A rapid LC-MS/MS method with 2-nitrosopyridine derivatization to determine fat-soluble vitamins, revealing a biphasic bile acids association in clinical cohorts

Fat-soluble vitamins (A, D, and E) are essential micronutrients whose absorption critically depends on bile acid-mediated solubilization. However, studies associating bile acids and these vitamins remain limited, partly due to methodological challenges. Therefore, we developed a novel and rapid LC-MS/MS method for the simultaneous quantification of these vitamins in serum, based on 2-nitrosopyridine (PyrNo) click derivatization. This strategy employs Diels-Alder cycloaddition for vitamin A/D and acid-catalyzed oxidative dehydrogenation for vitamin E, enabling one-step protein precipitation followed by a 15-min room-temperature reaction. The method significantly enhanced sensitivity (>350-fold for vitamin D), allowing analysis within 6 min, and demonstrated excellent performance: linearity (R > 0.99), accuracy (96.1-110.2%), precision (RSD ≤ 4.9%), with lower limits of quantification of 0.1 µmol/L for retinol, 2 nmol/L for 25-hydroxyvitamin D₂ and D₃, and 2 µmol/L for α-tocopherol. Application of this method to a clinical cohort of 277 patients, covering a broad spectrum of total bile acid (TBA) concentrations, revealed a previously unrecognized biphasic relationship between TBA and vitamin status. Within the normal TBA group (≤10 µmol/L), vitamin A deficiency was associated with lower median TBA, and a TBA cutoff of 3.7 µmol/L showed predictive ability for combined vitamin A/E deficiency (AUC = 0.6057). Importantly, when patients with elevated TBA (>10 µmol/L) were included, vitamin A/D levels peaked in the intermediate TBA range (3.7-10 µmol/L) and were lower in both the low (<3.7 µmol/L) and high (>10 µmol/L) TBA groups. This work provides both a major analytical advancement and novel clinical insights into bile acid-mediated vitamin homeostasis.