Ultrafast Quantitation of Fat Soluble Vitamins A and E in Human Serum Using the Agilent RapidFire High-Throughput Mass Spectrometry System

Significance of the topic

The quantitation of fat-soluble vitamins A (retinol) and E (α-tocopherol) in human serum is critical for clinical diagnostics, nutritional studies, and therapeutic monitoring. Traditional HPLC and LC/MS/MS assays often involve lengthy sample preparation, significant solvent consumption, and limited throughput. Rapid, robust methods support high-volume laboratories and emerging applications in metabolomics and clinical research.

Objectives and overview of the study

This study aimed to develop and validate an ultrafast SPE/MS/MS workflow using the Agilent RapidFire high-throughput mass spectrometry system. Key goals included minimizing sample preparation steps, achieving reliable quantification over clinically relevant concentration ranges, and dramatically increasing sample throughput compared to conventional LC/MS/MS protocols.

Methodology and sample preparation

A dilute-and-shoot workflow was implemented in a 96-well format. Human serum samples, calibration standards (0.1–5.0 µg/mL for vitamin A; 0.4–20 µg/mL for vitamin E), and quality controls were spiked with deuterated internal standards, followed by protein precipitation with acetonitrile and 1:10 dilution in water. Antioxidants (EDTA, BHT) and light-protected vessels preserved analyte stability. The processed plate was directly analyzed via online SPE and rapid APCI MS/MS.

Instrumentation used

• Agilent RapidFire 365 high-throughput SPE system
• Agilent 6460 Triple Quadrupole Mass Spectrometer with APCI source
• MassHunter Acquisition and Quantitative Analysis software
• Reversed-phase C18 RapidFire cartridges

Main results and discussion

Calibration curves exhibited excellent linearity (R2 > 0.995) across target ranges for both vitamins. Intra- and interday accuracy ranged from 95 % to 105 %, with coefficients of variation below 10 % at QC levels. No significant carryover was detected in blank injections following the highest calibrators. The system achieved an injection-to-injection cycle time of 15 seconds, equating to over 240 samples per hour. Reproducibility was demonstrated by > 2,000 sequential injections, yielding stable responses (CV ≈ 8–9 %).

Benefits and practical applications

The described method delivers > 10× higher throughput than conventional LC/MS/MS assays, reduces solvent and sample volume requirements, and streamlines sample preparation. Clinical laboratories and research facilities can efficiently monitor vitamin status in large cohorts, support nutritional studies, and integrate the workflow into automated platforms.

Future trends and applications

Emerging directions include expansion of high-throughput SPE/MS/MS to additional small-molecule panels, integration with robotic liquid handlers, and coupling with data-driven diagnostics and precision nutrition. Advances in cartridge chemistries and multiplexing will further accelerate sample throughput and broaden assay portfolios.

Conclusion

The Agilent RapidFire SPE/MS/MS method enables rapid, accurate quantitation of vitamins A and E in human serum with minimal preparation and exceptional throughput. Its robustness, precision, and speed make it a valuable tool for clinical research, routine testing, and high-volume analytical environments.

References

• Youssef M., Stroman M., Miller V.P. Ultrafast Quantitation of Fat Soluble Vitamins A and E in Human Serum Using the Agilent RapidFire High-Throughput Mass Spectrometry System. Application Note 5991-5293EN, Agilent Technologies, 2014.