Validation of a new fully automated assay of Thiamine-PyroPhosphate and Pyridoxal-5’-Phosphate in whole blood using LC-MS/MS

Importance of the topic

Thiamine Pyrophosphate (TPP) and Pyridoxal-5’-Phosphate (PLP) are the active forms of vitamins B1 and B6 and serve as essential coenzymes in energy metabolism and amino acid transformations. Deficiencies in these analytes can arise from inadequate diet, malnutrition, disease states or pharmacological treatments, underscoring the need for reliable monitoring in clinical settings.

Objectives and study overview

The study aimed to establish and validate a fully automated, high-throughput UHPLC-MS/MS assay for quantifying TPP and PLP in whole blood. The focus was on minimizing sample preparation steps while achieving analytical performance suitable for routine clinical diagnostics.

Methodology and Instrumentation

• Sample preparation was performed on a CLAM-2030 online with a Shimadzu Nexera X3 UHPLC system coupled to an LCMS-8050 triple quadrupole mass spectrometer.
• Blood samples were frozen, thawed, subjected to protein precipitation, filtration and transferred automatically for analysis.
• Chromatography used an XBridge BEH C18 column (3.0 x 75 mm) at 30 °C with a binary gradient of aqueous and organic mobile phases at 0.6 mL/min and 20 μL injection volume.
• Mass spectrometry was conducted in positive ESI mode with fast polarity switching and MRM transitions optimized for TPP, PLP and their deuterated internal standards.

Main results and discussion

• Linearity was excellent for both analytes over clinically relevant ranges (r2 > 0.999 for TPP 12–341 nmol/L and PLP 17–1592 nmol/L).
• Intra- and inter-assay precision at low, high and pooled concentrations yielded CVs below 4% for both TPP and PLP.
• Accuracy studies showed recoveries within ±3.4% across concentration levels.
• Correlation between automated and manual sample preparation demonstrated strong agreement using Passing-Bablock regression.

Benefits and practical applications

The fully automated workflow reduces hands-on time and human error, enabling high sample throughput and consistent data quality. This method supports clinical laboratories in routine monitoring of vitamin status for patient care and research applications.

Future trends and potential applications

• Integration with laboratory information management systems to streamline data handling.
• Multiplexing additional water-soluble vitamins and metabolites in a single run.
• Adaptation for point-of-care devices or decentralized testing environments.
• Implementation of high-resolution mass spectrometry for improved specificity.

Conclusion

The validated UHPLC-MS/MS assay with automated sample preparation provides a reliable, accurate and efficient tool for quantifying TPP and PLP in whole blood, addressing the growing demand for vitamin analysis in clinical diagnostics.