An analytical methodology for the quantitative profiling of fat-soluble antioxidants in maternal blood, umbilical cord blood and meconium

The determination of fat-soluble antioxidants represents a significant analytical challenge, particularly when dealing with biological matrices available only in limited quantities, such as those collected from preterm infants. This paper presents the development, validation, and application of a multianalyte high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of 21 fat-soluble antioxidants (including vitamins and carotenoids) in meconium, cord plasma, and maternal plasma samples. All the analytes were separated by non-aqueous reversed phase chromatography, detected by atmospheric pressure chemical ionization, acquiring in Scheduled/Multiple Reaction Monitoring mode to improve sensitivity. Their extraction was performed with hexane, after protein precipitation with cold ethanol, followed by a “lipid freezing” clean-up. Recoveries ranged between 63 and 100 % with relative standard deviations ≤ 18 %. For all matrices, limits of detection and lower limits of quantitation were in the ppb and ppm-ppb range, respectively. The developed approach enabled the study of the distribution of 21 fat-soluble antioxidants, which play a protective role against oxidative stress at various levels within the human body. To better interpret the data obtained from an unconventional matrix such as meconium, the results were compared with those from cord plasma and maternal plasma, revealing, in most cases, a common trend across the 24 cases under study.