Simultaneous Determination Of Vitamin Research Articles

Simultaneous Determination of Vitamin E and γ-Oryzanol in Rice Bran Oil via HPSEC-PDA without Sample Pretreatment.

Vitamin E (tocopherols and tocotrienols) and γ-oryzanol are two minor constituents of rice bran oil (RBO) and are known to be potential bioactive compounds. The content of γ-oryzanol, a unique antioxidant found only in RBO, is a key factor in determining the retail price of the oil. Limitations of conventional HPLC columns for vitamin E and γ-oryzanol analysis are the alteration of these components and the time-consuming need for pretreatment of samples by saponification. High-performance size exclusion chromatography (HPSEC) equipped with a universal evaporative light scattering detector (ELSD) is a versatile tool for screening optimum mobile phase conditions because components of the sample can be separated and detected in the same run. In this work, the RBO components (triacylglycerol, tocopherols, tocotrienols, and γ-oryzanol) assessed on a single 100-Å Phenogel column using ethyl acetate/isooctane/acetic acid (30:70:0.1, v/v/v) as the mobile phase provided baseline separations (R s >1.5) with a total run time of 20 min. The HPSEC condition was then transferred to determine the content of tocopherols, tocotrienols, and γ-oryzanol in RBO products using a selective PDA detector. The limit of detection (LOD) and limit of quantification (LOQ) of α-tocopherol, α-tocotrienol, and γ-oryzanol were 0.34 and 1.03 μg/mL, 0.26 and 0.79 μg/mL and 2.04 and 6.17 μg/mL, respectively. This method was precise and accurate, with a percentage of relative standard deviation (%RSD) of the retention time of less than 0.21%. The intra-day and inter-day variations were 0.15-5.05% and 0.98-4.29% for vitamin E and γ-oryzanol, respectively. The recoveries of tocopherols, tocotrienols, and γ-oryzanol ranged between 90.75% and 107.98%. Thus, the developed HPSEC-ELSD-PDA method is a powerful analytical tool for determining the vitamin E and γ-oryzanol present in oil samples without requiring any sample pretreatment.

Simultaneous analysis of vitamin D and K in processed food products via ultra high- performance liquid chromatography (UHPLC)

This study highlights a simplified extraction and method development of Ultra High-Performance Liquid Chromatography for simultaneous determination of vitamin D and K in selected processed food products. Simultaneous analysis of vitamin D and K was achieved via gradient elution of methanol with ultra-pure water (95:5–100:0–95:5), at programmed flow rate of (1.0–0.2–1.0 mL/min) for left pump and constant 1 mL/min for right pump within 25 min on HPLC Dionex Ultimate 3000 Series system using two Dionex Acclaim Polar Advantage II C18 columns at 30 °C and acquisition wavelength of 265 nm. The limits of detection and quantification ranged between 0.05 and 0.12 µg/mL and 0.16–0.41 µg/mL; for both vitamins. The method showed good linearity (R2: 0.996–0.999) and high accuracy (85.35–106.36%) for both vitamin D and K. Recovery above 70% was achieved for vitamin D, whereas lower recovery of 60–70% was obtained for vitamin K. The proposed analytical and extraction procedure was advantageous for sample purification and improved detection of trace vitamin levels from selected cereal and flour products.

Simultaneous determination of Vitamin A, 25-hydroxyl vitamin D3 α-tocopherol in small biological fluids by liquid chromatography-tandem mass spectrometry

