Rapid determination of lipophilic vitamins in human serum by ultra‐high performance liquid chromatography using a fluorinated column and high‐throughput miniaturized liquid–liquid extraction

A high‐throughput miniaturized liquid–liquid extraction procedure followed by a simple ultra‐high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core–shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λ_ex = 325, 295 nm and λ_em = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 μM, α‐tocopherol 0.38 μM, β‐tocopherol 0.18 μM, γ‐tocopherol 0.14 μM, and δ‐tocopherol 0.01 μM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.     

Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

Fat‐soluble vitamins play a pivotal role in the progression of atherosclerosis and the development of cardiovascular disease. Therefore, plasma monitoring of their concentrations may be useful in the diagnosis of these disorders as well as in the process of treatment. The study aimed to develop and validate an HPLC–MS/MS method for determination of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease. The analytes were separated on an HPLC Kinetex F5 column via gradient elution with water and methanol, both containing 0.1% (v/v) formic acid. Detection of the analytes was performed on a triple‐quadrupole MS with multiple reaction monitoring via electrospray ionization. The analytes were isolated from plasma samples with liquid–liquid extraction using hexane. Linearity of the analyte calibration curves was confirmed in the ranges 0.02–2 μg/mL for retinol, 0.5–20 μg/mL for α‐tocopherol, 5–100 ng/mL for 25‐hydroxyvitamin D2 and 2–100 ng/mL for 25‐hydroxyvitamin D3. Intra‐ and inter‐assay precision and accuracy of the method were satisfactory. Short‐ and long‐term stabilities of the analytes were determined. The HPLC‐MS/MS method was applied for the determination of the above fat‐soluble vitamin concentrations in patient plasma as potential markers of the cardiovascular disease progression.     

Determination of target fat‐soluble micronutrients in rainbow trout's muscle and liver tissues by liquid chromatography with diode array‐tandem mass spectrometry detection

This paper describes an analytical approach, based on LC‐diode array detector‐MS/MS (LC‐DAD‐MS/MS), for characterizing the fat‐soluble micronutrient fraction in rainbow trout (Oncorhynchus mykiss ). Two different procedures were applied to isolate the analytes from liver and muscle tissue: overnight cold saponification to hydrolyze bound forms and to simplify the analysis; matrix solid‐phase dispersion to avoid artifacts and to maintain unaltered the naturally occurring forms. Analytes were separated on a C₃₀ analytical column by using a nonaqueous reversed mobile phase compatible with the atmospheric pressure chemical ionization. Compared to other works, the most relevant advantage of the here illustrated method is the large amount of information obtained with few analytical steps: nine fat‐soluble vitamins (3,4‐dehydroretinol, retinol, cholecalciferol, ergocalciferol, α‐tocopherol, γ‐tocopherol, δ‐tocopherol, phylloquinone, and menaquinone‐4) and eight carotenoids (all‐trans ‐lutein, all‐trans ‐astaxanthin, all‐trans ‐zeaxanthin, all‐trans ‐β‐cryptoxanthin, all‐trans ‐canthaxanthin, all‐trans ‐ζ‐carotene, all‐trans ‐β‐carotene, and all‐trans ‐γ‐carotene) were quantified after the method validation, while other untargeted carotenoids were tentatively identified by exploiting the identification power of the LC‐DAD‐MS/MS hyphenation.     

Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance liquid chromatography

A method of sample clarification and high-performance liquid chromatography specifically developed to permit precise and rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in serum and plasma is reported. Serum proteins were denatured by the addition of acetonitrile containing alpha-tocopherol acetate, the internal standard; the vitamins were subsequently extracted into an organic matrix consisting of ethyl acetate-butanol (1:1); no solvent evaporation step was required. The three vitamins of interest were eluted from a reversed-phase C18 column with an isocratic mobile phase methanol-water (95:5); detection was accomplished by measuring ultraviolet absorption at 280 nm. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from spiked aqueous samples averaged 100.0, 100.0 and 98.8%, respectively. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from plasma and serum relative to water were 102.6, 96.9 and 96.5%, respectively. Retinol and alpha-tocopherol were stable in the extraction matrix for up to 3.5 h, and were stable in heparinized plasma stored at room temperature for two days. Oxalate, citrate and EDTA caused significant losses of retinol and alpha-tocopherol, while vitamin levels in serum and heparinized plasma were similar. Limits of detection for retinol and alpha-tocopherol were 60 ng/ml and 0.9 micrograms/ml, respectively. Each run required 12 min. Same-day coefficients of variation were 3.5 and 3.6% for retinol and alpha-tocopherol, respectively (n = 11). Between-day coefficients of variation for retinol and alpha-tocopherol were 4.8 and 5.5%, respectively (n = 5). This method permits simple, rapid, sensitive, selective and precise determination of retinol and alpha-tocopherol using 0.5 ml of serum or heparinized plasma.     

