Simultaneous Analysis of Water-soluble and Fat-soluble Vitamins in Fish by Novel SFC-MS/MS Method

Importance of the Topic

The analysis of water- and fat-soluble vitamins in fish is critical for nutritional profiling, food quality control, and regulatory compliance. Fish are a major dietary source of essential vitamins, and rapid, accurate determination ensures consumer safety, supports nutritional studies, and informs aquaculture practices.

Study Objectives and Overview

This study aimed to develop a novel supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) method for the simultaneous quantitation of 17 vitamins (eight water-soluble and nine fat-soluble) in fish samples. By employing a single chromatographic system, it overcomes limitations of traditional dual-method approaches.

Methodology

• Sample preparation: Separate extraction workflows for water-soluble vitamins (WSV) and fat-soluble vitamins (FSV) using combinations of methanol, hexane, ethyl acetate, and acidified solvents. Final extracts diluted and filtered prior to analysis.
• Chromatography: Raptor Biphenyl column (100×2.1 mm, 2.7 μm) with gradient elution of supercritical CO₂ and methanol (0.1% ammonium formate), total run time 6.5 min.
• Detection: Electrospray ionization in positive mode, multi-reaction monitoring (MRM) with three transitions per analyte for quantitation and confirmation.

Used Instrumentation

• Nexera UC SFC system.
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer.

Key Results and Discussion

• Achieved simultaneous baseline separation of 17 vitamins in 6.5 min.
• Limits of quantitation ranged from 0.05 to 13.5 ng/mL in matrix with linearity (R² ≥ 0.996) over relevant concentration ranges.
• Extraction recoveries in fish matrix between 52.9% and 134.3%; matrix effects evaluated, with strong enhancement for vitamin B1 and suppression for several fat-soluble vitamins.
• Repeatability (RSD) below 20% for most vitamins at 50 ng/mL, except vitamin K2 (~22%).
• Application to non-spiked seabass detected vitamins B1, B5, B6, B3 acid, B3 amide, E, and E acetate with concentrations from 0.004 to 12.7 μg/g.

Benefits and Practical Applications

This rapid SFC-MS/MS method streamlines vitamin profiling in fish, reducing analysis time and solvent consumption. It supports food industry QA/QC, nutritional research, and regulatory monitoring of dietary supplements and aquaculture products.

Future Trends and Potential Applications

• Expansion to other food matrices and complex biological samples.
• Integration with high-throughput screening for nutritional databases.
• Adaptation to emerging vitamins and metabolites using advanced SFC stationary phases.

Conclusion

A robust, high-speed SFC-MS/MS technique has been established for comprehensive analysis of water- and fat-soluble vitamins in fish. The method exhibits strong performance in terms of sensitivity, reproducibility, and matrix handling, offering a valuable tool for nutritional and quality control laboratories.

References

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