A sensitive method for the simultaneous analysis of vitamin A (VA), 25-hydroxyl vitamin D3 (25-OH VD3) and α-tocopherol (VE) in children plasma by liquid chromatography-tandem mass spectrometry (LC–MS/MS) has been developed and validated. Sample preparation chose the solid phase extraction. 100 μL of plasma was mixed with 300 μL ethanol contained 4 μL isotope-labelled analytes. After a series operation, the supernatant was applied to the solid phase extraction (SPE) plate (HLB μElution plate). The eluate was evaporated, and reconstituted in 100 μL methanol. And then, 6 μL reconstituted sample was injected into LC–MS/MS. Quantitative analysis was carried out by multiple reaction monitoring (MRM) with a positive mode electrospray (ESi + ). Separations of VA, 25-OH VD3 and VE were performed on an Acquity UPLC reversed-phase Phenyl-Hexyl analytical column (CSH, 2.1 × 50 mm, 1.7 μm). Gradient elution was used at a flow rate of 0.4 mL/min with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid, 5 mM ammonium formate in acetonitrile. The total time of analysis was 10 min. The method had a limit of quantification (LOQ) of 10.03, 1.20, and 0.04 ng/mL for VA, 25-OH VD3 and VE in methanol, respectively. The linear calibration curves were fitted over the range of 0.14–14.32 μg/mL, 1.80–180.29 ng/mL, and 6.03–602.99 ng/mL for VA, 25-OH VD3 and VE in methanol. The correlation coefficients were greater than 0.998 for all analytes. The recoveries for all analytes were between 80 and 120% with the inter- and intra-day precisions (presented as relative standard deviation, RSD%) less than 10.0%. Analysis of VA, 25-OH VD3 and VE in recurrent respiratory tract infection children plasma and anemic infants’ fingertip blood was then carried out using this method and statistical analysis of the data with statistic package for social science 20.0 (SPSS 20.0). Using this method, multiple fat-soluble vitamins could be detected at the same time. Solid phase extraction was used to simplify sample pretreatment. μElution plate used here could reduce the sample volume, only 100 μL sample was used in this method, and 6 μL reconstituted sample was injected into LC–MS/MS. This makes the method appropriate for larger sample pretreatment, and suitable for children, especially infants and newborns’ sample detection, in whom the circulation blood was low.

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.

Electrochemical determination of vitamin C in the presence of NADH using a CdO nanoparticle/ionic liquid modified carbon paste electrode as a sensor

A highly sensitive sensor based on a carbon paste electrode modified with a CdO nanoparticle and 1-methyl-3-butylimidazolium bromide was used for the voltammetric analysis of vitamin C in the presence of nicotin amide adenine dinucleotide. The highly sensitive sensor shows excellent enhancement and electrocatalytic activity towards vitamin C. The variation of pH shows the number of protons is equal to number of electrons for vitamin C electrooxidation. The electrooxidation peak current of vitamin C and nicotin amide adenine dinucleotide increased linearly with their concentration in the ranges of 0.07–480μmolL−1 vitamin C and 0.5–700μmolL−1 nicotin amide adenine dinucleotide. The limits of detection for vitamin C and nicotin amide adenine dinucleotide were 0.03μmolL−1 and 0.1μmolL−1, respectively. Simultaneous determination of vitamin C and nicotin amide adenine dinucleotide was investigated by using the square wave voltammetry technique. The proposed sensor showed good stability, sensitivity, selectivity, and reproducibility and can be used for some important food sample analyses.

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.

Quantification of the level of fat-soluble vitamins in feed based on the novel microemulsion electrokinetic chromatography (MEEKC) method

Simultaneous quantification of liposoluble vitamins is not a new area of interest, since these compounds co-determine the nutritional quality of food and feed, a field widely explored in the human and animal diet. However, the development of appropriate methods is still a matter of concern, especially when the vitamin composition is highly complex, as is the case with feed designated for laboratory animals, representing a higher health and microbiological status. A method combining microemulsion electrokinetic chromatography (MEEKC) with liquid-liquid extraction was developed for the determination of four fat-soluble vitamins in animal feed. A separation medium consisting of 25 mmol L⁻¹ phosphate buffer (pH 2.5), 2-propanol, 1-butanol, sodium dodecyl sulfate and octane allowed the simultaneous determination of vitamins A, D, E and K within a reasonable time of 25 min. The polarity of the separation voltage was reversed in view of the strongly suppressed electro-osmotic flow, and the applied voltage was set at 12 kV. The fat-soluble vitamins were separated in the order of decreasing hydrophobicity. It was proved that the proposed MEEKC method was sufficiently specific and sensitive for screening fat-soluble vitamins in animal feed samples after their sterilization.

