Fast, simple, and sensitive high-performance liquid chromatography method for measuring vitamins A and E in human...

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Vitamins A and E are crucial fat-soluble vitamins involved in numerous physiological functions.
• Accurate monitoring of vitamin A (retinol) and vitamin E (α-tocopherol, γ-tocopherol) levels is essential for diagnosing deficiencies and guiding treatment.
• Existing methods may lack the speed, sensitivity, or simplicity required for routine clinical analysis.

Purpose of the Study:

• To develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous measurement of retinol, α-tocopherol, and γ-tocopherol in human plasma.
• To establish reference intervals for these vitamins in a healthy adult population.
• To provide a reliable analytical tool for clinical laboratories.

Main Methods:

• Vitamins were extracted from human plasma using hexane.
• Separation was achieved on a reversed-phase column with methanol as the mobile phase.
• Retinol was detected via UV absorption, while tocopherols were detected by fluorescence emission.

Main Results:

• The HPLC method demonstrated a rapid cycle time of 4.0 minutes per sample.
• Excellent analytical measurement ranges were established: 0.03-5.14 mg/L (retinol), 0.32-36.02 mg/L (α-tocopherol), and 0.10-9.99 mg/L (γ-tocopherol).
• Intra-assay and total coefficients of variation were consistently below 6.0% for all analytes, indicating high precision and accuracy. Method traceability to NIST standard reference materials was confirmed. Established reference intervals were: retinol 0.30-1.20 mg/L, α-tocopherol 6.0-23.0 mg/L, and γ-tocopherol 0.3-3.2 mg/L.

Conclusions:

• A fast, simple, and sensitive HPLC method for measuring major forms of vitamins A and E in human plasma has been successfully developed and validated.
• The method's efficiency and accuracy support its use in clinical settings for vitamin status assessment.
• Established reference intervals provide a valuable benchmark for interpreting patient results and managing vitamin deficiencies.