Simultaneous Detection and Quantitation of 14 Fat-Soluble Vitamins and Carotenoids by LC/MS/MS Triple-Quadrupole

Importance of the Topic

Fat-soluble vitamins and carotenoids play critical roles as antioxidants and essential nutrients in human health. Accurate analysis of these compounds in dietary supplements ensures product quality, label claim verification, and consumer safety.

Objectives and Study Overview

This study presents a single method for simultaneous detection and quantitation of 14 fat-soluble vitamins and selected carotenoids in a complex multivitamin tablet. Using triple quadrupole LC/MS/MS, the method was evaluated for sensitivity, specificity, and accuracy to support quality control and nutrition label establishment.

Methodology

Samples were ground and extracted with DMSO followed by ethanol, centrifuged, and diluted. Chromatographic separation was achieved on a reversed phase column with a 12 min gradient using 0.1% formic acid in water and methanol. Detection employed positive electrospray ionization in dynamic MRM mode. Calibration covered 0.2 to 500 ng/mL using isotopically labeled internal standards or postmatrix spiking to correct for matrix effects. Method performance criteria included retention time tolerance, ion ratio, calibration linearity R2 > 0.99, and recovery between 70 and 130%.

Instrumentation

• Agilent 1290 Infinity II LC system with multisampler G7167B, high speed pump G7120A, and multicolumn thermostat G7116B
• Agilent 6470 triple quadrupole LC/MS (G6470B) with Jet Stream ESI source
• Agilent MassHunter Workstation and Quantitative Analysis software (v10.1 to 10.2)

Main Results and Discussion

Using methanol as the organic modifier improved ionization compared to acetonitrile, enhancing sensitivity. The method provided clear separation and no carryover for all analytes. Calibration curves showed residuals below 30% and R2 values above 0.99. Matrix-matched spiking confirmed accurate quantitation. Analysis of a commercial multivitamin tablet demonstrated all claimed fat-soluble vitamins met or exceeded label values, with postspike recoveries within acceptance.

Benefits and Practical Applications

• Single-run analysis reduces assay complexity and time
• Enhanced sensitivity and selectivity compared to traditional LC-UV or microbiological assays
• Reliable quality control and support for nutrition label claims in supplement production

Future Trends and Opportunities

Further integration of automated sample preparation, expansion to water-soluble vitamin panels, coupling with high-resolution mass spectrometry, and online extraction techniques can broaden scope and efficiency. Harmonization of methods across regulatory bodies will support broader adoption.

Conclusion

The developed LC/MS/MS method enables rapid, sensitive, and accurate simultaneous quantitation of 14 fat-soluble vitamins and carotenoids in a complex supplement matrix. Its high throughput, specificity, and robust performance make it well suited for routine quality control and nutritional labeling.

References

• National Research Council Diet and Health Implications for Reducing Chronic Disease Risk The National Academies Press 1989
• Young A J Lowe G L Carotenoids Antioxidant Properties Antioxidants 2018 7 28
• Agilent Technologies Simultaneous Detection and Quantitation of 14 Water-Soluble Vitamins in a Supplement by LC/MS/MS Triple-Quadrupole Application Note 5994-3016EN 2021