Age-related reference intervals for serum phytosterols in children by gas chromatography-mass spectrometry and its application in diagnosing sitosterolemia

Serum phytosterol profiles are essential for the diagnosis and management of sitosterolemia. However, pediatric reference interval (RI) studies are scarce and various mass spectrometry (MS) approaches for phytosterol analysis still face multiple limitations. Therefore, an optimized gas chromatography (GC)-MS assay and age-related RIs in children are both required.Cholesterol and phytosterols (sitosterol, campesterol, cholestanol, stigmasterol, and sitostanol) were simultaneously determined by optimized GC-MS and performance was verified by the lower limit of quantification (LLOQ), linearity, precision, recovery, matrix effects, and method comparison. Healthy children (247 males and 263 females) were recruited, sex and age dependence were assessed using quantile regression (2.5th percentile and 97.5th percentile), and RIs were established according to Clinical and Laboratory Standards Association guideline C28-A3. These RIs were validated in 19 patients with sitosterolemia and 23 patients with hypercholesterolemia.The optimized method shortened the sample processing time by approximately 60 min. Among the five phytosterols, all precision, recoveries (ranging from 89.97% to 104.94%), and relative matrix effects (%CV: ranging from 0.08% to 13.88%) met the specifications. GC-MS showed good agreement with lower cholesterol concentrations compared to conventional enzymatic methods. No significant differences between males and females were observed for all phytosterols, but age dependency was found and age-related RIs were established accordingly. Five phytosterols were significantly higher than RIs in patients with sitosterolemia.We established age-related RIs for five phytosterols in children based on an optimized GC-MS assay, providing a screening tool for the diagnosis of sitosterolemia in children.