Development, validation, and four-year clinical application of a novel, rapid, sensitive, and high-throughput UPLCMS/MS method for the simultaneous determination of vitamins A and E in 50 μL of serum.

In this study, a novel, sensitive, accurate, and high-throughput ultrahigh-performance liquid chromatographytandem mass spectrometry (UPLCMS/MS) method is developed to quantify vitamins A and E in only 50 μL of serum, overcoming volume-related detection challenges. Following extraction by acetonitrile protein precipitation and 96-well solid-phase extraction, the samples were analyzed using a BEH phenyl column (2.1 × 100 mm, 1.7 μm) with a mobile phase comprising 0.1 % formic acid in water and methanol. Using matrix-matched calibration (1.0 ×10-2-2.0 µg/mL for vitamin A and 0.1-20.0 µg/mL for vitamin E) with isotope-labeled internal standards (IS), the method demonstrated excellent performance: the mean IS-normalized matrix effects were 87.99-107.22 % (RSD < 10 %), the recovery was 96.06-102.93 % (RSD < 15 %), the accuracy was within 10 % bias, and the precision RSD was < 10 %. Analysis of 2137 healthy infants (aged <1 year) revealed that more than 97.9 % had vitamin A and E levels within the Mayo Clinic reference ranges, with sex-independent median levels of 0.3 μg/mL (95 % confidence interval: 0.17-0.47) and 10.8 mg/L (95 % confidence interval: 5.60-18.5), respectively, suggesting their potential utility as local reference values. No sex differences in vitamin A or E levels were observed across clinical symptoms (all p > 0.05), and significant group disparities were revealed: vitamin A levels were significantly lower in the poor sleep group and the fever/cough group than in the health examination group (both p < 0.001), while no difference was found in the rash/eczema group (p > 0.05). Similarly, vitamin E levels were lower in the fever/cough group (p < 0.05) than in the health examination group, but no differences were observed in the poor sleep or rash/eczema groups (p > 0.05).