B Vitamin Analysis in Dietary Supplements on the Agilent 1290 Infinity III LC System

Importance of the topic

Reliable quantification of B vitamins in dietary supplements is essential to verify nutritional claims, ensure consumer safety, and maintain product quality. The varied chemical structures and water solubilities of vitamins B1, B2, B5, B6, B7, B9, and B12 require a robust analytical approach that delivers consistent results across diverse supplement formulations.

Objectives and study overview

This application note describes the development and implementation of an ion-pairing reversed-phase liquid chromatography (LC) method with UV detection on the Agilent 1290 Infinity III LC System for simultaneous analysis of seven B vitamins. It also assesses the benefits of the Agilent InfinityLab Assist automation tool for system preparation (“Make Ready”) and shutdown (“Standby”) tasks in routine laboratory workflows.

Methodology and instrumentation

The method employs an Agilent Poroshell 120 EC-C18 column (2.1×100 mm, 2.7 µm) with mobile phases consisting of 50 mM sodium phosphate buffer (pH 3.07) containing 5 mM hexane sulfonate and methanol in a gradient program. Key instrumentation and software include:

• Agilent 1290 Infinity III High-Speed Pump and Multisampler with Sample Thermostat
• Agilent 1290 Infinity III Multicolumn Thermostat and Level Sensing module
• Agilent 1290 Infinity III Diode Array Detector with Max-Light Cartridge Cell
• Agilent InfinityLab Assist control software for automated tasks
• Agilent OpenLab CDS (v2.8 or later)

Sample preparation involved grinding tablets or effervescent tablets, dissolving in ultrapure water, and filtering before injection (5 µL). Vitamin standards (5 µg/mL each) were used to confirm retention times and detector response at optimized wavelengths (210 nm, 246 nm, 268 nm).

Main results and discussion

The optimized method achieved baseline separation of all target B vitamins with retention times between 3 and 9 minutes. Representative chromatograms demonstrated clear resolution in both aqueous standards and real supplement extracts. Automation of the “Make Ready” task reduced system preparation time by over 20 minutes compared to manual procedures, while maintaining equilibration reproducibility (RSD < 0.11% for standard mode). The “Standby” task configuration prevented salt precipitation by flushing buffer residues and allowed controlled shutdown of modules, further reducing idle time and solvent use.

Benefits and practical applications

• Enhanced reproducibility through automated equilibration monitoring
• Significant time savings for daily instrument setup and shutdown
• Reduced solvent consumption and energy usage during idle periods
• Minimized operator variability and errors in routine analysis
• Applicability to quality control environments in food, pharmaceutical, and supplement industries

Future trends and potential applications

Advances in LC automation and real-time monitoring are expected to further streamline high-throughput analysis of vitamins and other nutritional markers. Integration with mass spectrometry detection, predictive maintenance algorithms, and cloud-based data analytics will support method scalability, green chemistry initiatives, and remote instrument management. Such developments will broaden the applicability of automated ion-pairing LC in regulatory, clinical, and industrial settings.

Conclusion

The presented ion-pairing LC-UV method on the Agilent 1290 Infinity III system reliably quantifies seven B vitamins in diverse supplement matrices. Automation via Agilent InfinityLab Assist for “Make Ready” and “Standby” tasks delivers reproducible system preparation, significant time savings, and reduced resource consumption, enhancing laboratory efficiency and data quality.

References

1. Agilent Technologies. Best Practices for Using an Agilent LC System; Publication SD-29000194, 2016–2022.
2. Agilent Technologies. Agilent InfinityLab Assist: A Local User Interface to Control and Automate Your HPLC System; White Paper 5994-7572EN, 2024.
3. Dhale M.; Singh R.; Sharma R.; Arora S. Quantification of All B Vitamins in a Single Run Using Ion-Pair Modified Liquid Chromatography with UV Detection. Journal of Food Composition and Analysis 2023, 123, 105602.