Development of coupled ultrasound‐assisted and reversed‐phase dispersive liquid–liquid microextraction before high‐performance liquid chromatography for the sensitive determination of vitamin A and vitamin E in oil samples

A new approach based on the ultrasound‐assisted reversed‐phase dispersive liquid–liquid microextraction technique is developed for the extraction and determination of vitamin A and vitamin E from oil matrices before high‐performance liquid chromatography analysis. A methodology based on the full factorial design is carried out to choose the significant parameters. Then the significant factors affecting the extraction efficiency including pH, volume of extraction solvent, and volume of disperser solvent are optimized using a Box–Behnken design. After analyzing the results obtained, the optimum conditions were: pH 4.5, 80–20 μL of the ethanol/water solvent mixture as extraction solvent, 110 μL of 1,4‐dioxane as the disperser solvent, and a sonication time of 10 min. For validation of the developed method, the linear dynamic range, repeatability, limit of detection, and recoveries were obtained under the optimum conditions. The detection limits of the method were 1.6 and 2.3 ng/mL for vitamin A and vitamin E, respectively. The extraction recovery percentages for the studied drugs were above 91%, with acceptable relative standard deviation. The proposed methodology was successfully applied for the determination of the vitamins in different oil samples.     

Combination of ultracentrifugation and solid‐phase extraction with subsequent chromatographic analysis of α‐tocopherol in erythrocyte membranes

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid‐phase extraction for the determination of α‐tocopherol in human erythrocyte membranes by high‐performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d ‐mannitol, 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid and calcium chloride. The α‐tocopherol was then extracted from the erythrocyte membranes by solid‐phase extraction with n‐hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP‐18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α‐tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α‐tocopherol in real samples from patients. The combined ultracentrifugation and solid‐phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid–liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.     

Improvement of α‐tocopherols long‐term efficiency by modeling its heterogeneous natural environment in polyethylene

The natural antioxidant vitamin E (α‐tocopherol) is of interest to use in packaging applications to decrease the amount of toxic products migrating into food and drugs. We have earlier shown that the long‐term efficiency of α‐tocopherol in polyethylene (PE) films is poor. α‐Tocopherol is located in the lipid phase of the cell in vivo and it has been revealed that it is more efficient in a polar substrate. PE is more hydrophobic and homogenous than the heterogeneous and hydrophilic lipid phase. Three different additive systems were investigated to model α‐tocopherols heterogeneous natural environment in PE. Two of these had carboxylic acid groups, EAA and polyTRIM/PAA core‐shell particles (Core), and the third, oat starch, had no carboxylic acid groups. The materials were thermally aged and characterized by chemiluminescence (CL), FTIR, chromatography, and thermal analysis. The EAA system as well as the Core system improved the antioxidant properties of α‐tocopherol in PE, and the Core system had the best performance. We know that starch has stabilizing properties in PE, but it had no effect on the efficiency of α‐tocopherol. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 1660–1666, 2006     

Quantitative profiling of biomarkers related to B‐vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

Vitamins B₂ and B₆ serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large‐scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B₆ forms (pyridoxal 5′‐phosphate, pyridoxal, 4‐pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3‐hydroxykynurenine, xanthurenic acid and 3‐hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope‐labelled internal standards was added to 60 µL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable‐bond C8 column by a gradient‐type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75–123% and within‐day and between‐day coefficients of variance (CVs) were 2.5–9.5% and 5.4–16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B‐vitamin status and inflammation, and may prove useful in large‐scale epidemiological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of vitamins A (retinol) and E (alpha-tocopherol) in foods by liquid chromatography: collaborative study

A collaborative study was conducted for the determination of vitamins A and E. Existing AOAC liquid chromatographic (LC) methods are suited for specific vitamins A and E analytical applications. This method differs from existing methods in that it can be used to assay samples in all 9 sectors of the food matrix. Standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters and tocopherol esters to retinol and tocopherol, respectively. Retinol and alpha-tocopherol are quantitated on separate LC systems, using UV detection at 313 or 328 nm for retinol, and fluorescence detection (excitation 290 nm, emission 330 nm) for alpha-tocopherol. Vitamin concentrations are calculated by comparison of the peak heights or peak areas of vitamins in test samples with those of standards.     

