Qi Huang (Presenter)
Quantalytical Labs
Bio: A medicinal chemist being pooled into bioanalytical world, is utilizing some of his synthetic chemistry and rational drug design instinct to design and develop novel MS sensitizing reagents for quantification of low abundant analytes from biological matrices. Founding chemist of Quantalytical Labs Inc., a Senior Chemist at SPEware Corp., formerly a Senior Scientist at Amgen Inc.
Authorship: Qi Huang1,2, Philip Dimson1,2, John Laycock2
1. Quantalytical Labs Inc, 2. SPEware Corp.
| Short Abstract The physiologically active forms of Vitamin D, 1,25-dihydroxyvitamin D2/3 (DHVD) are important biomarkers for a number of disease states. With recent advancements in LC-MS/MS technology, reliable measurements of DHVD are still challenging due to low abundance and poor ionization capacity, even with the aid of PTAD, a Cookson type derivatization reagent. There were reports of quantification of DHVD with a combination of immuno-affinitive capture and PTAD derivatization. In this presentation, we will report quantification of plasma DHVD with a simple SPE and quick derivatization with an in house developed novel crown-ether based PTAD reagent. |
Long Abstract
The physiologically active forms of Vitamin D, 1,25-dihydroxyvitamin D2/3 (125DHVD), which are produced in kidney, are important regulators of calcium metabolism in human. They are important biomarkers for a number of disease states, including osteoporosis and diabetes. Traditionally, 25-hydroxyvitamin D2 and D3 (25HVD) are measured as surrogate markers as abundant levels are present (ng/mL range) in the blood circulation with long half-life. When the kidney functions are compromised, 25HVD are not indicators of vitamin D deficiency, since they are not the biological active forms. Therefore it is necessary to determine the presence of 125DHVD in the blood.
Due to very low levels (low pg/mL) of 125DHVD in circulation, their quantitation is a challenge. Traditionally, 125DHVD are quantified via radio-immuno-assays (RIA). RIAs lack the specificity to distinguish between 125DHVD2 and 125DHVD3. With recent advancements in LC-MS/MS technology, reliable measurements of 125DHVD are still challenging due to low abundance and poor ionization capacity, even with PTAD, a Cookson type derivatization reagent1. In order to improve the sensitivity for adequate quantification of 125DHVD, there have been reports with complexed sample preparations, such as combination of immunoaffinity extraction-solid phase extraction-PTAD derivatization-LC-MS/MS2, or multiple solid phase extractions with Amplifex™ (a quaternary ammonium based PTAD) derivatization-LC-MS/MS3.
We would like to share the discovery of a new Cookson type reagent, MB409™. With a crown-ether moiety, MB409™ greatly enhanced ionization capacity of the derivatized vitamin D. Here we report a simple sample preparation process along with derivatization with MB409™, followed by quantification via LC-MS/MS on an API-4000 MS spectrometer coupled with an Agilent 1200 binary HPLC pump. Application of 200 uL of plasma sample with one SPE followed by a quick MB409™ derivatization produced quantification sensitivity for both 125DHVD2 and 125DHVD3 with linear response range from low pg/mL to ng/mL on an API 4000 MS spectrometer.
By utilizing an automated SPE workstation and a liquid handler, the entire quantification process (SPE extraction, elution, derivatization, and direct injection to LCMS) can be automated to create an efficient work flow in the laboratories.
References & Acknowledgements:
1. Cookson RC. Organic Syntheses. 1988
2. Hedman CJ, Wiebe DA, Dey S, Plath J, Kemnitz JW, Ziegler TE. Development of a Sensitive LC/MS/MS Method for Vitamin D Metabolites: 1,25 Dihydroxyvitamin D2&3 Measurement Using a Novel Derivatization Agent.Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 2014;0:62-67. doi:10.1016/j.jchromb.2014.01.045.
3. Daniel Blake, Optimising the Derivatisation of Vitamin D Metabolites Within Clinical Research Using Amplifex™ Diene Reagent, Chromatography Today, 2014, 6, 6-8.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SPEware Inc, Quantalytical Labs Inc, |
| Board Member | no | |
| Stock | yes | SPEware Inc, Quantalytical Labs Inc |
| Expenses | no |
IP Royalty: yes
IP Desc:Quantalytical Labs Inc,
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |