Extraction of Vitamin D Metabolites from Human Plasma Using ISOLUTE SLE+ for LC–MS–MS Analysis

August 2, 2011

The Application Notebook

The Application Notebook, The Application Notebook-08-02-2011, Volume 0, Issue 0

Biotage Application Note

This application note describes a simple and robust procedure for the extraction of vitamin D metabolites from human plasma at clinically significant concentrations using ISOLUTE SLE+ supported liquid extraction plates. Supported liquid extraction is an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation.


Vitamin D deficiency causes bone softening diseases like rickets but has also been linked to cancer, M.S., arthritis, diabetes, Parkinson's and Alzheimer's disease. The accurate sampling and subsequent analysis of Vitamin D levels has become highly regulated and of significant clinical concern, with more sensitive, simplified and robust techniques required worldwide.

Extraction Conditions

This application note outlines a high throughput procedure using ISOLUTE SLE+ supported liquid extraction plates (part #8200200-P01) for processing 100 μL plasma samples. Method parameters have been optimized to maximize analyte recovery and minimize ion suppression.

Sample: Human plasma (100 μL).

Sample pre-treatment: Add plasma (100 μL) to a mixture of HPLC grade water (50 μL) and isopropanol (propan-2-ol) (50 μL) and mix for 10 seconds.

Sample loading: Load pre-treated sample (200 μL total volume) onto the ISOLUTE SLE+ plate, leave the samples to absorb for 5 minutes under gravity and then apply a pulse of vacuum for 2–5 seconds if not fully absorbed onto sorbent.

Analyte elution: Apply heptane (500 μL), wait 5 min to allow the solvent to soak, apply a short pulse of vacuum if solvent not fully absorbed. Repeat application of heptane (500 μL), allow to soak for 5 min and then apply another short pulse of vacuum.

Post extraction: Evaporate eluate to dryness without heat and reconstitute in 0.1% formic acid in HPLC grade water/0.1% formic acid in methanol (20:80 v/v, 100 μL). Cap samples and vortex gently for 60 seconds.


All results show recoveries above 85% with RSDs below 10%, Figure 1 shows the recoveries of all the relevant Vitamin D metabolites. LOQ values are 25 ng/mL with an LOD at 10 ng/mL.

Figure 1: Mean % recoveries of Vitamin D metabolites from human plasma using SLE+ procedure.


This procedure provides for a simplified extraction of Vitamin D metabolites optimized for LC–MS–MS analysis. The use of ISOLUTE SLE+ reduces the need for any off-line steps, incorporating a protein binding disruption step into the process, significantly decreasing sample preparation time.


This application note is based on the poster 'Vitamin D and Metabolites: Evaluation of Supported Liquid Extraction (SLE) prior to LC-MS/MS Analysis.', L Williams et al., presented at MSACL, San Diego, California, USA, 5–9 February 2011 (available from www.biotage.com/applications).

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