Analysis of Vitamins Using an SFC/UHPLC Hybrid System with a Triple Quadrupole LC/MS for Quantification

Significance of the Topic

Food and agricultural testing demands reliable quantification of vitamins in complex matrices. A single platform capable of analyzing both fat- and water-soluble vitamins enhances laboratory efficiency, reduces instrument footprint, and delivers orthogonal separation mechanisms crucial for thorough compositional analysis.

Objectives and Study Overview

The study evaluates an Agilent 1260 Infinity II SFC/UHPLC hybrid system coupled with an Agilent 6470A triple quadrupole LC/MS to quantify seven fat-soluble and eight water-soluble vitamins. Fat-soluble vitamins are separated under supercritical fluid chromatography (SFC) conditions, while water-soluble B-vitamins are analyzed by reversed-phase UHPLC. Performance metrics such as sensitivity, precision, and linearity are benchmarked against dedicated standalone systems.

Instrumentation Used

• Agilent 1260 Infinity II SFC/UHPLC Hybrid System (SFC control module, binary and quaternary pumps, multisampler, diode array detector, multicolumn thermostat, automated valve drives, isocratic pump with SFC/MS splitter)
• Agilent 6470A Triple Quadrupole LC/MS with Jet Stream technology operated in dynamic MRM mode
• Columns: ZORBAX SB-C18 (3.0×100 mm, 1.8 µm) for SFC; ZORBAX SB-Aq (3.0×100 mm, 1.8 µm) for UHPLC
• Software: OpenLab CDS ChemStation; MassHunter Data Acquisition, Qualitative, Quantitative, and MRM optimizer

Main Results and Discussion

• SFC Mode (fat-soluble vitamins): All seven analytes eluted within 1.0–2.8 min using a four-minute gradient with CO₂/methanol modifier. LOQs ranged from 2 to 15 ppb, retention time RSD ≤0.25 %, area RSD ≤3.8 %, and correlation coefficients >0.999.
• UHPLC Mode (water-soluble vitamins): Eight B-vitamins were resolved in a seven-minute gradient on an SB-Aq column with water/ammonium formate and methanol phases. LOQs spanned 0.3 to 10 ppb, retention time RSD ≤0.24 %, area RSD ≤2.7 %, and linearity >0.999.
• The hybrid system’s performance matched that of standalone SFC and UHPLC instruments, enabling seamless mode switching without compromising data quality.

Benefits and Practical Applications

• Streamlined workflow enabling sequential SFC and UHPLC analyses on a single platform
• Enhanced laboratory throughput and reduced maintenance requirements
• Applicable to food safety, nutritional quality control, and agricultural product screening
• High sensitivity and precision support trace-level vitamin quantification in complex samples

Future Trends and Potential Applications

• Integration with automated multidimensional chromatography for expanded analyte coverage
• Adoption of advanced ionization techniques and high-resolution mass spectrometry for improved selectivity
• Real-time online monitoring in manufacturing and bioprocessing environments
• Extension to other micronutrients and bioactive compounds in food and environmental matrices

Conclusion

The Agilent 1260 Infinity II SFC/UHPLC hybrid system coupled with a triple quadrupole LC/MS delivers reliable, high-throughput quantification of both fat- and water-soluble vitamins. This approach achieves low detection limits, excellent precision, and linearity equivalent to standalone instruments, offering a versatile solution for comprehensive vitamin analysis.

References

• Naegele E. Orthogonal Chromatographic Separations using the Agilent 1260 Infinity II SFC/UHPLC Hybrid System. Agilent Technologies Application Note 5991-8276EN, 2017.
• Rambla-Alegre M.; Dunkle M.N.; Vanhoenacker G.; David F.; Sandra P.; Vollmer M. Analysis of Antioxidants in vegetable oils using the Agilent 1260 Infinity II SFC/UHPLC Hybrid System with MS detection. Agilent Technologies Application Note 5991-1546EN, 2017.