Determination of water-soluble vitamins in multivitamin dietary supplements and in artichokes by micellar electrokinetic chromatography

Several procedures of extraction with solvents for the simultaneous determination of vitamin C and some vitamins belonging to the B group (thiamine, riboflavine, nicotinic acid and nicotinamide) in multivitamin preparations and in artichokes (Cynara cardunculus subsp. scolymus [L.] Hegi) were developed. Different experimental conditions were used, in terms of heat treatment, composition and pH of the extraction mixture, with particular attention to high-temperature steps; purification of the extracts with solid phase extraction and stabilisation through lyophilisation were discussed. Analyses of the extracts were conducted by capillary electrophoresis in micellar electrokinetic chromatography modality. Borate buffer at pH 8.2 was used, and sodium dodecyl sulphate was added to the background electrolyte as surfactant. A range of linearity was determined and calibration curves were plotted for all the analytes.

The simultaneous determination of vitamins A, E and β-carotene in bovine milk by high performance liquid chromatography–ion trap mass spectrometry (HPLC–MSn)

Bovine milk is considered to be a good dietary source of vitamins A, E and β-carotene. This paper describes a robust method for the simultaneous determination of these compounds using high performance liquid chromatography–ion trap mass spectrometry (HPLC–MSn) as the determinative step. The compounds were separated with a 5 μm Polaris 2.1 × 150 mm C18-A column and an aqueous-methanol mobile phase and the levels determined using the MS2 fragments (vitamin A, m/z 213 and m/z 199, vitamin E m/z 165 and β-carotene m/z 413). The HPLC–MSn was operated in the positive ion APCI mode. The method was validated using repeatability studies, duplicate analyses, recovery experiments, proficiency study data and comparison with previously validated high performance liquid chromatographic ultraviolet/visible (HPLC–UV/Vis) and fluorescence (HPLC–Fl) procedures used in our laboratory. Expanded measurement uncertainties were similar for HPLC–MSn, HPLC–UV/Vis and HPLC–Fl, e.g. vitamin A 45 μg/100 ml, HPLC–MSn ±8%, HPLC–UV ±9%; vitamin E 150 μg/100 ml, HPLC–MSn ±11%, HPLC–Fl ±9%; β-carotene 12 μg/100 ml, HPLC–MSn ±18%, HPLC–Vis ±21%.

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.

SIMULTANEOUS ANALYSIS OF VITAMIN C AND ASPIRIN IN ASPIRIN C EFFERVESCENT TABLETS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY–PHOTODIODE ARRAY DETECTOR

High performance liquid chromatography–photodiode array (HPLC–PDA) method has been employed for the simultaneous determination of vitamin C (ascorbic acid) and aspirin (acetylsalicylic acid) in aspirin C effervescent tablets. Chromatographic separation of ascorbic acid (AA) and acetylsalicylic acid (ASA) was performed by reversed–phase using a Betasil C18 column with particle size of 3 µm, 150 mm × 4.6 mm I.D. Optimum separation was achieved in isocratic mode with a binary mobile phase (mixture of water and acetonitrile with 0.1% of formic acid, 75:25, v/v) at a flow rate of 1.0 mL min−1. Recovery of the target compounds was obtained more than 98% with good quality parameters: linearity (r 2 > 0.997), limit of detection (LOD) and limit of quantification (LOQ) values between 1.4 × 10−3 and 5.1 × 10−4 mg mL−1, and intra-day and interday precisions with relative standard deviation (RSD) lower than 2.1%. The content of the analyzed samples ascorbic and acetylsalicylic acid was found 238.23 and 393.49 mg, respectively, which are very close to the label claimed by the manufacturer in aspirin C effervescent tablets. Therefore, this method can be proposed for routine analysis of these compounds in aspirin C effervescent tablets.

