Publication Date

2011

Document Type

Thesis

Committee Members

David Cool (Advisor), Khalid Elased (Committee Member), Sharath Krishna (Committee Member), Marvin Miller (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Vitamin D is an essential micronutrient required for maintenance of the skeletal system and its deficiency leads to diminished availability of calcium for maintaining bone mineralization. Vitamin D deficiency (VDD) during pregnancy and/or in early infancy can lead to rickets and a fragile bone state resulting in fractures with minimal forces. Such multiple unexplained fractures (MUF) present in infants at less than 6 months of age are often misdiagnosed as cases of child abuse. Therefore, it is important to evaluate the status of vitamin D in early infancy. The analysis of dried blood spots (DBS) provides a unique screening method for early and late identification of diseases.

The objective of this study was to develop a mass spectrometry (MS) method to analyze vitamin D levels in newborn DBS. We used a methanol-hexane solvent system for the extraction of vitamin D analytes from filter paper spots. The use of methanol-hexane solvent system was evaluated using desipramine HCl as a positive control. We were able to extract desipramine HCl from standard spots spiked with concentrations 8 -5000 ng/ml. We applied this extraction method to control and test DBS with slight modification involving preincubation of DBS in methanol and sodium hydroxide. Our MS results have shown qualitative separation of 25 (OH) D2 (m/z - 413.2) and 25 (OH) D3 (m/z - 401.2) with SNR > 3. The identity of vitamin D analytes was confirmed using MS/MS analysis showing the presence of respective fragment peaks (m/z - 336.2 and 365.2). For quantitative estimation i.e. SNR > 10, we derivatized vitamin D analytes in DBS using 4-Phenyl-1,2,4- triazoline-3,5-dione (PTAD) in a Diels-Alder conjugation reaction. However, both 25 (OH) D2 and 25 (OH) D3 show the formation of identical product ion (m/z- 298.0) making it difficult to quantitate them separately.

In addition to MS method development, we also tried to evaluate genetic mutations in vitamin D metabolizing enzymes and vitamin D receptor as a possible cause of VDD in these infants. We extracted genomic DNA and RNA from DBS for mutation scanning using melt curve analysis However, the outcome of such analysis was not successful owing to lack of integrity in genetic material extracted from DBS. In summary we developed a MS method for qualitative detection of vitamin D and also attempted to test genetic mutations as a cause of VDD in infants. Further experiments are necessary to evaluate the use of alternative options of liquid chromatography/gas chromatography (LC/GC) coupled with MS and/or use of atmospheric pressure chemical ionization (APCI) as source of ionization for quantitative assessment of vitamin D.

Page Count

109

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2011

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