standard cholecalciferol was performed on the monolithic column Chromolith Performance RP-18e column (100 × 4.6 mm) with mobile phase methanol/water (98:2, v/v) at a flow rate of 2.0 mL min−1 and at temperature 30 °C. The detector was set at 290 nm. Under the optimum chromatographic conditions, standard calibration curve was measured with good linearity—correlation coefficient for vitamin E acetate (r = 0.9992; n = 6) between the peak areas and concentrations of vitamin E acetate. Accuracy of the method defined as a mean recovery was in the range 96.4–103.6 % for all dietary supplements. The intraday method precision was satisfactory and relative standard deviation of sample analysis including preparation and determination of different dietary supplements was in the range 1.1–3.6 %. The developed method has shown high sample throughput during sample preparation process and short time (3.5 min) of analysis.

Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25- Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography

A simple and reliable High Performance Liquid Chromatography (HPLC) method for simultaneous determination of vitamin D-2 (VD-2), vitamin D-3 (VD-3), 25-hydroxyvitamin D-2 [25 (OH) VD-2], and 25-hydroxyvitamin D-3 [25(OH) VD-3] in human plasma was developed and validated. Plasma samples were deproteinized with a mixture of methanol and 2-proponol, and extracted with hexane. After evaporation, the residue was dissolved in methanol:water (9.6:0.4, v/v), centrifuged, and then clear solution was injected onto Zorbax C18 column. The mobile phase (gradient elution mode) consists of methanol, acetonitrile, and water (pH = 3.0); the eluents were monitored by photodiode array detector (wavelength set at 265 nm). The relationship between the concentration of VD-2, VD-3, 25(OH) VD-2, 25(OH) VD-3 in plasma and their peak area ratio to the IS was linear over the range of 5 - 100 ng/mL. The coefficients of variation for inter-day and intra-day assay were all ≤ 9.7% and biases ≤ 13.1%. Mean extraction recoveries of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 from plasma were all over 80%. The method was applied for the determination of vitamin D levels in plasma obtained from healthy subjects. Further, it was used to assess the stability of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 in plasma under various conditions encountered in the clinical laboratory.

Reverse-FIA with spectrophotometric detection method for determination of vitamin C

Simple reverse flow injection analysis (rFIA) manifold with spectrophotometric detection was developed for an indirect determination of ascorbic acid. Parameters such as stability, accuracy and precision were established for the method and evaluated statistically to assess the applications of the method. Ascorbic acid in this procedure accelerates dediazoniation reaction of formed diazonium ions; hence its quantity can be determined by monitoring the derivatization product from coupling unreacted diazonium ion with phenol to give an azo dye (coupling reaction). The rFIA design was based on the injection of sodium nitrite into an acidic p-aminoacetophenon carrier stream in which diazonium ion was formed. This ion was inhibited by ascorbic acid stream before coupling with the phenol-Na2CO3 stream. Under optimum conditions, ascorbic acid acts in accordance with the Beer’s law at two concentration ranges 0.4–6.5 μg ml−1 (R = 0.9995) and 7.0–20.0 μg ml−1 (R = 0.9949), with detection limits of 0.25 μg ml−1. The developed method was applied to the determination of vitamin C in pharmaceutical formulations which produced satisfactory results compared with the standard methods reported in the British Pharmacopoeia.