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Ion pair liquid chromatography method for the determination of thiamine (vitamin B1) homeostasis
Author(s) -
Basiri Babak,
Sutton James Michael,
Hanberry Bradley S.,
Zastre Jason A.,
Bartlett Michael G.
Publication year - 2016
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3544
Subject(s) - chemistry , chromatography , thiamine , thiamine pyrophosphate , tetrabutylammonium hydroxide , reagent , analyte , high performance liquid chromatography , calibration curve , detection limit , biochemistry , cofactor , organic chemistry , enzyme
A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion‐pairing agent. The separation was performed on a Phenomenex Kinetex EVO C 18 column with a gradient of a phosphate‐buffered aqueous solution of the ion‐pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0–4000 n m for both cells and the media where complete recovery of the analytes was also achieved. Copyright © 2015 John Wiley & Sons, Ltd.