Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 877, Issue 20-21, Pages 1882-1886Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2009.05.013
Keywords
Thiamin; Thiamin diphosphate; Determination; HPLC; Preterm infants; Pediatric diagnostics
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An improved and easy to use method for the determination of thiamin diphosphate (TDP) in 100 mu l of whole blood was developed. The small sample volume makes it possible to assess the nutritional status of vitamin B-1 in infants and even in preterm infants. Sample preparation comprises the extraction of TDP from whole blood by hemolysis, protein precipitation with trichloroacetic acid, and subsequent centrifugation. Potassium ferricyanide is used for pre-column derivatization of TDP to its fluorescent thiochrome derivative. Chromatographic separation was carried out using a reversed-phase column and an isocratic elution which consisted of a phosphate buffer and acetonitrile. TDP was detected fluori metrically and quantified by external standardization. Method validation showed a high precision, almost complete recovery, and a high sensitivity. The lower limit of detection and the lower limit of quantification were 0.2 ng/ml and 4 ng/ml, respectively. Linearity was demonstrated over the expected concentration range of 4-400 ng/ml. in conclusion, we present a convenient HPLC method for the determination of TDP which is precise, sensitive and suitable for pediatric diagnostics. (C) 2009 Elsevier B.V. All rights reserved.
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