A Reliable Semiautomated Method for the Determination of Total Thiamine in Whole Blood by the Thiochrome Method with High-Performance Liquid Chromatography

Abstract

A sensitive and reliable method for the determination of total thiamine (vitamin B₁) in whole blood has been developed which is suited for routine analysis. After extraction, and enzymatic hydrolysis of thiamine phosphate esters, thiamine is separated from interfering compounds by a fully automated high-performance liquid chromatographic (HPLC) system. Thiamine is detected fluorometrically as thiochrome. By calculating the concentration of thiamine on-line with the aid of a computer, it is possible to complete one analysis within four hours. Routine thiamine determinations can be carried out in a series of 120 samples within 48 hours.

The within-assay and between-assay coefficients of variation of the analysis of total thiamine in whole blood were 4·2 and 4·4%, respectively. The between-assay analytical recovery of thiamine diphosphate added to blood samples was 99·9 ± 11·7% (mean ± SD). The HPLC method described has been applied also to the analysis of thiamine in plasma and erythrocytes. In agreement with other reports, it was found that about 80% of total thiamine of whole blood is present in the erythrocytes. Reference values of thiamine in whole blood of the human were found in the range 95–155 nmol/l.

References

Sauberlich

. Biochemical alterations in thiamine deficiency. Their clinical interpretation. Am J Clin Nutr 1967; 20: 528–42.

Sauberlich

Dowdy

Skala

. Laboratory tests for the assessment of nutritional status. Critical Reviews in Clinical Laboratory Sciences. 1974; 4: 22–30.

Brin

. Erythrocyte as a biopsy tissue for functional evaluation of thiamine adequacy. JAMA 1964; 187: 186–96.

Leveille

. Modified thiochrome method for the determination of urinary thiamine. Am J Clin Nutr 1972; 25: 273–4.

Difco supplementary literature 0247. Difco Labs, Detroit, USA. January 1977.

van de Weerdhof

Wiersum

Reissenweber

. Application of liquid chromatography in food analysis. J Chromatogr 1973; 83: 455–60.

Pentinnen

. Differences in thiochrome fluorescence produced by thiamine and its mono-, di-, and triphosphate esters. Acta Chem Scand 1976; B30: 659–63.

Haugen

. The determination of thiamine and thiamine phosphates in blood and tissues by the thiochrome method. Scand J Clin Lab Invest 1961; 13: 50–6.

Bergmeyer

. Methoden der enzymatische Analyse Verlag Chemie, Weinheim, 1974; 3e Auflage: 335.

10.

Baker

Thomson

Frank

Leevy

. Absorption and passage of fat- and water-soluble thiamin derivatives into erythrocytes and cerebrospinal fluid of man. Am J Clin Nutr 1974; 27: 676–80.

11.

Smits

. PhD thesis, University of Utrecht, The Netherlands, 1949.

12.

Rümke

Bezemer

. Methoden voor de bepaling van normale waarden. II. Nieuwe methoden. Ned Tijdschr Geneeskd 1972; 116: 1559–68.

13.

Gregory

. Comparison of high-performance liquid chromatographic and Saccaromyces uvarum methods for the determination of vitamin B, in fortified breakfast cereals. J Agric Food Chem 1980; 28: 486–9.

14.

Toepfer

Polansky

. Microbiological assay of vitamin B₆ and its components. J Assoc Off Anal Chem 1970; 53: 546–50.

15.

Ishii

Sari

Sanemori

Kawasaki

. Analysis of thiamine and its phosphate esters by high performance liquid chromatography. Anal Biochem 1979; 97: 191–5.

16.

Kimura

Fujita

Nishida

Itokawa

. Differential fluorimetric determination of picogram levels of thiamine, thiamine monophosphate, diphosphate and triphosphate using high performance liquid chromatography. J Chromatogr 1980; 188: 417–9.