Fast, simple, and sensitive high-performance liquid chromatography method for measuring vitamins A and E in human blood plasma (original) (raw)
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Journal of Pharmaceutical and Biomedical Analysis, 2007
A fast (15 min) and simple HPLC method for determination of all-trans-retinol and α-, γand δtocopherols in human plasma has been developed. The assay utilized 200 μl of plasma to which 20 μl of internal standard solution (retinol acetate) was added followed by 200 μl of water, 400 μl of ethanol and 800 μl of hexane. The hexane layer was collected, evaporated, the residue dissolved in 200 μl of methanol and analyzed on a Zorbax Eclipse XDB-C18 column using a step gradient with a polar organic mobile phase composed of acetonitrile and methanol and variable wavelength fluorescence detection. The quantification limits for all-trans-retinol and γ-tocopherol were 20 ng/ ml and for αand δ-tocopherols 500 ng/ml and 10 ng/ml, respectively. The procedure was validated and applied to the analysis of plasma samples from the Baltimore Longitudinal Study of Aging.
Chromatographia, 2010
A versatile isocratic reversed-phase liquid chromatographic/ultraviolet-visible detection method for simultaneous determination of all-trans-retinol and a-tocopherol in human serum was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a Kromasil 100 RP 18 (150 9 4.6 mm, 5 lm) analytical column protected by a Perkin Elmer RP 18 (30 9 4.6 mm, 10 lm) guard cartridge. The mobile phase, methanol-water (96:04 v/v) was pumped at a flow rate of 2.2 mL min -1 and the column eluents were monitored at the wavelength of 292 nm using retinyl acetate (1.0 lg mL -1 ) as the internal standard for both analytes. Sample preparation was based on protein precipitation and stabilization with 2,6-bis(1,1dimethylethyl)-4-methylphenol/ethanol and a two step extraction process using n-hexane followed by dichloromethane as extraction solvents. Sample size was kept 20 lL and separation of analytes was achieved in less than 7 min. The present method demonstrated acceptable values for specificity/selectivity, linearity within the expected concentration range, recovery, precision, sensitivity, stability of solutions, robustness, and system suitability specifications and tests. The method was used for monitoring all-trans-retinol and a-tocopherol concentrations in human serum samples and could also be applied to other sample matrices such as brain slices and cosmetic products if attention is paid to the extraction procedure.
Journal of Chemistry, 2013
A method is described here for the simultaneous determination of retinol,α-tocopherol, lycopene, andβ-carotene in human plasma. The effectiveness of various protein precipitants and extraction solvents was tested. After adequate sample preparation, the samples were injected directly into the HPLC system. The separation was realized on an analytical reversed-phase column with a UV-Vis detection. The analytical performance of this method was satisfactory. The intraassay and interassay coefficients of variation were below 10%. The recoveries were as follows: 97.0% (CV 2.4%) for retinol, 94.6% (CV 1.7%) forα-tocopherol, 91.9% (CV 3.6%) for lycopene, and 93.9% (CV 4.2%) forβ-carotene. The levels of selected fat-soluble vitamins in plasma of patients with cardiovascular disease were measured and discussed.
Rapid communications in mass spectrometry : RCM, 2011
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 b...
Direct liquid chromatography method for retinol, α- and γ-tocopherols in rat plasma
Journal of Chromatography B, 2004
An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50 l, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery ® C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.
Anal Bioanal Chem, 2007
Retinol and a-tocopherol are biologically active compounds often monitored in blood samples because of their evident importance in human metabolism. In this study a novel ultra-performance liquid chromatographic (UPLC) method used for determination of both vitamins in human serum has been compared with conventional HPLC with particulate and monolithic C 18 columns. In UPLC a sub-two-micron particle-hybrid C 18 stationary phase was used for separation, in contrast with a five-micron-particle packed column and a monolithic column with a highly porous structure. Methanol, at flow rates of 0.48, 1.5, and 2.5 mL min −1 , respectively, was used as mobile phase for isocratic elution of the compounds in the three methods. Detection was performed at 325 nm and 290 nm, the absorption maxima of retinol and a-tocopherol, respectively. Analysis time, sensitivity, mobile-phase consumption, validation data, and cost were critically compared for these different chromatographic systems. Although cost and mobile-phase consumption seem to make UPLC the method of choice, use of the monolithic column resulted in almost the same separation and performance with a slightly shorter analysis time. These methods are alter-natives and, in routine laboratory practice, more economical means of analysis of large numbers of biological samples than use of a traditional particulate column.
