An HPLC-UV Method to Assess Human Plasma 25(OH)D₃

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Diagnostics

Background:

• Accurate assessment of vitamin D status is crucial for public health.
• 25-hydroxyvitamin D3 (25(OH)D3) is the primary indicator of vitamin D status.
• Validated analytical methods are essential for reliable vitamin D testing.

Purpose of the Study:

• To validate a High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method for quantifying 25-hydroxyvitamin D3 (25(OH)D3).
• To establish the method's linearity, precision, accuracy, and robustness for assessing vitamin D status.

Main Methods:

• HPLC-UV analysis using an Acclaim™ 120 C18 column.
• Preparation of 25(OH)D3 calibration curves from 5 to 50 ng/mL.
• Extraction of vitamin D from plasma samples using acetonitrile and formic acid.
• Validation conducted according to FDA guidelines.

Main Results:

• Excellent linearity for the 25(OH)D3 calibration curve (R² = 0.9989).
• Detection and quantification limits of 1.1703 ng/mL and 3.5462 ng/mL, respectively.
• High percentage recovery rates (92.2% to 97.1%) indicating method accuracy.

Conclusions:

• The validated HPLC-UV method is reliable for quantifying 25(OH)D3 in human plasma.
• This method can be effectively used to assess vitamin D status.
• The method's robustness was confirmed through analysis of temperature and flow rate variations.