Overview
The aim of this application note is to introduce a fast and effective sample preparation technique using Novum Simplified Liquid Extraction (SLE) products that overcomes the challenges observed in a traditional liquid-liquid extraction (LLE). This rapid cleanup is then paired with an LC/MS/MS method using a Kinetex 2.6 μm F5 core-shell HPLC column, resulting in >80% recoveries.
Sample Pre-treatment Step
To 200 μL human serum spiked with 30 μL 25-OH-Vitamin D3-d3 (1 μg/mL in water) add 190 μL of 2 % Ammonium hydroxide in water and vortex for 30 seconds.
Extraction Procedure
Sample Loading
- Load the sample from pre-treatment step onto the Novum SLE 3 cc tube (Part No. 8B-S138-5BJ) and apply a short and gentle pulse of vacuum (~5-10 sec at 5” or less of Hg) until the sample has completely entered the media.
NOTE: Avoid prolonged or excessive vacuum. The sample must not leave the Novum bed.
- Wait for 5-6 minutes.
NOTE: Inadequate or excessive wait period will lead to variable recoveries and poor precision.
Elution
- Dispense 3x 600 μL of ethyl acetate onto the Novum SLE sorbent and collect the solvent under force of gravity.
- Apply vacuum at 5” of Hg (or lower) for 20-30 secs to complete the extraction.
Dry Down
- Evaporate extracted samples to complete dryness under slow stream of N2 and room temperature.
- Reconstitute the dry residue in 200 μL of 0.1 % Formic acid in Water/0.1 % Formic acid in Methanol (30:70).
>> Download the full Application Note as PDF
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