Extraction of 25-hydroxy Vitamin D from Serum Using ISOLUTE® PLD+ Prior to LC-MS/MS Analysis

Significance of the Topic

The quantification of 25-hydroxy vitamin D in serum is critical for assessing vitamin D status in clinical and research settings. Reliable extraction and clean-up of serum samples prior to LC-MS/MS analysis ensure accurate measurement by minimizing matrix effects from proteins and phospholipids. The ISOLUTE PLD+ approach addresses the need for a rapid, reproducible protocol with high analyte recovery and low interferences.

Objectives and Overview

This application note describes the development and validation of a workflow for extracting 25-hydroxy vitamin D2 and D3 from human serum using ISOLUTE PLD+ protein and phospholipid removal plates, followed by UPLC-MS/MS analysis. The goals include maximizing recovery, reducing matrix effects, achieving rapid chromatographic separation, and demonstrating method robustness through calibration curves and external quality assessment.

Used Instrumentation

• Waters Acquity UPLC system with ACE EXCEL 2 C18-PFP column (100 × 2.1 mm, 2 µm).
• Waters Quattro Premier XE triple quadrupole mass spectrometer with electrospray ionization.
• Biotage ISOLUTE PLD+ 96-well plates for protein and phospholipid removal.
• Biotage SPE Dry or TurboVap systems for sample concentration.

Methodology

The sample preparation involves:

1. Pre-treatment: Add internal standard (d6-25-hydroxy vitamin D3, 30 ng/mL equivalent) to 100 µL serum and incubate for one hour.
2. Protein and phospholipid removal: Apply 400 µL acetonitrile to each well of the ISOLUTE PLD+ plate, load the serum mixture, mix by repeated aspiration/dispense, and apply 0.2 bar vacuum for extraction.
3. Post-extraction processing: Evaporate the eluate at 40 °C under air or nitrogen using SPE Dry or TurboVap, then reconstitute in 100 µL of 30/70 2 mM ammonium formate (0.1% formic acid) aqueous/methanol.

Chromatography:

• Mobile phases: (A) 2 mM ammonium formate/0.1% formic acid in water; (B) same buffer in methanol.
• Gradient elution: 25/75 to 0/100 A/B over 3 min at 0.4 mL/min, column at 40 °C, sample at 20 °C.
• Injection volume: 15 µL.

MS/MS conditions:

• Desolvation 450 °C, source 150 °C, collision cell pressure 3.76×10⁻³ mbar.
• MRM transitions: 25-OH D2 (395.5>269.5; qualifier 395.5>119.2), 25-OH D3 (383.5>257.5; qualifier 383.5>107.2), d6-25-OH D3 (389.6>263.5).

Main Results and Discussion

The method achieved baseline separation of 25-hydroxy vitamin D2 and D3 in under three minutes. Calibration curves in PBS/BSA and calibrated serum standards exhibited excellent linearity (r²>0.99) over 1–100 ng/mL. Recoveries exceeded 70% with RSD below 10%. External quality assessment via DEQAS samples met acceptance criteria, with all results within ±25% of the trimmed mean.

Benefits and Practical Applications

• Enhanced extract cleanliness relative to protein precipitation alone, reducing ion suppression.
• High-throughput compatibility with 96-well format and automation on Biotage Extrahera.
• Rapid analysis cycle suitable for routine clinical and research laboratories.
• Robust quantitation for diagnostics, QA/QC, and industrial analytics.

Future Trends and Potential Applications

Future developments include integration of automated liquid-handling platforms for increased throughput and consistency. The PLD+ workflow can be extended to other lipophilic analytes and biomarkers. Advances in UHPLC and high-resolution mass spectrometry promise multiplexed analysis of vitamin D metabolites and related compounds.

Conclusion

The ISOLUTE PLD+ protocol combined with UPLC-MS/MS provides a robust, efficient method for quantifying 25-hydroxy vitamin D in serum, delivering clean extracts, reliable recovery, and fast analysis suitable for diverse analytical settings.