Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC–fluorimetry

Mathew, Elizabeth Mary and Sakore, Pradnya and Lewis, Leslie and Kalaivani, Manokaran and Rao, Pragna and Moorkoth, Sudheer (2019) Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC–fluorimetry. Biomedical Chromatography, 33 (5). pp. 2-10. ISSN 0269-3879

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Abstract

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full‐spot analysis of a volumetrically deposited 40 μl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C18 column (5.0 μm, 250 × 4.6 mm) using 150mM phosphate buffer pH 7– acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34– 118.89% with recoveries >80%. Bland–Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.

Item Type: Article
Uncontrolled Keywords: Dried blood spot; HPLC–fluorimetry; infants; thiamine deficiency; thiamine diphosphate.
Subjects: Allied Health > MCOAHS Manipal > Medical Laboratory Tech
Medicine > KMC Manipal > Biochemistry
Medicine > KMC Manipal > Paediatrics
Pharmacy > MCOPS Manipal > Pharmaceutical Quality Assurance
Depositing User: KMC Library
Date Deposited: 12 Feb 2020 04:31
Last Modified: 12 Feb 2020 04:31
URI: http://eprints.manipal.edu/id/eprint/154858

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