Extraction of Fat-Soluble Vitamins from Human Serum Using ISOLUTE® SLE+ Prior to UHPLC/MS-MS Analysis

Importance of Topic

The accurate quantification of fat-soluble vitamins in human serum is critical for clinical diagnostics, nutritional monitoring, and pharmaceutical research. Vitamins A, D, E and K regulate essential physiological processes and their serum levels inform on deficiency or toxicity states. A robust extraction and assay protocol ensures reliable data for healthcare decision-making and quality control purposes.

Study Objectives and Overview

This application note aimed to establish a streamlined workflow for extracting multiple fat-soluble vitamins from human serum using ISOLUTE SLE+ plates, followed by UHPLC–MS/MS detection. Key objectives included:

• Achieving high recoveries (>90%) with low variability (RSD <10%) for a panel of retinol, β-carotene, 25-hydroxyvitamins D2/D3, α-tocopherol, phylloquinone and menaquinone-4.
• Demonstrating automation compatibility on the Biotage Extrahera platform and comparing performance with manual processing.
• Defining chromatographic and mass spectrometric parameters that deliver baseline separation and sensitive detection across broad concentration ranges.

Used Instrumentation

• Waters Acquity UPLC system with Restek Raptor Biphenyl column (100×2.1 mm, 2.7 μm).
• Waters Quattro Premier XE triple-quadrupole mass spectrometer with electrospray ionization.
• Biotage ISOLUTE SLE+ 400 μL supported liquid extraction plates.
• Biotage Extrahera™ automated sample processor.
• Biotage SPE Dry 96 sample concentrator.

Methodology and Sample Preparation

Serum samples (100 μL) were spiked with deuterated 25-OH-Vitamin D3 internal standard and pre-treated with a spiking solution or calibrator, followed by 1-hour equilibration in the dark. A mixture of propan-2-ol and heptane (1:3, v/v) was added to disrupt protein binding and increase analyte solubility. After a 5-minute incubation, the suspension was loaded onto ISOLUTE SLE+ plates. Analytes were eluted in two 500 μL heptane fractions, dried under nitrogen at room temperature, and reconstituted in 150 μL propan-2-ol prior to injection.

Chromatographic and MS Conditions

Gradient elution used mobile phases of ammonium acetate/formic acid in water (A) and a methanol/propan-2-ol blend (3:1, B). The flow rate was 0.4 mL/min at 40 °C. Injection volume was 10 μL. The MS monitored optimized MRM transitions for each vitamin in two acquisition windows to maximize sensitivity. The method achieved baseline separation of all analytes within 7 minutes.

Main Results and Discussion

Manual extraction recoveries exceeded 89% for all analytes with RSDs ≤10%. Automated processing on the Extrahera delivered slightly lower recoveries (71–84%) but maintained RSDs below 6%. Calibration curves were linear across clinically relevant ranges (e.g., retinol 80–4 000 ng/mL, α-tocopherol 800–40 000 ng/mL) with r² values >0.99. Representative chromatograms demonstrated clean baselines and minimal matrix interference. Vitamin K isoforms exhibited modest ion suppression, highlighting the benefit of targeted sample volume adjustment or additional internal standards.

Benefits and Practical Applications

The described protocol provides:

• High throughput via automated liquid handling and rapid UHPLC–MS/MS analysis.
• Reproducible quantitation suitable for clinical labs, nutritional studies, and QA/QC in pharmaceutical development.
• Flexible sample batching and compatibility with existing laboratory infrastructure.

Future Trends and Opportunities

Advances may include integration of higher-sensitivity mass analyzers to lower detection limits for Vitamin K, miniaturization of extraction formats, and development of multiplexed assays combining fat- and water-soluble vitamin panels. Automation software improvements could further reduce hands-on time and streamline data processing. Emerging lipidomic workflows may also incorporate this protocol as part of comprehensive metabolic profiling.

Conclusion

The ISOLUTE SLE+ extraction combined with UHPLC–MS/MS enables reliable, high-throughput measurement of fat-soluble vitamins in human serum. The method yields excellent recoveries, low variability, and broad dynamic ranges, supporting diverse applications in research and clinical diagnostics.

References

No external literature references were provided in the original text.