NIACIN (VITAMIN B3) - HPLC Analysis via Photochemical Post-Column Derivatization

Significance of the Topic

Niacin (vitamin B3) plays a pivotal role in energy metabolism as a precursor to NAD+ and NADP+. Its stability under heat, light, and oxygen makes it a reliable marker for nutritional analysis.

Objectives and Study Overview

This study presents an HPLC method with photochemical post-column derivatization for sensitive and selective quantification of nicotinic acid in food, optimized from EN 15652:2009. Three hydrolysis options (acidic, enzymatic, and acidic/alkaline) are evaluated to liberate niacin from complex matrices.

Methodology and Instrumentation

The protocol involves:

• Sample preparation by acidic, enzymatic, or combined acidic/alkaline hydrolysis to convert nicotinamide to nicotinic acid.
• Isocratic HPLC on a Phenomenex Luna 5 µm C18 column (150 × 2.0 mm) at 30–40 °C.
• Mobile phase: 0.035 mol/L KH2PO4 aqueous buffer, degassed by helium or vacuum.
• Flow rate of 1.0 mL/min and injection volume of 20 µL.
• Post-column UV photochemical reactor (254 nm, 1 mL volume) to generate fluorescent derivatives.
• Fluorescence detection at 322 nm (excitation) and 380 nm (emission) with a pressure-resistant cell.

Main Results and Discussion

The optimized method delivers reproducible retention times and baseline separation of niacin derivatives. Photochemical derivatization yields high sensitivity and low background noise. Alkaline treatment ensures full conversion of nicotinamide to nicotinic acid, simplifying quantification and minimizing matrix effects.

Benefits and Practical Applications

• Chemical-free derivatization enhances safety and reduces reagent costs.
• Flexible hydrolysis options adapt to various food matrices.
• Compliance with EN 15652 ensures regulatory acceptance.
• Well-suited for routine quality control and nutritional labeling in food laboratories.

Future Trends and Potential Applications

Emerging developments include integration with mass spectrometry for structural confirmation, miniaturized photochemical reactors for greener analysis, and automation for high-throughput screening in industrial contexts.

Conclusion

This HPLC method with photochemical post-column derivatization offers a robust, sensitive, and standardized approach for niacin determination in food, meeting international quality standards and supporting reliable nutritional assessment.

Instrumentation Used

• HPLC system with isocratic pump and autosampler.
• Phenomenex Luna 5 µm C18 column (150 × 2.0 mm).
• Photochemical reactor UVE, 1 mL, 254 nm.
• Fluorescence detector (Exc. 322 nm, Em. 380 nm, up to 7 bar).

Reference

1. European Committee for Standardization EN 15652:2009. Detection of Vitamin B3 (Niacin) by HPLC.
2. Direct notice by Kantonales Laboratorium Thurgau, Switzerland.