Simultaneous assay of serum vitamin A and vitamin E by high performance liquid chromatography using time-switched UV and fluorimetric detectors

An HPLC method utilizing a UV and a fluorimetric detector linked in series is described. By use of a simple integrator-controlled time-switched relay, analysis of serum vitamin A and E is accomplished on the same chromatogram and at optimum sensitivity for each detector. A single internal standard (retinyl acetate) monitored only by the UV detector permits measurement of both vitamins over a wide linear range. Precision of the assays is satisfactory, both on a within-day and on a day-to-day basis. Recoveries of both vitamins are virtually 100% whilst sensitivity is 2 μg/L (retinol) and 0.05 mg/L (α-tocopherol).     

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.     

Three‐phase hollow‐fiber microextraction combined with ion‐pair high‐performance liquid chromatography for the simultaneous determination of five components of compound α‐ketoacid tablets in human urine

The determination of α‐ketoacid concentration is demanded to evaluate the absorption and metabolic behavior of compound α‐ketoacid tablets taken by chronic kidney disease patients. To eliminate the interference of endogenous substance of urine and enrich the analytes, a three‐phase hollow‐fiber liquid‐phase microextraction combined with ion‐pair high‐performance liquid chromatography method was established for the determination of d ,l ‐α‐hydroxymethionine calcium, d ,l ‐α‐ketoisoleucine calcium, α‐ketovaline calcium, α‐ketoleucine calcium, and α‐ketophenylalanine calcium of compound α‐ketoacid tablets in human urine samples. The extraction parameters, such as organic solvent, pH of donor phase and acceptor phase, stirring rate, and extraction time were optimized. Under the optimal conditions, the obtained enrichment factors were up to 11‐, 110‐, 198‐, 202‐, and 50‐fold, respectively. The calibration curves for these analytes were linear over the range of 0.1–10 mg/L for α‐ketovaline calcium, d ,l ‐α‐ketoisoleucine calcium, and α‐ketoleucine calcium, 0.5–10 mg/L for d ,l ‐α‐hydroxymethionine calcium, and α‐ketophenylalanine calcium with r > 0.99. The relative standard deviations (n = 5) were less than 6.27% and the LODs were 100.7, 10.0, 5.8, 7.8, and 8.6 μg/L (based on S/N = 3), respectively. Good recoveries from spiked urine samples (92–118%) were obtained. The proposed method demonstrated excellent sample clean‐up and analytes enrichment to determine the five components in human urine.     

Simultaneous quantification of vitamins A, D3 and E in fortified infant formulae by liquid chromatography-mass spectrometry

A novel method for the simultaneous quantification of Vitamins A, D3 and E in fortified infant formulae has been developed using isocratic normal-phase liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Food products were saponified and the vitamins were extracted by solid-phase extraction (SPE) on a Chromabond XTR cartridge. Quantification of Vitamins D3 and E were performed with Vitamin D2 and 5,7-dimethyltocol (DMT) as internal standards (IS), respectively while no IS was used for Vitamin A. Detection of the vitamins was made in the selected ion monitoring (SIM) mode. MS calibration curves were linear between 0.15 and 12 mg/l for Vitamin A, 5-400 microg/l for Vitamin D3 and 0.25-20 mg/l for Vitamin E with regression coefficient r2 > 0.996 and the limits of detection were below 1.4 ng. The repeatability (CV) obtained on a reference dietetic infant formula was 2.3% for Vitamin A, 2.6% for Vitamin E and 5.9% for Vitamin D3. The between-day variations (CV) over 6 days were in the ranges of 2.4-6.9% for the three vitamins. The mean recoveries from a reference infant formula spiked with all three vitamins ranged from 96 to 105% with a relative standard error less than 9%. The applicability of the method was demonstrated by analyzing a set of infant formula and infant cereals; similar results were obtained with the LC-MS method and reference HPLC methods.     

Determination of vitamins A, D and E in a small volume of human plasma by a high-throughput method based on liquid chromatography/tandem mass spectrometry

We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies.     

Determination of B-group vitamins in Turkish honey using ultra-performance liquid chromatography with electrospray ionization coupled to tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (UPLC–ESI–MS/MS) for analysis of B-group vitamins in honey has been presented. Aim of this study is the characterization of different types of Turkish honeys according to B-group vitamins. Vitamins were determined in 17 different types of Turkish honey samples by UPLC–ESI–MS/MS. Heather honey samples were distinguished among the studied honeys with the richest vitamin content with 286.10?mg/kg, and it is followed by sunflower honey and thyme honey with the total vitamin contents of 206.01 and 163.27?mg/kg, respectively. The presence of vitamin B₁ (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (nicotinamide, B₃N and nicotinic acid, B₃H), vitamin B₆ (pyridoxine), and vitamin B₉ (folic acid) was detected in all the honey samples analyzed. Moreover, vitamin B₅ (pantothenic acid) was observed in most of them. Vitamin B₁₂ (cyanocobalamin) and vitamin B₇ (biotine) were not detected in the studied honey samples. Turkish honey samples showed efficacious vitamin content for the consumers.     

