Analysis of retinol, α -tocopherol, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC-MS/MS method (original) (raw)
Related papers
Journal of Chromatography B, 2020
Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC-MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid-liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triplequadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (< 20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status. renin-angiotensin-aldosterone system, and vascular walls [6]. Concentrations of 25-hydroxyvitamin-D2 (25D2), and 25-hydroxyvitamin-D3 (25D3) are used for evaluating vitamin D status in the
Journal of separation science, 2014
Vitamins A and E are fat-soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high-performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α-tocopherol and γ-tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed-phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03-5.14, 0.32-36.02, and 0.10-9.99 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. Intr-aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Techn...
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...
Modern analytical methodologies in fat and water soluble vitamins
John Wiley and Sons eBooks, 2000
Michael F. Holick 2.1. Origins of Vitamin D 2 and Vitamin D 3 2.2. Metabolism and Biologic Functions of Vitamin D 2.3. Measurement of Vitamin D Bioactivity 2.4. Modern Methods for Determining the Vitamin D Content in Blood 2.5. Strategies for Determining Circulating Concentrations of 25-Hydroxyvitamin D 2.6. Strategies for Determining Circulating Concentrations of 1,25-Dihydroxyvitamin D 2.7. Measurement of 24,25-Dihydroxyvitamin D and 25,26-Dihydroxyvitamin D 2.8. Clinical Utility for Assays for Vitamin D and Its Metabolites 2.8.1. Vitamin D 2.8.2. 25-Hydroxyvitamin D 2.8.3. 1,25-Dihydroxyvitamin D 2.8.4. Other Metabolites 2.9. Conclusion References
Journal of Chromatography B-analytical Technologies in The Biomedical and Life Sciences, 2004
In the present study, a simple and rapid reversed-phase HPLC procedure has been developed for the simultaneous determination of eight fat-soluble vitamins (retinol, menadione, menaquinone, δ-tocopherol, cholecalciferol, α-tocopherol, α-tocopherol acetate and phylloquinone) in biological fluids: blood serum and urine. The analytical column, Phenomenex Luna C18 (150mm×4.6mm) 3μm, was operating at ambient temperature. Mobile phase consisted of a mixture of CH3OH–CH3CN
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.