Modern Chromatographic Analysis of Vitamins, Revised and Expanded

Modern Chromatographic Analysis Of Vitamins: Revised And Expanded
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Rockwood, William L. Roberts, Sonia L. Whittington, A.

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Among the advantages of this method are its high sensitivity and specificity. Vitamin D is a fat-soluble vitamin that is involved in calcium and phosphorus homeostasis. Vitamin D deficiency is associated with osteoporosis and osteomalacia in adults and rickets in children. Measurements of 25OH-vitD are commonly performed in patients with osteoporosis, osteomalacia, and abnormal concentrations of calcium and phosphorus, and elevated concentrations of parathyroid hormone PTH.

Vitamin D is also measured in patients with malabsorption of vitamin D, celiac disease, cystic fibrosis, and Crohn disease. The objectives of this study were to develop a highly sensitive and specific method for 25OH-vitD 2 and 25OH-vitD 3 , to determine concentrations of 25OH-vitD in the serum of healthy adults in samples collected during winter and summer and to assess the relationship between the concentrations of 25OH-vitD and PTH in healthy subjects.

All standards were prepared in methanol.

Modern Chromatographic Analysis Of Vitamins: Revised And Expanded - CRC Press Book

All other reagents were purchased from Sigma, St Louis, MO, and were of the highest purity commercially available. The plate was vortex-mixed, and the samples were analyzed. The mobile phase for the first-dimension separation was delivered at a flow rate of 0. Mobile phase for the analytic separation was delivered at a flow rate of 0.

A switching valve was installed between the columns; effluent from the CN column was directed to the analytic column from 2. The quadrupoles Q1 and Q3 were tuned to unit resolution, and the mass spectrometer conditions were optimized for maximum signal intensity of 25OH-vitD 2 25OH-vitD 3.

Two mass transitions were monitored for each analyte and the internal standard. All data were acquired and processed with Analyst 1.

Calibration was performed with every batch of samples. Evaluation of method performance included imprecision, limit of detection LOD , limit of quantitation LOQ , upper limit of linearity, method comparison, recovery, carryover, and ion suppression.

Three Gurus of Supercritical Fluid Chromatography

Samples for evaluation of linearity were prepared in 0. Samples for evaluation of the linearity and sensitivity were analyzed in duplicate over 3 days. To evaluate the robustness of the method, we analyzed more than 3, patient samples. A decrease in the intensity of the baseline in the mass transitions of 25OH-vitD indicated ion suppression. Samples used for the method comparison were residual serum and plasma samples submitted for routine testing of 25OH-vitD.

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The results were evaluated by Deming regression. Assessment of specimen suitability was performed by analyzing blood from 16 people collected in lithium heparin, sodium heparin, sodium citrate, potassium EDTA, serum, plasma separator, and serum separator tubes. A set of samples was collected in winter February; 72 samples from women and 58 from men , and another set of samples was collected in summer July—August; 77 samples from women and 53 from men. The median age of men was 31 years range, 19—59 years , and the median age of women was 30 years range, 19—65 years.

All studies with samples from human subjects were approved by the University of Utah Institutional Review Board Salt Lake City ; informed consent was obtained from all participants. Within-run, between-run, and total imprecision data are shown Table 1. Assessment of the specificity for 25OH-vitD 2 and 25OH-vitD 3 in patient samples was performed through evaluation of the ratios of concentrations determined from the 2 sets of mass transitions of the analytes and the internal standards.

Evaluation of the stability of 25OH-vitD 2 and 25OH-vitD 3 in plasma samples indicated good stability under the conditions evaluated. In samples prepared in methanol, we observed partial degradation of 25OH-vitD 2 and 25OH-vitD 3 after storage at room and refrigerated temperatures. Degradation products were partially resolved from the peaks of 25OH-vitD 2 and 25OH-vitD 3 , and their intensities increased after extended storage at room and refrigerated temperatures and exposure to light. To determine the adequacy of vitamin D in a local population and to evaluate the association between concentrations of 25OH-vitD and PTH, we analyzed samples from apparently healthy adult volunteers.

An association between the concentrations of 25OH-vitD and PTH Figure 4 was evaluated for the entire data set and for the median values of the data grouped by the ranges of the 25OH-vitD concentrations Figure 4.

Modern Chromatographic Analysis Of Vitamins: Revised And Expanded (Chromatographic Science Series)

The majority of published methods for 25OH-vitD use dehydrated parent ions or loss of water as product ions. Association between concentrations of hydroxyvitamin D 25OH-vitD and parathyroid hormone PTH in the serum of healthy adults A and median concentrations and central 90th percent of the distribution of PTH concentrations depending on the concentration of 25OH-vitD B. Ionization of 25OH-vitD using atmospheric pressure chemical ionization was considerably more efficient than electrospray ionization.

The commonly used single water loss product ions were not used in the method because they produced high background noise with consequent lower sensitivity.

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October Retrieved 25 October Micronutrient Information Center. Retrieved 7 November Journal of Biological Chemistry. MedlinePlus, U. Journal of Nutritional Science and Vitaminology. Fundamentals of Biochemistry: Life at the Molecular Level 2nd ed.

Advanced nutrition and human metabolism. Encyclopedia of Dietary Supplements. Retrieved 29 August Boston: Elsevier. European Food Safety Authority. Modern Nutrition in Health and Disease 10th ed. Dietary reference intakes: The essential guide to nutrient requirements. Journal of Nutritional Biochemistry.

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Current state of bioanalytical chromatography in clinical analysis

Fragment ions were simi- lar to those observed by van Breemen et al. Poor et al. Natural product Schmitz et al. Yuan Zhang.

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