Analysis of High- and Low-Dosed Vitamins in a Single Run Using the Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector Solution

Importance of the topic

The ability to quantify both high- and low-concentration vitamins in a single HPLC run addresses a common challenge in food analysis where main and trace components often require separate analyses. The Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector (HDR DAD) expands linear dynamic range by over 30×, enabling streamlined workflows and improved sensitivity.

Study Objectives and Overview

This application note evaluates the HDR DAD solution for simultaneous separation and quantification of seven water-soluble vitamins (C, B1, B2, B6, B12, niacinamide, pantothenic acid) in effervescent tablets. Key goals included demonstration of linearity, sensitivity and stability of vitamin B12 under typical sample conditions.

Instrumentation Used

• Agilent 1260 Infinity Binary Pump
• Agilent 1260 Infinity Standard Autosampler with cooler
• Agilent 1260 Infinity Diode Array Detectors 1 & 2 with 60 mm and 3.7 mm Max-Light flow cells
• Agilent 1260 Infinity Column Compartment (40 °C)
• Agilent OpenLAB CDS ChemStation

Methodology

Tablet samples were extracted in water/ethanol, filtered and analyzed on a ZORBAX RRHT Eclipse Plus C18 column (4.6×150 mm, 1.8 µm). A gradient of phosphate buffer (pH 3) and methanol (3→50 % over 11 min), 1.2 mL/min flow, and wavelength channels at 220, 265 and 360 nm were applied. The HDR DAD tool clusters two detectors with different path lengths and combines signals to maintain linearity from trace to main component levels.

Main Results and Discussion

The HDR DAD extended the linear range typically from 0.6×10⁻⁶ to 6.7 AU/cm, compared to conventional DAD limits of 7×10⁻⁶ to 2 AU/cm. One injection sufficed to quantify vitamins spanning four orders of magnitude: vitamin C (60 mg/tablet, 6,000 ng injected) and vitamin B12 (2 µg/tablet, 0.2 ng injected). Detector comparisons confirmed that single-cell DADs could not quantify both analytes simultaneously. A notable challenge was vitamin B12 degradation (~50 % over three injections), suggesting measures such as light exclusion and vial filling to minimize oxygen exposure.

Benefits and Practical Applications

• Combines main and trace analysis in one run, reducing time and solvent use
• Improved quantification accuracy across wide concentration ranges
• Enhanced sensitivity for low-dose analytes without compromising overload of high-dose components

Future Trends and Potential Applications

Emerging approaches may include integration of protective sample environments (e.g., inert gas), application to other complex matrices such as biological or environmental samples, and coupling with mass spectrometry for confirmatory analysis.

Conclusion

The Agilent 1200 Infinity HDR DAD effectively enables single-run simultaneous quantification of high- and low-dosed vitamins, simplifying workflows and ensuring reliable data across broad concentration ranges. Adoption of this technology can significantly enhance throughput and data quality in nutritional and food testing laboratories.

References

1. Kirchner U., Degenhardt K., Raffler G. Determination of Vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an Immunoaffinity column: First Action 2011.09. J. AOAC Int. 95(4):2012.
2. Amidzic R. et al. RP-HPLC Determination of vitamins B1, B3, B6, and B12 in multivitamin tablets. J. Serb. Chem. Soc. 70(10):1229–1235 (2005).
3. Yamada K. et al. Degradation of vitamin B12 in dietary supplements. Int J Vitam Nutr Res. 78(4–5):195–203 (2008).