A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood

Background and aims: The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection.

Methods: Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76).

Results: Linearity for both analytes was above 0.99 (r²) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method.

Conclusions: Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.