High-Throughput Determination of Thiamine in Whole Blood and Erythrocytes

Importance of the topic

The accurate determination of thiamine (vitamin B1), particularly its bioactive form thiamine diphosphate (TDP), is critical for assessing nutritional status and preventing deficiency diseases such as beriberi. Reliable high-throughput methods are essential for large-scale population studies, clinical research, and quality control in nutrition and public health.

Study objectives and overview

This application note presents the development and validation of an ultrahigh-performance liquid chromatography method with fluorescence detection (UHPLC-FLD) integrating online precolumn derivatization. The workflow targets TDP quantification in whole blood and erythrocytes, enabling rapid analysis of a nationally representative sample of Cambodian women and children to evaluate thiamine status.

Methodology and instrumentation used

Sample Preparation and Derivatization:

• Protein precipitation with 10% trichloroacetic acid (TCA).
• Removal of TCA by liquid–liquid extraction with water-saturated methyl tert-butyl ether.
• Online precolumn derivatization in the autosampler injector using potassium ferricyanide in NaOH and methanol.

Instrumentation:

• Agilent 1290 Infinity LC system with binary pump, autosampler, column compartment, and fluorescence detector.
• Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT column (3.0 × 50 mm, 1.8 µm) with C18 guard cartridge.
• Fluorescence detection at excitation 375 nm and emission 435 nm.
• OpenLab CDS ChemStation Edition software.

Chromatographic Conditions:

• Gradient elution using dibasic sodium phosphate buffer/methanol (90:10 to 30:70).
• Flow rate 0.6 mL/min, column temperature 25 °C, injection volume 15 µL.

Main results and discussion

Performance characteristics:

• Limit of detection (LOD): 10.0 nmol/L; limit of quantification (LOQ): 30.2 nmol/L.
• Linearity over 32.9–263.6 nmol/L with R² ≥ 0.9978.
• Recovery 95–106% across two control levels; repeatability RSD < 4%.

Comparison of offline and online derivatization demonstrated extended sample stability (> 72 h vs. < 16 h) and reduced manual handling, minimizing degradation and improving throughput.

Benefits and practical applications of the method

The automated UHPLC-FLD approach offers:

• High throughput, enabling analysis of over 1,400 samples with minimal operator intervention.
• Enhanced precision and reproducibility by fresh derivatization immediately prior to injection.
• Robust selectivity for TDP over other thiamine forms, supporting reliable nutritional assessment.

It is suitable for clinical research, large-scale epidemiological surveys, and routine QA/QC in nutritional laboratories.

Future trends and potential applications

Further developments may include:

• Extension to other water-soluble vitamins and metabolites using similar online derivatization strategies.
• Miniaturized or multiplexed autosampler configurations for even higher throughput.
• Integration with mass spectrometric detection to expand analyte coverage and specificity.

Conclusion

The described UHPLC-FLD method with online precolumn derivatization provides a fast, sensitive, and reproducible tool for thiamine analysis in whole blood and erythrocytes. Its high throughput and automated workflow make it ideal for large population studies and clinical monitoring of vitamin B1 status.

Reference

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