SBIR-STTR Award

After almost a century of research there are still critical questions regarding the role of vitamin D in health and disease. Beyond the well established function of vitamin D in bone health, evidence is emerging that it may be a risk factor in chronic diseases ranging from autoimmune diseases and cancer to cardiovascular disease and type II diabetes. One complication in ongoing research is the lack of reliable measurement technology with sufficient selectivity and sensitivity to determine physiological levels of vitamin D and its metabolites in tissues, blood, and food. Commercially available kit assays provide high throughput analysis of 25(OH)D, but not of vitamin D, and inter-laboratory performance is poor. Additionally, these kits are unable to accurately and separately measure 25(OH)D3 and 25(OH)D2, as they are confounded by cross-reactivity with catabolic 24,25(OH)2D metabolites. On the other hand, HPLC coupled with mass spectrometry (LC-MS) offers both increased sensitivity and selectivity and minimizes interferences commonly seen from complex food matrices. Several LC-MS/MS methods have been developed for measuring vitamin D metabolites in plasma that reported Limits of Quantification (LOQ) ranging from 0.17 to 6.5 ng/mL. While promising, the premature fragmentation of vitamin D molecules in ion sources contributes to the higher variability of these methods and high LOQ. To overcome this problem, a specific vitamin D derivatization reaction based on Diels-Alder cycloaddition was developed. Using PTAD derivatization and methylamine, LOQs of 10 - 20 pg/mL have been accomplished for five vitamin D metabolites. Although a significant improvement, this method does not achieve the sensitivity of immuno- assays and does not allow for simultaneous analysis of multiple samples in a single chromatographic run. A solution to these problems that will be developed in this proposal is to increase both the selectivity and sensitivity with which the components of vitamin D are determined. This will be achieved with a new liquid chromatography-mass spectrometry approach in which the 9,10- secosteroid components of vitamin D are derivatized with a new reagent that both differentially codes components isotopically according to sample origin and greatly increases their ionization efficiency. Taken together this will enable multiple samples to be analyzed simultaneously at the level comparable to immuno-assays (1-5 pg/mL) while still allowing molecular discrimination between vitamin D2, vitamin D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3.

Public Health Relevance:
A new quantification strategy and reagents for the analysis of vitamin D and its different forms is being developed that will facilitate broader understanding of the various bio- regulatory functions unique to this hormone-like vitamin.

Thesaurus Terms:
(3 Beta,5z,7e)-9,10-Secocholesta-5,7,10(19)-Trien-3-Ol;9,10-Secocholesta-5,7,10(19)-Trien-3-Ol, (3beta,5z,7e)-;Acetonitriles;Address;Alder;Alder Plant;Alnus;Antibodies;Assay;Autoimmune Diseases;Bioassay;Biologic Assays;Biological Assay;Blood;Blood Plasma;Blood Serum;Body Tissues;Calciol;Cancers;Cardiovascular Diseases;Chemotherapy-Hormones/Steroids;Cholecalciferol;Chromatography, High Performance Liquid;Chromatography, High Pressure Liquid;Chromatography, High Speed Liquid;Chronic Disease;Chronic Illness;Code;Coding System;Cognitive Discrimination;Complex;Complication;Coupled;Cyanomethanes;D 2, Vitamin;Detection;Diabetes Mellitus, Adult-Onset;Diabetes Mellitus, Ketosis-Resistant;Diabetes Mellitus, Non-Insulin-Dependent;Diabetes Mellitus, Noninsulin Dependent;Diabetes Mellitus, Slow-Onset;Diabetes Mellitus, Stable;Diabetes Mellitus, Type 2;Diabetes Mellitus, Type Ii;Discrimination;Discrimination (Psychology);Disease;Disorder;Endocrine Gland Secretion;Ergocalciferol;Ethane Nitriles;Family;Flame Ionization;Food;Goals;Hplc;Health;High Pressure Liquid Chromatography;Hormones;Institutes;Lc/Ms;Laboratories;Mody;Malignant Neoplasms;Malignant Tumor;Mass Spectrum;Mass Spectrum Analysis;Maturity-Onset Diabetes Mellitus;Measurement;Measures;Methods;Methyl Cyanides;Methylamines;Molecular;Niddm;Non-Insulin Dependent Diabetes;Non-Insulin-Dependent Diabetes Mellitus;Performance;Phase;Photometry/Spectrum Analysis, Mass;Physiologic;Physiological;Plasma;Programs (Pt);Programs [publication Type];Proteins;Reaction;Reagent;Relative;Relative (Related Person);Reporting;Reproducibility;Research;Reticuloendothelial System, Blood;Reticuloendothelial System, Serum, Plasma;Risk Factors;Role;Running;Sampling;Scheme;Secosteroids;Serum;Serum, Plasma;Solutions;Source;Specificity;Spectrometry, Mass;Spectroscopy, Mass;Spectrum Analyses, Mass;Spectrum Analysis, Mass;T2d;T2dm;Technology;Therapeutic Hormone;Time;Tissue Sample;Tissues;Type 2 Diabetes;Type Ii Diabetes;Vit D;Variant;Variation;Vitamin D;Vitamin D 3;Vitamin D2;Vitamin D3;Vitamins;Work;Adult Onset Diabetes;Aminomethane;Antibody;Autoimmune Disorder;Base;Bone Health;Calciferol;Cardiovascular Disorder;Chronic Disease /Disorder;Chronic Disease/Disorder;Chronic Disorder;Cross Reactivity;Cycloaddition;Diene;Disease /Disorder;Disease/Disorder;Gene Product;High Performance Liquid Chromatography;High Throughput Analysis;Ion Source;Ionization;Ketosis Resistant Diabetes;Liquid Chromatography Mass Spectrometry;Malignancy;Mass Spectrometry;Maturity Onset Diabetes;Methanamine;Methylamine;Methylamine Bisulfite;Monomethylamine;Neoplasm /Cancer;Neoplasm/Cancer;Noninsulin Dependent Diabetes Mellitus;Pi Bond;Premature;Programs;Reversed Phase Chromatography;Scale Up;Social Role