Direct determination of tocopherols in paprika and paprika oleoresin by liquid chromatography

A method for the determination of tocopherols and tocopherol esters, quantified as tocopherol acetate, in paprika and paprika oleoresin is reported. Paprika samples were extracted with ethyl acetate and aliquots of the extracts were directly injected into a liquid chromatograph. Reverse phase high-performance liquid chromatography with spectrophotometric detection at 280 nm was used. Gradient elution was applied, allowing the determination of tocopherols and tocopherol esters in the presence of carotenoids. The method does not need previous separation steps, so is useful for the routine determination of vitamin E in commercial samples.     

Convenient Preparation of 2,7,8‐Trimethyl‐6‐hydroxychroman‐2‐carboxylic Acid (γ‐Trolox)

The title chroman is useful in synthesis and as a water‐soluble analog of γ‐tocopherol, a member of the vitamin E family. This new synthesis of γ‐trolox proceeds via selective aromatic demethylation of Trolox, the more easily available 2,5,7,8‐tetramethyl homolog compound. This route is shorter than the previous synthesis, avoids the use of cyanide and methoxybutadiene, and requires no chromatography.     

Small‐Molecule Modulation of Soft‐Matter Frank–Kasper Phases: A Method for Adding Function to Form

Incorporation of small amounts of α‐tocopherol (vitamin E) in blends with the cellobiose–triazole‐linked atactic poly(4‐methyl‐1‐pentene) (CB‐aPMP) sugar–polyolefin conjugate can be used to exert external control over thermotropic phase behavior and provide access to non‐canonical soft matter Frank–Kasper A15 and σ phases. These results establish a paradigm that can be used for the further design and development of scalable quantities of soft matter FK phases of increased structural complexity and functional capability.     

Solid-phase extraction of vitamins A and E from animal feeds: A substitute for liquid--liquid extraction

A substitution was developed for the laborious liquid-liquid extraction method according to European Economic Community legislation for isolation of Vitamins A and E from feeds. A rapid method for the isolation of the 2 vitamins by means of solid-phase extraction on OASIS columns gave reproducible results comparable to those of the European Union-recommended method. The vitamins in the feed were extracted with a minor modification of the official method (KOH in ethanol). After filtration, a portion was applied onto the OASIS column and then washed. The vitamins were eluted in ethyl acetate. The eluate evaporated under nitrogen, and the precipitate was dissolved in methanol before liquid chromatography analysis. Recovery for this method was 80% for Vitamin A and 110% for Vitamin E, as calculated as the result for National Institute of Standards and Technology (NIST) Infant Formula. Day-to-day reproducibility for the method was 21 and 11% of the average results for Vitamins A and E, respectively.     

Functionalized nanoparticles based solid‐phase membrane micro‐tip extraction and high‐performance liquid chromatography analyses of vitamin B complex in human plasma

Iron nanoparticles were prepared by a green method following functionalization using 1‐butyl‐3‐methylimidazolium bromide. 1‐Butyl‐3‐methylimidazole iron nanoparticles were characterized using FTIR spectroscopy, energy dispersive X‐ray fluorescence, X‐ray diffraction, scanning electron microscopy and transmission electron microscopy. The nanoparticles were used in solid‐phase membrane micro‐tip extraction to separate vitamin B complex from plasma before high‐performance liquid chromatography. The optimum conditions obtained were sorbent (15 mg), agitation time (30 min), pH (9.0), desorbing solvent [water (5 mL) + methanol (5 mL) + sodium hydroxide (0.1 N) + acetic acid (d = 1.05 kg/L, pH 5.5), desorbing volume (10 mL) and desorption time (30 min). The percentage recoveries of all the eight vitamin B complex were from 60 to 83%. A high‐performance liquid chromatography method was developed using a PhE column (250 × 4.6 mm, 5.0 μm) and water/acetonitrile (95:5, v/v; pH 4.0 with 0.1% formic acid) mobile phase. The flow rate was 1.0 mL/min with detection at 270 and 210 nm. The values of the capacity, separation and resolution factor were 0.57–39.47, 1.12–6.00 and 1.84–26.26, respectively. The developed sample preparation and chromatographic methods were fast, selective, inexpensive, economic and reproducible. The developed method can be applied for analyzing these drugs in biological and environmental matrices.