Automated solid-phase extraction coupled with heart-cutting two‑dimensional liquid chromatography for simultaneous quantification of thirteen fat-soluble vitamins in foods for special medical purposes.

Abstract

A method based on heart‑cutting two‑dimensional liquid chromatography coupled with automated solid‑phase extraction was developed and validated for the simultaneous determination of thirteen fat‑soluble vitamins, including vitamins A, D₂, D₃, four tocopherols (α‑, β‑, γ‑, δ‑tocopherol), α‑tocotrienol, four β‑carotene isomers (all‑trans‑, 9‑cis‑, 13‑cis‑, 15‑cis‑β‑carotene), and lutein-in foods for special medical purposes (FSMP). The method integrates automated SPE sample purification with a two‑dimensional chromatographic separation that employs a C30 column in the first dimension and a PFP column in the second dimension. A heart‑cutting valve‑switching strategy was implemented to selectively transfer the vitamin‑D‑containing fraction from the first to the second dimension, thereby overcoming matrix interference and enabling accurate trace‑level quantification. The method demonstrated excellent performance: limits of detection (LODs) ranged from 0.01 μg/L (vitamin A) to 0.05 mg/L (tocopherols and α‑tocotrienol) and 0.001-0.005 mg/L (β‑carotene isomers), with corresponding limits of quantification (LOQs) from 0.03 μg/L to 0.15-0.015 mg/L. Spike recoveries for all analytes were between 88.5% and 108.2%, with intra‑ and inter‑day precision (RSD) below 5.0%. When applied to commercial FSMP samples, results showed good agreement (relative deviations < 10%) with established Chinese national standard methods. This sensitive, robust, and highly automated 2D‑LC‑SPE platform provides a reliable, high‑throughput solution for quality control, nutrition‑label compliance verification, and regulatory monitoring of FSMP products.