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Determination of thiamin diphosphate in whole blood samples by high-performance liquid chromatography--a method
Robert Walter Körner1, Anne Vierzig, Bernhard Roth
1Department of Therapeutic Drug Monitoring, Institute of Pharmacology, University of Cologne, Gleuelerstrasse 24, D-50931 Cologne, Germany.
Insights
This study presents a new, simple HPLC method to measure thiamin diphosphate (TDP), or vitamin B1, in small blood samples. This technique is ideal for assessing vitamin B1 nutritional status in infants and newborns.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Analytical Chemistry
Background:
- Thiamin diphosphate (TDP) is the active form of vitamin B1, crucial for metabolic processes.
- Assessing vitamin B1 nutritional status, especially in vulnerable populations like infants, requires sensitive and accessible methods.
- Existing methods may require larger sample volumes, posing challenges for pediatric diagnostics.
Purpose of the Study:
- To develop and validate a user-friendly, high-performance liquid chromatography (HPLC) method for TDP determination.
- To enable TDP analysis using small whole blood volumes (100 microliters).
- To establish the method's suitability for pediatric nutritional status assessment, including preterm infants.
Main Methods:
- Sample preparation involved hemolysis, protein precipitation using trichloroacetic acid, and centrifugation.
- Pre-column derivatization of TDP to its fluorescent thiochrome derivative using potassium ferricyanide.
- Chromatographic separation on a reversed-phase column with isocratic elution (phosphate buffer/acetonitrile), followed by fluorimetric detection and external standardization.
Main Results:
- The method demonstrated high precision, near-complete recovery, and excellent sensitivity.
- The lower limit of detection (LOD) was 0.2 ng/ml, and the lower limit of quantification (LOQ) was 4 ng/ml.
- Linearity was confirmed over the concentration range of 4-400 ng/ml, suitable for clinical applications.
Conclusions:
- A convenient, precise, and sensitive HPLC method for TDP determination in whole blood has been successfully developed.
- The method's requirement for only 100 microliters of blood makes it highly suitable for pediatric diagnostics.
- This assay facilitates accurate assessment of vitamin B1 nutritional status in infants, including preterm infants.
Abstract:
An improved and easy to use method for the determination of thiamin diphosphate (TDP) in 100 microl 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 fluorimetrically 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.