Simultaneous Determination of Vitamin A and E in Infant Formula by HPLC with Photodiode Array Detection

The objective of this study was to develop a method to simultaneously quantify vitamins A and E in infant formula. To determine the vitamin A and E content, vitamin A and four different vitamin E isomers (<TEX>${\alpha}$</TEX>-, <TEX>${\beta}$</TEX>-, <TEX>${\gamma}$</TEX>-, and <TEX>${\delta}$</TEX>-tocopherol) were separated by high performance liquid chromatography with a photodiode array detector using a Develosil RPAQUEOUS RP-<TEX>$C_{30}$</TEX> column (<TEX>$4.6{\times}250$</TEX> mm, 5 <TEX>${\mu}M$</TEX>). The vitamin A and E contents in the certified reference material determined using this method were within the certified range of standard values. The limits of detection (LODs) and limits of quantitation (LOQs) for vitamin A were 0.02 and 0.06 <TEX>${\mu}g/L$</TEX>, respectively. LODs and LOQs for the vitamin E isomers ranged from 0.20 to 0.55 and from 0.67 to 1.81 <TEX>${\mu}g/L$</TEX>, respectively. Linear analyses indicated that the square of the correlation coefficient for the vitamin A and E isomers was 0.9997-0.9999. The recovery of vitamins ranged from 96.69 to 97.79%. The results demonstrate that this novel method could be used to reliably analyze vitamin A and E content in infant formula.

PII-81Evaluation of two restricted access media supports in HPLC method development for the simultaneous determination of vitamins A and E in human plasma

PII-81Evaluation of two restricted access media supports in HPLC method development for the simultaneous determination of vitamins A and E in human plasma

Simultaneous determination of vitamins C, B6 and PP in pharmaceutics using differential pulse voltammetry with a glassy carbon electrode and multivariate calibration tools

In this work, the artificial neural networks (ANN) and partial least squares (PLS) were applied to data obtained by differential pulse voltammetry for the determination of vitamins in synthetic and pharmaceutical samples. For calibration purposes, both synthetic and commercial samples were employed as standards. From the results it was possible to verify that ANN is the best method for modeling the data due to the fact that interactions among electro-active components result in non-linear response on the glassy carbon electrode. The results achieved for the determination of vitamins in pharmaceutical samples using ANN method provided a maximum value for relative error of 0.40% for VC, 8.3% for VPP and 9.1% for VB6. The proposed methodology is simple, rapid and can be easily used to control quality laboratories as an alternative analysis method.

Water-Soluble Vitamins

Water-Soluble Vitamins

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 (α-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 α-tocopherol, γ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol were 0.73, 0.86, 1.0, 1.2, 1.7 and 1.3 ng, respectively.

Simultaneous Determination of Vitamin A and β-Carotene in Dietary Supplements by Liquid Chromatography

Several liquid chromatography (LC) methods for analysis of vitamin A in foods and feeds have been previously reported but only a few have been applied in non-food matrixes. A validated LC method is needed for determination of vitamin A and beta-carotene in the various matrixes presented by dietary supplements. The performance of a reversed-phase method with methanol-isopropanol gradient elution was evaluated with standard retinyl derivatives and beta-carotene. The reversed-phase method is capable of separating retinol from other derivatives such as retinyl acetate, retinyl palmitate, and beta-carotene. Two types of extraction were used to extract the analytes from the dietary supplements: a hexane-methylene chloride extraction for soft-gel capsules containing beta-carotene, and a direct solvent extraction for dietary supplements in tablet form. The direct solvent extraction consisted of treatment with ethanol and methylene chloride following addition of hot water (55 degrees C). Results with the reversed-phase method for vitamin A and beta-carotene in the products examined (n = 8) indicated excellent method performance. The main form of vitamin A or beta-carotene in dietary supplements was the all-trans isomer. The reversed-phase method avoids saponification and is rapid, accurate, precise, and suitable for simultaneous determination of retinyl derivatives and beta-carotene in dietary supplements.

