Vitamin status B1, B2 and B6

Importance of the topic

B-vitamins such as B1, B2 and B6 serve as essential coenzymes in energy metabolism, neurotransmitter synthesis and redox reactions. Monitoring their status in whole blood enables early detection of deficiencies linked to neuropathy, diabetes complications and cognitive impairment, supporting timely intervention.

Objectives and overview

This study presents a validated LC-MS/MS workflow using a complete kit for simultaneous quantification of B1 (TPP), B2 (FAD) and B6 (PLP, PL) in human whole blood. The focus is on simplifying sample preparation, ensuring analytical accuracy and providing a ready-to-use solution for routine clinical laboratories.

Methodology and instrumentaion

The method relies on one-step protein precipitation using reagent P containing stable isotope-labelled internal standards (d3-TPP, 13C-FAD, d3-PLP, d3-PL). Key features:

• Sample volume: 50 µl whole blood
• Precipitation with 100 µl reagent P; mix and centrifuge
• Instrumentation: LC-MS/MS with triple quadrupole detector
• Chromatography: included analytical column; autosampler wash solution; mobile phases A and B
• Calibration: four-level calibrators; two-level lyophilized controls

Main results and discussion

The assay demonstrates clear chromatographic separation with retention times of 1.42 min (TPP), 1.47 min (PLP), 1.55 min (PL) and 2.46 min (FAD). Quantification shows linear response over a broad concentration range with signal intensities up to 1.4×106 cps. The one-step preparation reduces turnaround time and minimizes matrix effects, yielding precise and accurate results.

Benefits and practical applications

• Rapid, cost-effective workflow suitable for high-throughput clinical analysis
• Comprehensive kit reduces method development and validation efforts
• Stable isotope standards ensure robust quantification
• Applicable in diagnostic settings for neuropathy, diabetes and nutritional assessment

Future trends and applications

Expanding the panel to include additional vitamins and metabolites could further streamline nutritional profiling. Integration with automated sample handling and high-throughput MS platforms may enhance clinical throughput. Emerging techniques such as dried blood spot LC-MS/MS offer minimally invasive sampling options.

Conclusion

The presented LC-MS/MS assay provides a reliable and user-friendly solution for the simultaneous measurement of B1, B2 and B6 in whole blood. Its simplicity, accuracy and compliance with CE-IVD regulations make it a valuable tool for routine vitamin status monitoring in clinical laboratories.