A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood
Introduction
Pyridoxal-5-phosphate (P5P) and thiamine pyrophosphate (TPP) are the active coenzymes of the vitamins, B6 and B1 respectively. These water soluble vitamin derivatives play an important role in the metabolism of a whole suite of compounds including other vitamins, lipids, amino acids and in the Krebs Cycle for energy production as well [1]. Malnutrition and deficiencies in these compounds has been linked with several disease profiles one notable of which is cognitive function in the elderly [2]. Hence there is general interest in their analysis and the monitoring of their levels.
Recently, a variety of methods have been proposed for the analysis of both P5P and TPP by way of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [1], [3], [4]. The main advantage of this platform is the ability to have shorter run times, greater specificity and removes the need for derivatisation that is required for their analysis on traditional fluorescence based methods. However, for many laboratories that currently or wish to perform P5P and TPP testing, this may not be a feasible option due to financial constraints and/or lack of expertise in using LC-MS/MS. Hence, the majority of the analysis that is performed is done by conventional liquid chromatography with fluorescence detection (HPLC-FLD) and is demonstrated by 85% of participants in the 2020 Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) for whole blood vitamins using this method. As P5P and TPP do not inherently fluoresce a derivatisation step is required for their analysis. TPP detection is achieved by derivatisation to a thiochrome product with the use of alkaline potassium ferricyanide [5], [6], [7], [8]. For P5P the three main forms of derivatisation are its conversion to a semi-carbazone [9], [10], [11], with the use of cyanide [12], [13] or with post column infusions of chlorite [14], [15].
To our knowledge, there exist only two methods which have been reported allowing for the combined detection of both P5P and TPP via fluorescence. The first method is a readily available commercial kit supplied by the company, CHROMSYSTEMS which also produces independent assays for vitamin B1 and B6 separately [16]. As these are commercial kits, the reagents used are unknown. The other method is a recent application note produced from Phenomenex using a combination of a thiochrome and semi-carbazone derivative [17]. However, for the latter no correlations or participation in an external quality assurance (EQA) program was described to further assess performance. Therefore, due to the limited number of available combined assays, in this investigation, our aim was to develop a HPLC-FLD method that allows for the simultaneous analysis of both P5P and TPP within the one sample. We also wanted to compare our method’s performance against the CHROMSYSTEMS method and an EQA program.
Section snippets
Chemicals and reagents
P5P, TPP, trichloroacetic acid (TCA), potassium cyanide, potassium ferricyanide and dipotassium phosphate was obtained from Sigma-Aldrich (Castle Hill, Australia). The derivatisation mix, containing 10 mM potassium cyanide and 0.1% potassium ferricyanide in phosphate buffer (pH 8) was made fresh each assay. Good laboratory practice and extra caution was taken when using the cyanide containing compounds.
The Methanol used was Merck LiChrosolv Hypergrade for LC–MS or equivalent (Bayswater,
Results
Fig. 1 displays a variety of chromatograms for the compounds that were analysed. In Fig. 1A, which represents an example chromatogram of our method, an acceptable level of retention and baseline resolution is achieved for both analytes of interest (void volume: 0.40 min, P5P: 1.1 min and TPP: 3.0 min). No interfering peaks are seen at the retention times of the analytes of interest. Fig. 1B and 1C are example chromatograms of samples but where the mobile phases are at fixed pHs (3.5 and 7.5
Discussion
In this paper, we have developed a novel method for the quantification of P5P and TPP in whole blood using HPLC-FLD. This developed method has met, to a satisfactory extent, the performance parameters concerning precision and recovery. Furthermore the accuracy of the method was validated by successful results in a recent cycle from an EQA program and correlated well against the pre-existing method.
The mean bias of the assay against the commercial kit was more prominent for P5P than TPP
Conclusion
We present a novel method for the combined analysis of the vitamin B1 and B6 coenzymes, TPP and P5P using HPLC-FLD. This method can be considered as a more economical alternative in routine diagnostic laboratories that may not have the resources or expertise in establishing a LC-MS/MS method or would like to pursue an in-house method over the currently available commercial kit based assays.
CRediT authorship contribution statement
Van Long Nguyen: Conceptualization, Data curation, Investigation, Methodology, Validation, Writing - original draft, Writing - review & editing. Mustafa Darman: Conceptualization, Data curation, Investigation, Methodology, Validation. Andrea Ireland: Investigation, Methodology, Validation. Michael Fitzpatrick: Conceptualization, Data curation, Investigation, Methodology, Validation, Writing - review & editing.
Acknowledgements
The authors would like to acknowledge staff at the department of Chemical Pathology, Royal Prince Alfred Hospital with special mentions to the laboratory manager, Robert McQuilty and Director, Professor David Sullivan. We would also like to acknowledge the RCPAQAP for use of their proficiency testing data.
Ethical approval
The work described in this manuscript was a method comparison study. The samples used were from routine testing conducted within the laboratory department and hence ethical approval was not required.
References (18)
- et al.
Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI-MS/MS
J. Chromatogr. B
(2017) - et al.
A novel LC-MS/MS assay for vitamin B1, B2 and B6 determination in dried blood spots and its application in children
J. Chromatogr. B
(2019) - et al.
The analysis of thiamin and its derivatives in whole blood samples under high pH conditions of the mobile phase
J. Chromatogr. B
(2005) - et al.
Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status
J. Chromatogr.
(1985) - et al.
Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation
J. Chromatogr. B
(2003) - et al.
Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay
Anal. Biochem.
(2002) - et al.
Clinical analysis of vitamin B(6): determination of pyridoxal 5'-phosphate and 4-pyridoxic acid in human serum by reversed-phase high-performance liquid chromatography with chlorite postcolumn derivatization
Anal. Biochem.
(2004) - et al.
Simultaneous determination of underivatized vitamin B1 and B6 in whole blood by reversed phase ultra high performance liquid chromatography tandem mass spectrometry
PLoS ONE
(2015) - et al.
A complex interplay of vitamin B1 and B6 metabolism with cognition, brain structure, and functional connectivity in older adults
Front. Neurosci.
(2017)