Simultaneous determination of fat-soluble vitamins A, D and E and pro-vitamin D 2 in animal feeds by one-step extraction and high-performance liquid chromatography analysis

Traditionally, alkaline saponification for the extraction of vitamins and step-wise HPLC analyses have been widely used for analysis of fat-soluble vitamins in animal feeds. The objective of present study was to develop an analytical method using one-step extraction and simultaneous determination of the vitamins A, D and E and pro-vitamin D 2 in animal feeds by HPLC. Various analytical conditions were tested which included sample particle size, extraction solvents, the ratio of solvent to sample, extraction approach, extraction time, N 2 protection, prepurification with Sep-Pak cartridge and detection wavelength. The accuracy of the developed method has been proven by comparison with the AOAC method. The reproducibility of the developed method has been substantiated by repeated recovery experiments. The detection limit for the four vitamins was 10 ng/g of feed sample. One of the important properties of the present method is rapidity and ease of use.

Simultaneous determination of vitamin C and its carbamylated derivatives by high-performance liquid chromatography with post-column derivatization

A highly sensitive method for the simultaneous determination of ascorbate (AsA), dehydroascorbate (DHA), 2,3-diketogulonate (2,3-DKG), carbamyl ascorbate (CAA) and carbamylated dehydroascorbate derivative (CDA) was developed by high-performance liquid chromatography with post-column derivatization. The successful separation of these substances was achieved by an adsorption chromatography using poly(ethylene glycol) copolymer as a packing material in the separation column. For the detection, each substance was boiled with benzamidine in alkaline solution, producing fluorescence products. Both CAA and CDA were alkaline-labile, degrading to AsA and 2,3-DKG, so that these carbamylated derivatives could be detected in a similar manner as AsA and 2,3-DKG, respectively. The detection limits for quantitative determination of these substances were less than 0.5 μ M, and the coefficients of variation of the peak areas were in the range of 2.2 to 2.8%. The usefulness and practicability of the present method were verified by application to the determination of these substances in plant leaves soaked in 0.5 M sodium cyanate solution.

Inhibition of NF-kappa B transcriptional activity by alpha-tocopheryl succinate.

The role of vitamin E in cell regulation in addition to its function as an antioxidant has attracted attention. The effects of alpha-tocopherol (T) and alpha-tocopheryl succinate (TS) on transcriptional activation of the tumor necrosis factor alpha (TNF-alpha) gene and nuclear factor kappa B (NF-kappa B) activation were examined. Two stable transformants were used: TR-1 cells derived from THP-1 cells transfected with a vector contains the human TNF-alpha promoter (1.4-kb) joined to the human placental alkaline phosphatase (PLAP) coding sequence, and B164 cells derived from the same cell line but carrying the vector containing the human beta-actin promoter (4.3-kb) as a control. The transfectants were cultured in the presence of TS, followed by stimulation with lipopolysaccharide (LPS). After stimulation, PLAP activity secreted into the culture medium was measured. TS reduced TNF-alpha transcriptional activity in a concentration-dependent manner, while no effect was observed on that of the beta-actin promoter. Gel shift assay revealed that THP-1 cells pretreated with TS and then with LPS showed inhibition of NF-kappa B activity by 43% at 50 microM versus the TS-untreated group. Since TS did not affect activator protein-1 (AP-1) activity under the same conditions, the inhibitory effect of TS on NF-kappa B activation might be specific. However, T had no effect on the results of the gel shift assay. Vitamin E transportation was analyzed by simultaneous determination of vitamin E and its derivatives using HPLC. The vitamin E recovered from culture pellets showed almost the same amounts of T and TS transferred and was recovered in unchanged form. These observations indicated that TS inhibited NF-kappa B activation and/or translocation to the nuclei in its unchanged form under the culture conditions used here. These results suggested that vitamin E is involved in signal transduction via an effect distinct from its antioxidant function. To explain the lack of activity with T, it remains to be clarified whether physiological incorporation of T occurred.