Ultra-Sensitive Method for the Determination of 1,25 di-OH Vitamin D2 and 1,25 di-OH Vitamin D3 in Serum Using Supported Liquid Extraction Prior to LC-MS/MS

Importance of the topic

The active forms of vitamin D, 1,25 dihydroxyvitamin D2 and D3, are key regulators of calcium metabolism and cellular signaling. Accurate, ultra-sensitive measurement of these metabolites in serum is critical for clinical diagnostics, monitoring of metabolic bone diseases, and research into endocrine disorders.

Objectives and Study Overview

This application note describes a workflow to extract and quantify 1,25 di-OH vitamin D2 and D3 from human serum. The goal was to achieve a calibration range of 5–500 pg/mL, maximize sensitivity (LLOQ ~5 pg/mL), and ensure reproducible recoveries using supported liquid extraction followed by LC-MS/MS detection.

Methodology

Sample preparation combines serum with deuterated internal standard and 50% propan-2-ol to release protein-bound analytes. Supported liquid extraction is performed on ISOLUTE SLE+ 400 µL plates, loading by gravity and eluting with heptane. On-plate derivatization with PTAD in ethyl acetate/heptane and formation of methylamine complexes enhances MS response. Extracts are evaporated at ~40 °C and reconstituted in 70% methanol containing methylamine before analysis.

Instrumentation Used

• Shimadzu Nexera UHPLC system
• ACE Ultracore 2.5 Super C18 column (50 x 2.1 mm)
• AB Sciex 5500 triple quadrupole mass spectrometer
• Biotage Extrahera automation platform and TurboVap 96 evaporator

Main Results and Discussion

• Recovery: 99–104% with %RSD ≤3.6 for both analytes
• Linearity: 5–500 pg/mL (r² >0.994) with 1/x weighted regression
• Sensitivity: LLOQ ~5 pg/mL demonstrated by spiked charcoal-stripped serum
• Selectivity: No interference from 24,25 di-OH vitamin D3; baseline separation achieved

Benefits and Practical Applications

This method requires only 250 µL of serum and eliminates emulsions common in traditional liquid–liquid extraction. On-plate derivatization and methylamine complexation improve sensitivity with minimal additional cost. Automation on the Biotage Extrahera boosts sample throughput and consistency.

Future Trends and Opportunities

• High-throughput integration with automated sample handlers for large-scale studies
• Multiplexed assays including mono- and di-hydroxy vitamin D metabolites
• Pharmacokinetic applications in vitamin D analog development
• Comprehensive steroid profiling panels using similar workflows

Conclusion

The described SLE-LC-MS/MS protocol delivers robust, ultra-sensitive quantification of 1,25 di-OH vitamin D2 and D3 in serum. High recovery, reproducibility, and selectivity make it suitable for clinical research and quality control laboratories.