Analysis of Thiamine Pyrophosphate and Pyridoxal-5’-phosphate in whole blood using a fully automated sample preparation LC/MS/MS system

Significance of the topic

Thiamine pyrophosphate (TPP) and pyridoxal-5’-phosphate (PLP) serve as critical coenzymes in glucose metabolism and amino acid synthesis. Rapid and reliable quantitation in whole blood is essential for diagnosing B1 and B6 deficiencies and supporting high-throughput clinical workflows.

Objectives and study overview

This study evaluates a fully automated sample preparation and LC–MS/MS workflow for simultaneous quantitation of TPP and PLP. The aim is to eliminate manual handling, reduce variability, and increase throughput while maintaining analytical performance.

Methodology and instrumentation

Whole blood (25 µL) is processed inline using the CLAM-2000 automation unit coupled to a Shimadzu Nexera X2 UHPLC and LCMS-8045 tandem quadrupole mass spectrometer. Key steps include:

• Automated protein precipitation with precipitation reagent and deuterated internal standards.
• Filtration via disposable micro-vials under vacuum.
• Injection of 20 µL into a reversed-phase Shimpack GIST C18-AQ column at 30 °C.
• ESI-MS/MS detection using optimized transitions for TPP (425.0>122.2, 425.0>303.2) and PLP (248.1>150.2, 248.1>94.2).

Results and discussion

Linearity was confirmed over the range 39–890 nmol/L for TPP and 21–550 nmol/L for PLP with R² ≈ 0.997. Accuracy for six calibrators fell within 85–115% for both analytes. Precision assessed at low, medium and high levels yielded CVs below 3%. The overlapping preparation and analysis steps deliver one result per six minutes, supporting high sample throughput.

Benefits and practical applications

The integrated CLAM-2000–LCMS-8045 platform:

• Removes manual pipetting errors and variability.
• Shortens turnaround time for clinical laboratories.
• Maintains robust sensitivity and reproducibility for routine vitamin B1/B6 assessments.

Future trends and opportunities

Further developments may include multiplexing additional water-soluble vitamins, miniaturized automation for point-of-care settings and integration with data analytics for comprehensive nutritional profiling.

Conclusion

The fully automated sample preparation LC–MS/MS system offers a rapid, reliable and high-throughput solution for quantifying TPP and PLP in whole blood, meeting the demands of modern clinical chemistry without sacrificing analytical quality.