Most clinical assays begin with blood collection in a laboratory setting. The Gates foundation and many others have recognized and attempted to deal with the fact that in much of the world clinical testing is done in a primitive setting. The luxury of having a phlebotomist to draw blood with a laboratory to prepare serum or plasma for analysis is not universal at many points-of-care, even in America. The most significant component of this proposal is that the world will obtain a plasma extraction technology that produces a small, reproducible aliquot of plasma without an energy source or human intervention. Moreover, 2.5 or 6 ¿L plasma samples collected in this manner can be transported dry on a collection disc to a central laboratory by courier or mail. The technology proposed to accomplish this is based on blood fractionation by frontal loading of a miniature assemblage of membranes where plasma is transported through multiple membrane layers by capillary action. Blood cells are removed via a combination of adsorption and filtration as plasma migrates to a 6.4 mm diameter collection disc at the bottom of the membrane stack during roughly three min. Stripping (delaminating) the upper, cell bearing membrane layers from the system exposes the plasma loaded collection disc which dries in roughly 10 min. These dried samples can be transported from almost anywhere in to world to a laboratory for analysis within 24 hr. This plasma extraction technology is being developed and validated as component of a mass spectrometry (MS) analysis of vitamin D analogues. As part of the vitamin D test proposed and developed in Phase I, a reagent was developed (SecoSET) that derivatizes vitamin D species with high selectivity. The function of SecoSET is to impart a permanent positive charge to vitamin D species that increases their ionization efficiency in electrospray ionization MS and concomitantly reduces their LOQ.

Public Health Relevance Statement:


Public Health Relevance:
Blood tests are at the heart of modern health care, requiring a skilled phlebotomist to draw a blood sample by venipuncture and a well-equipped laboratory to prepare the blood for analysis. The significance of the work proposed here is that a new technology is being developed that circumvents this process, allowing a physician or individual to prepare their own sample by placing a drop of blood from a finger-stick on a small card that is transported to a laboratory by courier or mailed. Initial trails of this technology will be appliedin the development of a vitamin D test. This technology enables a new paradigm in that samples from large numbers of remote sites can be analyzed in a single, sophisticated central laboratory at reduced cost.

Project Terms:
Adsorption; Aliquot; Alkylation; Americas; Antibodies; base; Biological Assay; Biological Products; Blood; Blood capillaries; Blood Cells; blood fractionation; Blood specimen; Blood Tests; Caliber; capillary; Carrier Proteins; Cell membrane; Cells; Charge; Clinical; Collection; cost; Data; Development; Digestion; Dimensions; Drops; Drug Formulations; Energy-Generating Resources; Filtration; Fingers; Foundations; Healthcare; Heart; Human; Immune; Individual; Intervention; ionization; Laboratories; Mails; Marketing; Mass Spectrum Analysis; Membrane; Methods; Microfluidics; Monitor; Monoclonal Antibodies; new technology; operation; Peptides; Pharmaceutical Preparations; Phase; Physicians; Plasma; point of care; Preparation; Process; Process Assessment (Health Care); Production; Proteins; Proteomics; public health relevance; quality assurance; Reagent; research clinical testing; Sales; Sampling; Serum; Shipping; Ships; Site; Spectrometry, Mass, Electrospray Ionization; Speed (motion); Stream; Structure; System; Technology; Temperature; Testing; Text; Therapeutic Agents; therapeutic protein; Time; Trypsin; Validation; Venipunctures; Vitamin D; Vitamin D Analog; Work; Writing