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SELECTBIO Conferences Biomarkers - From Research To Commercialization


Estimation of Thiamine Diphosphate in Dried Blood Spot Samples Using HPLC Fluorimetry

Elizabeth Mary Mathew, Research Scholar, Manipal University

Thiamine deficiency in infancy and childhood leads to lactic acidosis, infantile beri beri, Wernicke’s encephalopathy and also may lead to seizures which may cause severe damage to brain .Thiamine deficiency if detected early in infancy can be treated with the thiamine supplementation and can prevent seizures, other disabilities and death.  Dried blood spot sampling technique is an innovative blood sampling technique helpful in sample collection from infants for mass new born screening. The present research reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from dried blood spots. The analyte on dried blood spots were extracted into methanol and separated and quantified by HPLC- fluorimetry on an Inertsil ODS 250 mm column using 150 mM phosphate buffer pH 7: acetonitrile (90:10, %v/v) as mobile phase. The column oven and autosampler temperature were set at 35°C & 4°C respectively.  The excitation and emission wavelength were 367 nm & 435 nm respectively. The lower limit of detection and the lower limit of quanti?cation were 23 ng/mL and 50 ng/mL, respectively. The method demonstrated linearity over 50-1000ng/mL, precision represented by % CV of  1.5 – 7.1 %. The analyte demonstrated stability in matrix at room temperature, 2-8° C, and -80°C. Bland Altman analysis of DBS sampling Vs whole blood demonstrated a bias of 12.30 ng/mL which shows that the method is comparable. This method can be applied for quantification of thiamine diphosphate levels in large population study such as in  new born screening.  

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