Analysis of Fat-soluble Vitamins Using the Nexera™ UC Supercritical Fluid Chromatograph

Importance of Topic

Fat-soluble vitamins (A, D, E, and K) play vital roles in vision, bone health, antioxidant defense, and blood coagulation. Because these compounds are not synthesized adequately in the human body, accurate quantitation in foods and nutraceuticals is essential to prevent deficiencies and related disorders. Analytical challenges arise from the structural similarities and isomeric diversity of these lipophilic molecules, demanding high resolution and sensitivity methods.

Objectives and Overview

This application note presents a simultaneous analytical workflow for twenty fat-soluble vitamins and related metabolites. The aim is to demonstrate the capability of a supercritical fluid chromatograph (SFC) coupled to a triple quadrupole mass spectrometer to achieve rapid separation, low detection limits, and robust quantitation for quality control in food and supplement matrices.

Methodology and Instrumentation

Chromatographic separation was performed on a Shim-pack UC-RP column (150 mm × 2.1 mm, 3 µm) using CO₂ as mobile phase A and methanol containing 0.1 % (w/v) ammonium formate as mobile phase B. A linear gradient from 5 % to 50 % B over 10 minutes (total run time 18 minutes) was applied at 0.8 mL/min and 40 °C, with back pressure held at 10 MPa. Detection employed an LCMS-8050 triple quadrupole MS in positive-ion ESI MRM mode. Key instrumental details:

• Supercritical Fluid Chromatograph: Nexera UC SFC
• Column: Shim-pack UC-RP, 150 × 2.1 mm, 3 µm
• Mobile phase A: CO₂; B: 0.1 % ammonium formate in methanol
• Gradient: 5 %–50 % B (0–10 min), re-equilibration to 5 % B by 18 min
• Flow rate: 0.8 mL/min; Column temp: 40 °C; BPR: 10 MPa
• MS: LCMS-8050; ESI(+); MRM transitions optimized for each analyte

Main Results and Discussion

Using 1 µmol/L standards, all twenty targets—including retinoids, ergocalciferols, tocopherols, tocotrienols, and quinones—were baseline-separated within 2–10 minutes. Calibration curves exhibited excellent linearity (R² > 0.995) across 0.01–10 µmol/L ranges, and limits of detection ranged from 0.4 to 47 nmol/L. Six-replicate analyses of 1 µmol/L standards showed retention time RSDs below 0.5 % and peak area RSDs under 6 %, confirming repeatability. Analysis of a supplement extract demonstrated co-detection of cholecalciferol and menaquinone with spike-and-recovery rates of 94.6 %–106 %, illustrating method accuracy in complex matrices.

Benefits and Practical Applications

The SFC-MS/MS method offers rapid analysis (18 min total run), high throughput, and excellent sensitivity for routine quality control of fat-soluble vitamins in food and dietary supplements. Its minimal solvent use and efficient separation of structurally similar isomers make it well suited for laboratories seeking reliable quantitation and reduced environmental impact.

Future Trends and Possibilities

Advancements in SFC column chemistries may further enhance resolution of challenging isomeric compounds. Integration with high-resolution MS and automated sample preparation will expand applications to broader lipidomics studies. Additionally, adaptation of this approach for on-line extraction and coupling with multi-dimensional separation could enable comprehensive profiling of vitamin derivatives in clinical and nutritional research.

Conclusion

This study demonstrates a robust, sensitive, and efficient SFC-MS/MS workflow for simultaneous quantitation of fat-soluble vitamins. The method achieves fast run times, low limits of detection, and high reproducibility, supporting its use in quality assurance and research environments.

Reference

• Shimadzu Corporation. Analysis of Fat-soluble Vitamins Using the Nexera UC Supercritical Fluid Chromatograph; Application No. C191; First Edition May 2019.