Extraction of Desmopressin from Serum Using EVOLUTE® EXPRESS WCX Prior to LC-MS/MS Analysis

Significance of the Topic

Desmopressin, a synthetic vasopressin analogue, is widely used in treating nocturnal enuresis, diabetes insipidus and certain clotting disorders. Accurate measurement in human serum supports therapeutic monitoring and pharmacokinetic studies. Solid phase extraction (SPE) prior to LC-MS/MS enhances sensitivity and selectivity by removing proteins, lipids and phospholipids.

Objectives and Overview of the Study

This work demonstrates a high-throughput SPE protocol using EVOLUTE EXPRESS WCX plates for desmopressin extraction from human serum coupled to LC-MS/MS quantification. Key goals:

• Maximize recovery and reproducibility over a concentration range of 0.2–200 ng/mL
• Minimize co-extracted interferences, especially phospholipids
• Optimize sample preparation and chromatographic conditions for robust analysis

Methodology and Instrumentation

Sample preparation involved: dilution of 200 µL serum with 10 mM ammonium acetate pH 6, SPE conditioning (methanol, buffer), sample loading, two aqueous/organic washes and elution with 2% formic acid in 70/30 methanol/water. Extracts were evaporated and reconstituted in 0.1% formic acid in 50/50 water/acetonitrile.

• UHPLC: Waters ACQUITY I-Class with ACE EXCEL C18-amide column (100×2.1 mm, 1.7 µm), gradient 0–4 min from 90/10 to 5/95 aqueous/organic, 0.3 mL/min, 40 °C.
• MS/MS: Waters Xevo TQ-S, ESI positive mode, MRM transitions m/z 535.3→328.2 (quant) and 535.3→120.1 (qual), desolvation 500 °C, source 150 °C.

Main Results and Discussion

Recovery exceeded 80% across all levels with RSD <10%. Chromatograms at 5 ng/mL showed sharp peaks and low background. Calibration curves were linear (r2>0.99) with LOQ of 0.2 ng/mL. Phospholipid monitoring via MRM of the m/z 184 ion demonstrated substantial removal compared to protein precipitation, confirming extract cleanliness.

Benefits and Practical Applications

• The SPE method delivers high throughput and automation compatibility for clinical and research laboratories.
• Enhanced sample cleanliness improves MS-based quantification accuracy and instrument uptime.
• Low LOQ and reproducible recoveries support pharmacokinetic profiling and therapeutic drug monitoring.

Future Trends and Possibilities

Integration with fully automated SPE-LC-MS workflows and potential miniaturization of elution volumes could further increase throughput and reduce solvent consumption. Expansion to multiplexed peptide assays and adaptation to other weak cation-exchange analytes represent promising applications.

Conclusion

The optimized EVOLUTE EXPRESS WCX SPE protocol combined with UHPLC-MS/MS offers a reliable, sensitive and automatable solution for quantifying desmopressin in serum. High recoveries, clean extracts and low detection limits make it suitable for clinical and bioanalytical settings.

Reference

• Biotage. Application Note AN887: Extraction of Desmopressin from Serum Using EVOLUTE EXPRESS WCX Prior to LC-MS/MS Analysis. 2017.