Simultaneous Analysis of Water-soluble and Fat-soluble Vitamins in Fish by Reversed-Phase LC-MS/MS Method

Significance of the Topic

Vitamins are essential micronutrients that humans cannot synthesize and must obtain through diet. Fish serve as a rich source for both water-soluble (B-group) and fat-soluble (A, D, E, K) vitamins. Traditional analyses often require separate chromatographic methods for each vitamin class. A unified, sensitive, and rapid method for simultaneous quantification enhances efficiency in food quality control, nutritional research, and regulatory compliance.

Study Objectives and Overview

This work aimed to develop a single reversed-phase LC-MS/MS method capable of quantifying 17 vitamins (9 water-soluble and 8 fat-soluble) in fish matrices. The approach sought to streamline sample preparation and chromatographic separation, allowing comprehensive profiling in a 15-minute run.

Methodology and Instrumentation

Sample Preparation:

• Fish samples (red snapper and seabass) were homogenized, frozen, and thawed prior to extraction.
• Water-soluble vitamins were extracted from 100 mg of homogenate using methanol, followed by hexane cleanup.
• Fat-soluble vitamins were extracted from 1 g of homogenate using ascorbic acid in methanol/water and hexane/ethyl acetate mixtures, then evaporated and reconstituted.

Chromatography and Detection:

• Shimadzu LCMS-8060 triple quadrupole with ESI in positive MRM mode.
• Kinetex PFP column (150×2.1 mm, 2.6 µm) at 40 °C, 0.4 mL/min flow.
• Gradient elution: 3% to 95% methanol (0.1% formic acid) over 15 min.
• MRM transitions optimized for quantifier and qualifiers, with Ar collision gas and nitrogen nebulizing, drying, and heating gases.

Main Results and Discussion

Calibration curves in fish matrix showed excellent linearity (R²>0.995) from low ng/mL to 100 ng/mL. Water-soluble vitamins achieved LODs of 0.01–0.15 ng/mL and LOQs of 0.02–0.45 ng/mL, while fat-soluble vitamins had LODs of 0.04–1.56 ng/mL and LOQs of 0.11–4.72 ng/mL. Repeatability (%RSD) ranged from 0.7% to 9.9%. Recovery studies yielded 45–66% for water-soluble and 61–100% for fat-soluble vitamins, with some matrix suppression observed for certain fat-soluble compounds. Application to seabass demonstrated detection of seven vitamins, confirming method robustness and sensitivity.

Benefits and Practical Applications

The proposed method reduces analysis time and solvent consumption by combining two extraction protocols and a single chromatographic run. It is suited for routine nutritional profiling, quality control in aquaculture, and compliance testing of fish‐derived products.

Future Trends and Opportunities

Further optimization may focus on enhanced extraction efficiency for challenging vitamins and expanded application to diverse food matrices. Coupling with high-resolution MS or automation could improve throughput and profiling of additional micronutrients.

Conclusion

A validated reversed‐phase LC-MS/MS method allows simultaneous quantification of 17 vitamins in fish with high sensitivity, precision, and throughput, supporting comprehensive nutritional assessments and quality control.

Reference

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