Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column

F. Batifoulier; M. A. Verny; C. Besson; C. Demigné; C. Rémésy

doi:10.1016/j.jchromb.2004.11.004

Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column

F. Batifoulier, M. A. Verny, C. Besson, C. Demigné, C. Rémésy

Pharmacology and Nutritional Sciences

Research output: Contribution to journal › Article › peer-review

53 Scopus citations

Abstract

A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO₄) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90-111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (-67%), brains (-50%), kidneys (-60%), followed by a complete recovery upon repletion.

Original language	English
Pages (from-to)	67-72
Number of pages	6
Journal	Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume	816
Issue number	1-2
DOIs	https://doi.org/10.1016/j.jchromb.2004.11.004
State	Published - Feb 25 2005

Bibliographical note

Funding Information:
This project has been sponsored by the French Ministry of Industry and Finance (Program ASG “Cereal Genotyping and Food Quality” no. 01.04.90.6058). Thanks to all the members and collaborators.

Keywords

RP-amide C16 column
Thiamine
Thiochromes

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry
Clinical Biochemistry
Cell Biology

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10.1016/j.jchromb.2004.11.004

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title = "Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column",

abstract = "A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO4) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90-111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (-67%), brains (-50%), kidneys (-60%), followed by a complete recovery upon repletion.",

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AU - Batifoulier, F.

AU - Verny, M. A.

AU - Besson, C.

AU - Demigné, C.

AU - Rémésy, C.

N1 - Funding Information: This project has been sponsored by the French Ministry of Industry and Finance (Program ASG “Cereal Genotyping and Food Quality” no. 01.04.90.6058). Thanks to all the members and collaborators.

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AB - A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO4) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90-111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (-67%), brains (-50%), kidneys (-60%), followed by a complete recovery upon repletion.

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Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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