Introduction
ISOLUTE® PLD+ Protein and Phospholipid Removal plates offer a substantial improvement in extract cleanliness compared to traditional protein precipitation techniques for bioanalytical sample preparation.
This application note describes a simple, effective ISOLUTE® PLD+ protocol for the extraction of 25-hydroxy vitamin D from serum, demonstrating high, reproducible analyte recoveries with low protein and phospholipid content in the extracts.
Figure 1. Structures of 25-hydroxy Vitamin DAnalytes
25-hydroxy vitamin D2, 25-hydroxy vitamin D3 and d6-25- hydroxy vitamin D3 as the internal standard.
Figure 2. Typical ISOLUTE® PLD+ procedureSample Preparation Procedure
Format: ISOLUTE® PLD+ Protein and Phospholipid Removal plate, part number 918-0050-P01
Sample Pre-treatment
Add 10 μL of ISTD (equivalent to 30 ng/mL) to the serum sample.
Mix. Allow to stand for ~1 hour for binding to occur.
Solvent Application
Apply 400 μL of Acetonitrile (MeCN) to each well of the ISOLUTE® PLD+ plate.
Sample Application
Add 100 μL of serum with ISTD and mix thoroughly via repeat aspirate/dispense steps.
Analyte Elution
Apply vacuum -0.2 bar or 3 psi positive pressure for approximately 5 minutes. For highly particulate laden samples increased pressure or vacuum conditions may be required.
Post Extraction
Dry the extract in a stream of air or nitrogen using a SPE Dry (40 °C at 40 L/min) or TurboVap
(40 °C at 1.0 bar).
Reconstitution
Apply 100 μL of 30/70 2 mM Ammonium Formate, 0.1% formic acid aq/MeOH.
>> Download the full Application Note as PDF
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