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

To meet the increasing clinical needs for 25-hydroxyvitamin D3 (25OH-D3) detection, the development of an efficient and accurate high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for plasma 25OH-D3 quantitation is important. Since 25OH-D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH-D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid-liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography-tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.