Analysis of Vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS

Abstract

Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13 ′ -hydroxychromanols (13 ′ -OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13 ′ -carboxychromanol (13 ′ -COOH), appear to be more bioactive than tocopherols in anti-infl ammatory and anticancer actions. To understand the potential contribution of metabolites to Vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying Vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with effi cacy of ≥ 89% and interday/intraday variation of 3-11% for major metabolites. To ensure accurate quantifi cation, short-chain, long-chain, and sulfated carboxychromanols are included as external/ internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed withγ-tocopherol or δ -tocopherol. Although plasma levels of 13 ′ -COOHs and 13 ′ -OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated Vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo.