Development, validation and comparison of four methods for quantifying endogenous 25OH-D3 in human plasma

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Biomedical Science

Background:

• Accurate quantification of 25-hydroxyvitamin D3 (25OH-D3) is essential for clinical applications.
• Endogenous compounds like 25OH-D3 present challenges in quantification due to the lack of a true plasma blank.
• Validating methods for endogenous compounds requires careful consideration of regulatory guidelines.

Purpose of the Study:

• To develop and validate efficient and accurate high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods for plasma 25OH-D3 quantitation.
• To address the challenges of quantifying endogenous 25OH-D3 by exploring various blank matrix strategies.
• To compare the performance of four distinct HPLC-MS methods for clinical monitoring.

Main Methods:

• Liquid-liquid extraction using methyl tert-butyl ether for sample preparation.
• Implementation of strategies including water blank, 5% human serum albumin in water blank, surrogate analyte, and background subtraction.
• Full validation of four liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods according to established criteria.
• Parallel quantification of 30 human plasma samples using the four validated methods.

Main Results:

• All four validated LC-MS/MS methods met the required criteria for clinical sample monitoring.
• Inter-method variability was low, with differences between any two methods <12.6%.
• The total relative standard deviation across all methods was consistently low, <5.2%.

Conclusions:

• Background subtraction and using 5% human serum albumin in water as a blank matrix are recommended for 25OH-D3 quantification.
• These preferred methods offer advantages in data quality, matrix similarity, cost-effectiveness, and practicality for clinical settings.
• The validated methods provide reliable tools for meeting clinical demands for 25OH-D3 detection.