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Reverse phase open column chromatography determination of urinary riboflavin
S Huang1, M J Caldwell, K L Simpson
1Department of Food Science and Nutrition, University of Rhode Island, Kingston 02881.
Summary
A new reverse phase open column chromatography method accurately measures urinary riboflavin (Vitamin B2) with excellent reproducibility and recovery. This technique offers a reliable alternative for riboflavin analysis in biological samples.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Clinical Chemistry
Background:
- Riboflavin (Vitamin B2) is essential for numerous metabolic processes.
- Accurate quantification of urinary riboflavin is crucial for assessing nutritional status and diagnosing deficiencies.
- Existing methods for riboflavin determination may have limitations in terms of cost, complexity, or sensitivity.
Purpose of the Study:
- To develop and validate a novel reverse phase open column chromatography method for the determination of urinary riboflavin.
- To assess the reproducibility, accuracy, and sensitivity of the developed method.
- To compare the performance of the new method with traditional fluorometric and High-Performance Liquid Chromatography (HPLC) techniques.
Main Methods:
- Reverse phase open column chromatography utilizing a C18 stationary phase and a MeOH/H2O/HOAc mobile phase.
- Direct application of urine specimens onto the column for riboflavin elution and measurement.
- Validation of the method through recovery studies and assessment of within-day and day-to-day reproducibility.
- Comparative analysis against traditional fluorometric and HPLC methods using linear regression.
Main Results:
- The method demonstrated high reproducibility with coefficients of variation for day-to-day reproducibility of 0.39% (low concentration) and 1.74% (high concentration).
- Excellent recovery rates for added Vitamin B2 were observed, ranging from 98.89% to 100%.
- The developed method showed strong linear correlation with both traditional fluorometric (r=0.984) and HPLC (r=0.999) methods.
- Quantification of riboflavin was achieved down to 0.05 micrograms/ml with linearity up to 10 micrograms/ml.
Conclusions:
- Reverse phase open column chromatography provides a reproducible and accurate method for urinary riboflavin determination.
- This technique offers a viable and potentially simpler alternative to existing fluorometric and HPLC methods.
- The method's sensitivity and linearity make it suitable for a wide range of riboflavin concentrations in urine samples.
