High Throughput Analysis of 25-OH-Vitamin D using Micro-flow LC/MS/MS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 257-280-Disorders of Vitamin D Metabolism & Action
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-279
Michael J . Y. Jarvis*1, Evelyn L McClure1 and Andrea Bozovic2
1AB Sciex, Concord, ON, Canada, 2UHN, Laboratory Medicine Program, Toronto, ON, Canada

For Research Use Only.  Not For Use In Diagnostic Procedures. Micro-flow liquid chromatography has become a compelling alternative to conventional HPLC for many analyses given its cost-reduction and time-saving potential. Lower solvent consumption resulting from the use of micro-flow rates (5-60 µl/min), as compared to a typical flow rate of 500 µl/min using conventional HPLC, significantly reduces solvent and waste disposal costs. In addition, high on-column linear velocities and low mixing and delay volumes allow for fast chromatography and thus higher sample throughput. These benefits are realized while sensitivity is maintained or enhanced as compared to conventional HPLC, making micro-flow LC an excellent fit for the analysis of Vitamin D given the sensitivity and throughput requirements of this assay.


Here we present the details of the analysis of 25-monohydroxy Vitamin D2 and 25-monohydroxy Vitamin D3 using the Eksigent ekspert™ microLC 200 system in combination with the AB SCIEX QTRAP® 4500 LC/MS/MS system. Chromatographic separation of the two compounds is achieved using a Halo C18 column (0.5 x 50 mm, 2.7 µm) and a water/methanol/formic acid gradient. Total runtime for the analysis is 3 minutes, which is achieved at a flow rate of 20 µl/min. To avoid introducing early-eluting matrix to the system, the first part of the gradient is diverted to waste thus improving robustness and column lifetime.


The Limit of Quantitation (LOQ) for the analysis was determined using ‘blank’ stripped serum and spiked serum and found to be below 4 ng/mL, and 5 ng/mL for 25-monohydroxy Vitamin D3 and 25-monohydroxy Vitamin D2, respectively. Accuracies were within 92-109% across the calibration range (1 – 100 ng/mL), with CVs below 15% based on n=5 for each calibration concentration. Serum samples from donors were also analyzed using the method described above, and the results indicate that method performance is comparable to that of a conventional HPLC method.


An LC-MS/MS method has been developed for the analysis of 25-hydroxyvitamin D2 and D3 using micro-flow LC, resulting in a reduction of solvent consumption and faster analytical run-times. The method enabled sensitive detection of 25-hydroxyvitamin D2 and D3 at less than 4ng/mL, and 5 ng/mL, respectively.

Disclosure: MJYJ: Employee, AB SCIEX. ELM: Employee, AB SCIEX. Nothing to Disclose: AB

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm