Determination of serum retinol by reversed-phase high-performance liquid chromatography (original) (raw)
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An isocratic liquid chromatographic method has been developed for rapid measurements of retinol, retinal, all-trans-retinoic acid and 13-cis-retinoic acid. Using 85% methanol and 15% 0.01 M sodium acetate buffer, pH 5.2, as the mobile phase and at a flow rate of 1.5 mL/min, retinol, retinal, all-trans-retinoic acid and 13-cis-retinoic acid were eluted from a 5 µm C18 reversed-phase column (4.6 mm I.D., 15 cm) in less than 14 min. Retinyl acetate, when desired, was eluted in 25.5 min under the above conditions. Each compound was detected at the most sensitive wavelength by coupling fluorescence and UV detection.
Rapid and Simple Measurement of Retinol in Human Dried Whole
We describe an improved method for the measurement of retinol in dried blood spots (DBS) on filter paper. Retinol in human DBS on filter paper was analyzed by normal phase HPLC after a simple extraction method. Retinol associated with its binding protein was eluted from the paper into aqueous solution facilitated by ultrasonic agitation. Retinol associated with retinol binding protein was denatured with acetonitrile, and then retinol was isolated in a single hexane extract and analyzed directly by HPLC. When analyzing DBS, the individual plasma volume of the spots was calculated by measuring the sodium content or by weighing the blood spots. The described method yielded low intra-and interassay variability (<6%), with sufficient sensitivity (detection limit, 0.1 mol/L) and good recovery (97% spike). Compared with matching plasma samples, DBS retinol consistently decreased 18 -23% during the 1st wk of storage. After 1 wk, retinol remained stable in the blood spots at 23°C for >3 mo. In conclusion, the analysis of retinol in DBS by HPLC is comparable to retinol analysis in serum. The variability of the method was reduced by using sodium concentration to estimate sample volume. Collection of DBS for retinol analysis is appropriate under field conditions, where it is difficult to centrifuge or freeze blood samples. J. Nutr. 132: 318-321, 2002.
When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68 mL min −1 and an column temperature of 37.10 • C. Detection was performed at 350 nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109 ng mL −1 of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.
Journal of Pharmaceutical Sciences, 1982
LIA rapid, specific, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) assay for the quantitative determination of all-trans-retinoic acid (I) or 13-cis-retinoic acid (11) in rat serum without extraction or lyophilization is described. Chromatographic separation from retinol, serum components, and retinol acetate standard was achieved on octadecylsilane-coated particles with acetonitrile-1% ammonium acetate as the eluent. Serum samples (100 pl) containing as little as 10 ng of retinoid were analyzed. Serum level profiles of rats dosed with the retinoids demonstrated the utility of the assay and indicated elimination half-lives of 0.58 and 0.92 hr for I and 11, respectively. Keyphrasea 0 High-performance liquid chromatography-assay for 13-cis-retinoic acid and all-trans-retinoic acid 0 13-cis-Retinoic acid-high-performance liquid chromatographic analysis 0 All-transretinoic acid-high-performance liquid chromatographic analysis
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.
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.
Vitamin A is very important for visual apparatus, for correct proliferation of epidermic cells, reproductive and immune cells. All natural sources of vitamin A in the diet are represented by provitamine A carotenoids. Vegetables and fruits provide the main sources of this compound. The knowledges about vitamin A are very important for understanding the relation between serum retinol and the content of vitamin A from the source of food and are also significant for preventing the toxic phenomena caused by overdose. Determination of vitamin A in substances, pharmaceutical forms, biological materials, has been performed by the HPLC method in the normal an in the reversed phase system, with ultraviolet (UV) detection. In this article we analysed the possible correlation between serum retinol values and the dynamic evolution of biochemical parameters (ALAT, ASAT, triglycerides, cholesterol, total bilirubin, indirect bilirubin, direct bilirubin, GGT, alkaline phosphatase and total protein)...
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.
Rapid and simple measurement of retinol in human dried whole blood spots
The Journal of nutrition, 2002
We describe an improved method for the measurement of retinol in dried blood spots (DBS) on filter paper. Retinol in human DBS on filter paper was analyzed by normal phase HPLC after a simple extraction method. Retinol associated with its binding protein was eluted from the paper into aqueous solution facilitated by ultrasonic agitation. Retinol associated with retinol binding protein was denatured with acetonitrile, and then retinol was isolated in a single hexane extract and analyzed directly by HPLC. When analyzing DBS, the individual plasma volume of the spots was calculated by measuring the sodium content or by weighing the blood spots. The described method yielded low intra- and interassay variability (<6%), with sufficient sensitivity (detection limit, 0.1 micromol/L) and good recovery (97% spike). Compared with matching plasma samples, DBS retinol consistently decreased 18-23% during the 1st wk of storage. After 1 wk, retinol remained stable in the blood spots at 23 degre...