Ion pair liquid chromatography method for the determination of thiamine (vitamin B1) homeostasis

Abstract

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 C18 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 nM for both cells and the media where complete recovery of the analytes was also achieved.

Vitamin B1 in critically ill patients: needs and challenges

“…A survey of participants enrolled in the RCPAQAP whole-blood programme revealed that only one of nine respondents included an internal standard [22]. Furthermore, from our literature search, only four publications reported the use of an internal standard [109,125,128,129].…”

“…Separation can be accomplished using gradient or isocratic techniques, with or without ion-pairing chemicals and on a range of columns including octadecyl carbon chain silica based (C 18 ), polymer, or amino. Sample preparation procedures include cloud point extraction (using the cloud phenomena of surfactants), solid phase extraction and derivitisation (which can occur pre-or post-column) [112,[127][128][129]. There are also an array of mobile phase solutions and chemicals for sample preparation with variations in pH levels from 2 to 13, even when identical chemicals are utilised.…”

“…Non-isotopic internal standards, acetylaneurine, pyrithiamine and theobromine, were used in the three HPLC-florescent published methods [125,128,129]. Only one of the three LC-MS/MS publications cited the use of an isotopic labelled (d3) internal standard [109].…”

Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue

“…In the present study, all samples were injected into the chromatographic system within 1 min. Comparing to data obtained by other study, 30 min can be considered a lower stability [41]. They found that TDP, TMP and B1 kept in a cell culture after derivatization were stable until 3 h, at room temperature.…”

“…One possible explanation for the difference between these results could be not only the kind of samples, cells and tissues, but also light effects on the compound stability. The derivatized samples were stored in the dark at room temperature [41]. Further systematic studies have to be done to test this hypothesis.…”