Biotage has produced an application note describing the extraction of the peptides oxytocin and vasopressin from serum using a fast, simple EVOLUTE EXPRESS load-wash-elute SPE procedure and achieving high reproducible extraction recoveries.
Introduction
Extraction from serum was performed using EVOLUTE EXPRESS ABN 96-well plates, utilizing a highly aqueous elution solvent, and minimizing co-extractable material in the form of proteins, lipids and phospholipids.
Method
The sample preparation procedure, UHPLC and MS conditions are given in the application note.
Results
Good retention and chromatographic peak shape was obtained using the C18–300 column.
Serum was spiked at various concentrations from 0.2–500 ng/mL for recovery determination. High reproducible recoveries > 70 % with corresponding RSDs < 10 % were demonstrated.
Calibration curves were generated using serum spiked at concentrations from 0.2-500 ng/mL. Good linearity, coefficients of determination (r2 > 0.99) and sensitivity were obtained. Serum matrix demonstrated low endogenous levels of the analytes which contributed to a slight intercept on the
calibration curves.
Extract Cleanliness
Phospholipid Removal
Phospholipids were investigated to provide an indication of extract cleanliness. The most abundant phospholipids were previously selected from full scan, SIR and precursor ion scanning experiments using MRM transitions monitoring the common 184 product ion.
Post Column Infusion
Extract cleanliness was also investigated using post-column infusion (PCI) experiments. Mobile phase and blank serum extracts were injected onto the LC-MS/MS setup whilst teeing in oxytocin and vasopressin. Infusion was used to determine regions of suppression for each technique.
SPE Considerations
- During method development, EVOLUTE® EXPRESS ABN was compared to both strong and weak cation exchange sorbents. EVOLUTE EXPRESS WCX (weak cation exchange) also provided good analyte recoveries. However, better extract cleanliness was obtained using the optimized elution solvent in combination with EVOLUTE EXPRESS ABN.
- Acidic pH conditions were used for sorbent conditioning (full SPE method) and sample pre-treatment in order to suppress ionization of the analyte, making it less polar, and improving initial analyte retention.
(For further SPE considerations, please consult the application note).
