What Sample Prep is Best for Vitamin D Analysis?

Low level testing of endogenous vitamin and hormone levels in biological samples is the driving force behind the need for more sensitive and specific analytical methods. One example is vitamin D analysis growing in popularity over recent years. Vitamin D deficiency can lead to health issues such as low bone density. High levels can also be problematic because it can lead to a buildup of calcium in the body. So, it’s important to be able to accurately monitor vitamin D levels. Lipids contained in biological samples are known to be detrimental in the analysis of 25-hydroxyvitamin D2 and D3 metabolites.

What Sample Prep Method Should I Use?

Traditional sample preparation techniques such as protein precipitation do not remove lipid interferences. Solid phase extraction can be effective, but it requires multiple steps and method development. Agilent’s Captiva EMR-Lipid is a much more rugged and reliable alternative. It is available in a 96-well plate format, which is key for high throughput clinical laboratories. This can also be paired with a Chrom Tech collection plate for streamlined automated analysis. Captiva EMR-Lipid provides highly selective and efficient lipid/matrix removal without the loss of analytes. The novel technology removes lipids based on size exclusion and hydrophobic interaction. The effective lipid removal ensures minimal ion suppression of 25-hydroxyvitamin D2 and D3 metabolites, which significantly improves the LC method reliability and ruggedness.

The 96-well plate (and 1 mL cartridge) formats contain a solvent retention frit, which allows in-well protein precipitation that streamlines sample preparation. The filter design promotes easy flow when paired with vacuum or positive pressure*. Here is the sample preparation methodology used in the study by Agilent Technologies:

*We recommend using a positive pressure manifold over a vacuum manifold. For more information, check out our positive pressure manifold product page.

Chromatography Analysis of 25-hydroxyvitamin D2 and D3 Metabolites

LC/MS/MS has gained wide acceptance for the separation and accurate quantification of vitamin D metabolites. Below you will find the LC and MS/MS parameters utilizing an Agilent Poroshell 120 EC-C18 column and guard column:

For detailed results, click here to review the application note provided by Agilent. Captiva EMR-Lipid proved to be simple to use and provided excellent results for removing phospholipids for vitamin D metabolite analysis.