What Sample Prep is Best for Vitamin D Analysis?

With growing demand for sensitive and specific analytical techniques, laboratories are increasingly focusing on accurate low-level detection of endogenous vitamins and hormones in biological samples. One of the most notable examples is vitamin D analysis. Maintaining proper vitamin D levels is vital: deficiency can lead to low bone density, while excessive amounts may cause harmful calcium accumulation. However, accurate quantification of 25-hydroxyvitamin D₂ and D₃ metabolites can be challenging, particularly because lipids in biological matrices often interfere with LC/MS detection.

Selecting the Right Sample Preparation Method

Traditional methods such as protein precipitation do not effectively remove lipids, which can cause ion suppression and reduce method sensitivity. While solid phase extraction (SPE) can eliminate lipids, it requires complex method development and multiple steps that slow down high-throughput workflows.

A more efficient and reliable alternative is Agilent’s Captiva EMR-Lipid — a rugged sample preparation solution designed for selective lipid and matrix removal without sacrificing analyte recovery. Available in a 96-well plate format, Captiva EMR-Lipid is ideal for high-throughput clinical and research laboratories. For fully automated workflows, pair it with a Chrom Tech collection plate to streamline sample handling.

Captiva EMR-Lipid leverages a dual-mechanism design — combining size exclusion and hydrophobic interaction — to efficiently remove phospholipids and other matrix components. This dramatically reduces ion suppression and enhances LC/MS reliability for 25-hydroxyvitamin D₂ and D₃ analysis, producing consistent, reproducible results.

The 96-well plate and 1 mL cartridge formats include a solvent retention frit that enables in-well protein precipitation to further simplify sample preparation. The frit design supports smooth flow under vacuum or positive pressure — though positive pressure manifolds are generally recommended for optimal control and consistency. Learn more on our Positive Pressure Processor product page.

LC/MS/MS Analysis of 25-Hydroxyvitamin D Metabolites

Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is now the preferred method for precise quantification of vitamin D metabolites. Agilent’s Poroshell 120 EC-C18 columns deliver exceptional efficiency and reproducibility for this application. For best performance, we recommend using the Poroshell 120 EC-C18 analytical column with the corresponding guard column to ensure longevity and consistent peak shape.

For a detailed look at method conditions and results, review Agilent’s full application note. The Captiva EMR-Lipid workflow demonstrated exceptional phospholipid removal and signal stability for vitamin D metabolite analysis — simplifying sample preparation without compromising data quality.