Quantification of fat-soluble vitamins in human serum by LC–MS/MS

Accurate measurement of fat-soluble vitamin levels in blood samples is essential for personalized nutrition and medicine. This study presents a rapid, accurate, and sensitive LC–MS/MS method for the simultaneous quantification of the major fat-soluble vitamins—vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1—in human serum. Serum sample preparation included protein precipitation and solid-phase extraction. Chromatographic separation was performed using a phenyl column and gradient elution of methanol with 0.1% formic acid. Using positive electrospray ionization mode for mass spectrometric detection, multiple reaction monitoring, and isotope-labeled internal standards for each analyte ensures accurate quantification. The chromatographic run time is only 4.5 min. The method demonstrated robust detection limits ranging from 0.015 to 0.1 ng/mL and recoveries between 93.22 and 106.79%. In addition, intra- and inter-day coefficients of variance ranged from 1.11 to 8.71%. This validated approach was successfully applied to actual serum samples, demonstrating its utility as a useful tool for assessing serum levels of fat-soluble vitamins. These results support advances in the safety and efficacy of vitamin therapy, making it a valuable tool for clinical and research applications.