Simultaneous Detection and Quantitation of 14 Water-Soluble Vitamins in a Supplement by LC/MS/MS Triple-Quadrupole

Importance of the Topic

The essential roles of water-soluble B vitamins in energy metabolism, nervous system function, and cellular health demand accurate measurement methods. Dietary supplements are complex matrices that challenge traditional assays in sensitivity, specificity, and throughput, highlighting the need for advanced analytical techniques such as LC/MS/MS for reliable quality control and label compliance.

Study Objectives and Overview

This application note presents the development and validation of a single-run LC/MS/MS triple quadrupole method to simultaneously detect and quantify 14 water-soluble B vitamins in a multivitamin tablet. The goals were to achieve high sensitivity, selectivity, and speed to support routine quality assurance in supplement manufacturing.

Methodology and Instrumentation

Sample preparation involved grinding tablets, extracting with 0.1% phosphoric acid containing EDTA and vitamin C, heating, and centrifugation. Quantitation strategies used isotopically labeled internal standards, matrix-matched calibration, and standard addition to correct matrix effects and ensure accuracy.

Used Instrumentation

• Agilent 1290 Infinity II LC system with multisampler, high-speed pump, and multicolumn thermostat
• Agilent 6470 triple quadrupole MS with Jet Stream ESI source
• Agilent InfinityLab Poroshell 120 Phenyl-Hexyl column (3.0×100 mm, 2.7 μm)
• MassHunter Workstation software for acquisition and data processing

Main Results and Discussion

• Linearity was demonstrated over 0.5–500 ng/mL for all vitamins with R² > 0.99.
• Dynamic MRM provided high specificity: retention time deviation <5% and ion ratio tolerance within 30%.
• The phenyl-hexyl column delivered enhanced selectivity through hydrophobic and π–π interactions, improving peak resolution.
• Postspike recoveries ranged from 70% to 130%, confirming method accuracy; folate quantitation used standard addition to address ion enhancement.
• All 14 B vitamins in the tested supplement met or exceeded the manufacturer’s label claims.

Benefits and Practical Applications

The single-run LC/MS/MS approach significantly reduces assay time and cost while delivering robust sensitivity and specificity. It is ideally suited for quality control, regulatory compliance, and nutrition label verification in the dietary supplement industry.

Future Trends and Possibilities

• Integration of high-resolution mass spectrometry for broader vitamin and metabolite profiling.
• Automation and miniaturization of sample preparation workflows for higher throughput.
• Extension to other supplement formats, food matrices, and clinical samples.
• Application in nutritional and pharmacokinetic studies to monitor bioavailability and metabolism.

Conclusion

The validated LC/MS/MS method offers a rapid, sensitive, and accurate solution for simultaneous quantitation of multiple water-soluble B vitamins in complex supplement matrices. Its adoption can enhance laboratory efficiency, data reliability, and consumer confidence in product labeling.

References

1. Kennedy DO. B Vitamins and the Brain: Mechanisms, Dose and Efficacy—A Review. Nutrients. 2016;8(2):68.
2. Henderson JW, Long WJ. Exceptional Selectivity of Agilent ZORBAX Eclipse Plus Phenyl-Hexyl Columns to Separate Estrogens. Agilent Technologies application note. 2008.
3. Zhao H, Zulkoski J, Mastovska K. Development and Validation of a Multiclass, Multiresidue Method for Veterinary Drug Analysis in Infant Formula and Related Ingredients Using UHPLC-MS/MS. J Agric Food Chem. 2017;65(31):7268–7287.
4. Agilent Technologies. Product Information: Nature’s Way Alive Max Potency Men’s Daily Multivitamin. 2020.