Bertrand van Zelst (Presenter)
Erasmus MC
Authorship: Bertrand van Zelst, Patrick Kooij, Yolanda de Rijke
Department of Clinical Chemistry, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, the Netherlands
| Short Abstract An LC-ESI-MS/MS method is described for the quantification of thiamine-pyro-phosphate (TPP) and pyridoxal-5-phosphate (PLP), the biologically active forms of vitamin-B1 and vitamin-B6. A linear gradient of 0.1% formic acid/methanol was used for simultaneous separation on a Symmetry C18 column. The method was linear until 5000 nmol/L for both analytes at an LLOQ of 12 nmol/L for TPP and 6 nmol/L for PLP. Inter-day and intra-day precision were <10.4% (TPP), <5.5% (PLP) and 5.5% (TPP), <3.8% (PLP) respectively. The relative matrix effect was 96.7% for TPP and 93.2% for PLP. Method-comparison of patient samples showed a correlation of y=1.01x-1.6 for PLP and y=1.00x+16.0 for TPP. |
Long Abstract
Introduction:
Vitamin B1 is used in several decarboxylation- and transketolase reactions and is also involved in the generation of ATP. Deficiency of vitamin B1 can lead to neurological diseases like beri-beri and Korsakoff syndrome. Vitamin B6 is a cofactor in numerous biologic processes such as amino acid metabolism. Malnutrition may lead to vitamin B6 deficiciency resulting in anemia, pellagra, and changes in mental status. The aim of this study was to develop and validate a method to simultaneously measure the concentration of the biologically active forms of vitamin B1 (thiamine-pyro-phosphate, TPP) and vitamin B6 (pyridoxal-5’-phosphate, PLP) in whole blood using an LC-ESI-MS/MS method.
Methods:
A stable isotope (TPP-d3 & PLP-d3) was added to the samples, followed by deproteinization with 10% TCA. After centrifugation, 20 µL of the supernatant was injected into the LC-ESI-MS/MS. Reversed phase chromatography was performed on a UPLC system using a WatersTM Symmetry C18 column with a gradient of 0.1% formic acid in methanol. TPP and PLP were measured on a Quattro Premier XE tandem MS with the mass transitions of 424.6>121.5 (TPP) and 247.8>149.8 (PLP) in the positive ion mode.
Results:
The chromatographic run lasted 2 minutes. The method was linear from 0-5000 nmol/L for TPP & PLP. The intra-assay CVs were 5.5% and 3.8% for TPP and PLP respectively. The inter-assay CVs for TPP and PLP were 10.4% and 5.5%. The relative matrix-effect was 96.7% for TPP and 93.2% for PLP. The mean recoveries for TPP and PLP were 98.8% and 94.0%. The lower limit of quantification was 12 nmol/L for TPP and 6 nmol/L for PLP. The comparison of the new LC-ESI-MS/MS method with our current LC-ESI-MS/MS method yielded the following equation for PLP: new = 1.01 [0.98-1.04] x old – 1.58 [-4.04-0.67] (r2=0.99). The comparison of the new LC-ESI-MS/MS method with our current HPLC-FL method for total vitamin B1 resulted in the following: new = 1.00 [0.89-1.12] x old + 16.0 [2.6-28.2] (r2=0.82).
Conclusion:
This LC-ESI-MS/MS method is characterized by simple sample processing and a short run time. The comparison with the current methods is excellent although a significant positive bias was detected for the comparison of TPP vs. total thiamine. This bias will be addressed in future investigations.
References & Acknowledgements:
The authors would like to acknowledge the assistance of Lisa Calton, Robert Wardle and Peter Batjoens (Waters Corporation).
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