Determination of Vitamin B6 in Blood Samples with Fluorescence Detection after Derivatization with Bisulfite

Significance of the topic

Vitamin B6 plays a critical role in amino acid metabolism, neurotransmitter synthesis and hemoglobin formation. Accurate determination in blood is essential for diagnosing deficiencies, monitoring therapy and ensuring patient safety.

Objectives and study overview

This study aims to develop a fast, sensitive and straightforward HPLC method with fluorescence detection for quantifying vitamin B6 in blood samples. A key feature is in-line derivatization with bisulfite, eliminating the need for post-column reagent addition.

Methodology

The method employs reversed-phase HPLC in ion-pair mode using a ProntoSIL 120-5 C18 AQ column (125×3 mm, with integrated precolumn). Mobile phase A consists of 100 mM KH2PO4, 100 mM NaClO4 and 0.5 g/L NaHSO3 in 97:3 water/ACN, and phase B is acetonitrile. A gradient from 0 to 50% B over 1.7 min, followed by re-equilibration, yields a total run time of 5 min. The flow rate is 1.2 mL/min, column temperature 30 °C, and injection volume 10 µL. Derivatization of vitamin B6 occurs directly in the mobile phase.

Used instrumentation

• HPLC system with ProntoSIL 120-5 C18 AQ column, 125×3 mm (Order No. 12XF184PSJ)
• Fluorescence detector RF-20AXs (excitation 290 nm, emission 400 nm, sensitivity high, gain ×16, acquisition rate 5 Hz, integration time 0.1 s)

Main results and discussion

The method produces a single, well-resolved peak for vitamin B6 in under 5 min, with no significant interferences from blood matrix components. Comparison of standard and sample chromatograms confirms specificity. High sensitivity and rapid equilibration support routine clinical analysis.

Benefits and practical applications

• Minimal sample preparation and no post-column reagent addition
• Short run time enabling high throughput
• High sensitivity for trace-level detection in clinical diagnostics
• Robustness and reproducibility suited for QA/QC environments

Future trends and potential applications

Potential enhancements include coupling with mass spectrometry for multiplexed assays, miniaturized column formats for portable testing and automated workflows for point-of-care diagnostics.

Conclusion

The presented HPLC-FL method with in-line bisulfite derivatization delivers a rapid, sensitive and reliable approach for vitamin B6 determination in blood, meeting the needs of clinical and quality-control laboratories.

References

No references provided.