Simultaneous Analysis of Vitamins A and E in Serum by UPLC-MS/MS for Clinical Research

Importance of the Topic

Accurate quantification of fat-soluble vitamins A (retinol) and E (α-tocopherol) in human serum is critical for assessing nutritional status, monitoring disease biomarkers, and supporting clinical research. Traditional HPLC-UV methods often require large sample volumes and lengthy preparation and run times, limiting throughput and efficiency.

Objectives and Study Overview

The study aimed to develop and validate a rapid, sensitive UPLC-MS/MS workflow for the simultaneous extraction and measurement of vitamins A and E in serum. Key goals included minimizing sample volume, reducing analysis time, achieving robust matrix cleanup, and demonstrating agreement with external quality schemes.

Applied Instrumentation

• ACQUITY UPLC I-Class (FTN) System
• ACQUITY UPLC HSS PFP Column (2.1 × 50 mm, 1.8 µm)
• Oasis PRiME HLB µElution 96-well Plate
• Xevo TQD Tandem Quadrupole Mass Spectrometer
• MassLynx v4.2 with TargetLynx XS Application Manager

Methodology and Sample Preparation

• Serum (100 µL) spiked with deuterated internal standards (2H8-vitamin A, 2H6-vitamin E).
• Protein precipitation using ethanol, dilution with water, centrifugation.
• SPE cleanup on Oasis PRiME HLB µElution plate, wash with 25% acetonitrile, elution with acetonitrile into glass inserts, dilution with water.
• Chromatography: 0.4 mL/min gradient of water/methanol with 2 mM ammonium acetate and 0.1% formic acid; 3.0 min total run time.
• MS/MS detection in positive ESI MRM mode with transitions 269.15→93.0/83.0 for retinol and 431.4→165.1/83.0 for α-tocopherol.

Results and Discussion

• No detectable carryover; high-concentration samples diluted 1:4 with 86–102% accuracy.
• Precision (n=25 over 5 days): CV ≤6.9% for both vitamins at multiple QC levels.
• Limit of quantification: 50 ng/mL (vitamin A, 7.3% CV) and 1.1 µg/mL (vitamin E, 14.7% CV).
• Linear dynamic range: 28–4800 ng/mL (vitamin A, r2 ≥0.997) and 2–51.6 µg/mL (vitamin E, r2 ≥0.991).
• Matrix effects minimal; phospholipid removal >96% compared to protein precipitation alone.
• Effective separation from endogenous/exogenous isobaric interferences (e.g., retinoic acid, retinyl palmitate, tocopherol isomers).
• External QA (UK NEQAS) Deming regression slopes 0.90–0.97, biases <11% versus trimmed mean values.

Benefits and Practical Applications

• High throughput: 3-minute UPLC run and low solvent consumption.
• Minimal sample volume suitable for neonatal or limited-volume clinical samples.
• Robust assay for nutritional monitoring, biomarker research, and QA/QC in routine laboratories.
• Streamlined phospholipid removal enhances reproducibility and extends column lifetime.

Future Trends and Applications

• Integration with automated sample-prep platforms for even higher throughput.
• Expansion of MRM panels to cover additional fat-soluble vitamers and metabolites.
• Application in large-scale epidemiological or intervention studies.
• Potential adaptation to point-of-care or near-patient testing environments.

Conclusion

The described UPLC-MS/MS method provides a rapid, sensitive, and reliable approach for simultaneous measurement of vitamins A and E in serum using minimal sample volume. Excellent precision, linearity, and matrix cleanup make it well suited for clinical research and high-throughput laboratory workflows.