Sensitive determination of vitamin B6 in blood samples via fluorescence detection

Significance of the Topic

The precise determination of vitamin B6 in blood is essential for assessing nutritional status and diagnosing metabolic disorders. Plasma pyridoxal 5′-phosphate levels correlate with tissue concentrations and lower nanomolar detection limits are often required for clinical studies.

Objectives and Study Overview

This study demonstrates a rapid UHPLC method integrating bisulfite derivatization within the mobile phase and fluorescence detection to quantify vitamin B6 in blood samples in under five minutes without postcolumn derivatization.

Methodology and Instrumentation

Sample preparation involved deproteinization of EDTA blood, centrifugation, and filtration; pure and spiked samples were analyzed alongside calibration standards (26–260 nmol per liter). The separation used a ProntoSIL 120-5 C18 AQ column (125 × 3 mm, 5 µm) with mobile phase A composed of phosphate buffer, perchlorate, and bisulfite in water/acetonitrile (97:3 v:v) and acetonitrile as mobile phase B. A linear gradient was applied at 1.2 milliliters per minute and detection was performed by fluorescence (excitation 290 nm, emission 400 nm).

Main Results and Discussion

The method achieved baseline separation of vitamin B6 with no postcolumn derivatization pump. Repeatability over five injections yielded relative standard deviations below 1 percent for retention time and peak area. Calibration curves were linear (r2 > 0.999) over 26 to 260 nanomolar, with a limit of detection of 1 nanomolar and quantification at 3 nanomolar. A brief washing step removed matrix components following analyte elution.

Benefits and Practical Applications

The approach offers rapid analysis, low detection limits, high robustness, and simplified workflows suitable for routine clinical laboratories and hospitals.

Future Trends and Applications

Further speed enhancements can be realized using shorter columns, smaller particle sizes, or miniaturized flow systems. Integration with automated platforms and expansion to related vitamers could broaden clinical and research applications.

Conclusion

The presented UHPLC-fluorescence method provides a fast, sensitive, and reliable tool for vitamin B6 analysis in blood, eliminating the need for separate derivatization and meeting clinical throughput demands.

References

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