Vitamin B1 and B6 have recently been included in the Dutch clinical guidelines for the general practitioner in the differential diagnosis of dementia. To keep up with the sharp rise in the number of requests, an LC–MS/MS method using stable isotopes as internal standards was developed. The active vitamers thiamine pyrophosphate (TPP) and pyridoxal-5′-phosphate (PLP) in whole blood are simultaneously measured with a short run time of 2 min. Whole blood is mixed with internal standard solution containing both TPP-d3 and PLP-d3, followed by deproteinization with a trichloroacetic acid (TCA) solution. A UPLC–MS/MS system from Waters™ was used for chromatographic separation and subsequent detection by electrospray ionization in the positive mode with mass transitions of 425.1 > 121.85 for TPP and 247.9 > 149.9 for PLP. The method is linear across the range of 12–4870 nmol/L for TPP and 6–4850 nmol/L for PLP. The mean intra-assay and inter-assay precision are 3.5% and 7.6% respectively for TPP and 3.4% and 6.1% for PLP. The relative matrix effect (TPP 97%, PLP 93%), recovery (TPP 99%, PLP 94%) and lower limit of quantification (TPP 12 nmol/L, PLP 6 nmol/L) meet the applied acceptance criteria. The comparison of the new LC-ESI–MS/MS method for TPP with our current HPLC-Fluorescence method for total thiamine yields the following equation: TPP LC–MS/MS = 0.97 × total thiamine HPLC – 10.61 (r2 = 0.94). The comparison of the new LC-ESI–MS/MS method for PLP with our current LC-ESI–MS/MS method results in PLP LC–MS/MS new = 1.01 × PLP LC–MS/MS old – 1.58 (r2 = 0.99). In conclusion, this LC–MS/MS based assay is characterized by simple sample processing with a short run time and comparison with the current methods is excellent. The new LC–MS/MS method is a convenient method to determine TPP and PLP in whole blood for both clinical routine and research applications.

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Keywords Liquid chromatography–tandem mass spectrometry (LC–MS/MS), Method validation, Pyridoxal-5′-phosphate (PLP), Thiamine pyrophosphate (TPP), Vitamin B1, Vitamin B6, Whole blood
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Journal Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Roelofsen-de Beer, R.J.A.C. (R. J.A.C.), van Zelst, B.D, Wardle, R. (R.), Kooij, P.G. (P. G.), & De Rijke, Y. (2017). Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI–MS/MS. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 1063, 67–73. doi:10.1016/j.jchromb.2017.08.011