Analytical and Bioanalytical Chemistry, 2007
Retinol and a-tocopherol are biologically active compounds often monitored in blood samples because of their evident importance in human metabolism. In this study a novel ultra-performance liquid chromatographic (UPLC) method used for determination of both vitamins in human serum has been compared with conventional HPLC with particulate and monolithic C 18 columns. In UPLC a sub-two-micron particle-hybrid C 18 stationary phase was used for separation, in contrast with a five-micron-particle packed column and a monolithic column with a highly porous structure. Methanol, at flow rates of 0.48, 1.5, and 2.5 mL min −1 , respectively, was used as mobile phase for isocratic elution of the compounds in the three methods. Detection was performed at 325 nm and 290 nm, the absorption maxima of retinol and a-tocopherol, respectively. Analysis time, sensitivity, mobile-phase consumption, validation data, and cost were critically compared for these different chromatographic systems. Although cost and mobile-phase consumption seem to make UPLC the method of choice, use of the monolithic column resulted in almost the same separation and performance with a slightly shorter analysis time. These methods are alter-natives and, in routine laboratory practice, more economical means of analysis of large numbers of biological samples than use of a traditional particulate column.
Journal of Chromatography B, 2002
A sensitive HPLC assay for all-trans-retinol, a-tocopherol, and g-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v / v) without I.S. addition. After removal of the precipitated protein, 20 ml aliquots of the supernatant (equivalent to 6.7 ml of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C S3 ODS2 column with a methanol-water step gradient (97:3 to 18 100) at 1.0 ml / min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for a-tocopherol and 250 ng for gand d-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.
Fast determination of retinol and α-tocopherol in plasma by LC
Journal of Pharmaceutical and Biomedical Analysis, 2002
A fast, selective and economical method for the determination of retinol and a-tocopherol is presented. Both vitamins are separated by high-performance liquid chromatography (HPLC) in less than 4 min using an isocratic elution with methanol. The robustness of the method was checked in real samples, obtaining relative standard deviation lower than 3%. The described method was satisfactorily applied to serum samples proceeding of patients enrolled in a METHADONE maintenance treatment program. The retinol concentrations in the serum of these patients fell into the normal interval of concentrations; however, the serum a-tocopherol contents were higher than the normal values.
Journal of the Brazilian Chemical Society, 2012
Um método para quantificação simultânea de licopeno, β-caroteno, retinol e α-tocoferol por cromatografia líquida de alta eficiência (HPLC) com detecção no visível/fluorescente e eluição isocrática foi otimizado e validado. O método consiste de extração líquido-líquido rápida e simples e posterior quantificação do sobrenadante extraído por HPLC. Alíquotas de plasma foram estocadas a-20°C por três meses para estudo da estabilidade. Aplicação metodológica foi realizada em amostras fornecidas por pintores e indivíduos não expostos a tintas (n = 75). O ensaio foi linear para todas as vitaminas analisadas (r > 0,99). Precisões intradia e interdia apresentaram coeficiente de variação (CV) menor que 5%. Exatidões variaram de 0,29 a-5,80% e recuperações entre 92,73 e 101,97%. Amostras de plasma e sobrenadante extraído foram estáveis por até 60 dias a-20°C. Foi demonstrada uma diminuição significativa nas concentrações de licopeno, β-caroteno e retinol em indivíduos expostos quando comparados com os não-expostos (p < 0,05). O método é simples, reprodutível, preciso, exato e sensível, e pode ser utilizado na rotina de laboratórios clínicos. A method for the simultaneous quantification of lycopene, β-carotene, retinol and α-tocopherol by high-performance liquid chromatography (HPLC) with Vis/fluorescence detection with isocratic elution was optimized and validated. The method consists of a rapid and simple liquid-liquid extraction procedure and a posterior quantification of extracted supernatants by HPLC. Aliquots of plasma were stored at-20°C for three months for stability study. The methodology was applied to samples from painters and individuals not exposed to paints (n = 75). The assay was linear for all vitamins (r > 0.99). Intra-and inter-run precisions were obtained with coefficient of variation smaller than 5%. The accuracies ranged from 0.29 to-5.80% and recoveries between 92.73 and 101.97%. Plasma samples and extracted supernatants were stable for 60 days at-20°C. A significant decrease of lycopene, β-carotene and retinol concentrations in plasma from exposed individuals compared to non-exposed individuals (p < 0.05) was observed. The method is simple, reproducible, precise, accurate and sensitive, and can be routinely utilized in clinical laboratories.