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-inflammatory 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 efficacy of ≥ 89% and interday/intraday variation of 3-11% for major metabolites. To ensure accurate quantification, 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 with γ-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.

Keywords: liquid chromatography tandem mass spectrometry; metabolism; sulfation; tocopherol; tocotrienol.

Fig. 1.

Structures of vitamin E forms.

Fig. 2.

Vitamin E (γT) metabolism and metabolites. Vitamin E forms are metabolized via ω-hydroxylation and oxidation to generate 13′-OH and 13′-COOH, which is subsequently metabolized via β-oxidation to form 11′-, 9′-, 7′-, 5′-, and 3′-COOH. Sulfation of intermediate carboxychromanols appears to take place in parallel with β-oxidation in response to supplementation of vitamin E forms.

Fig. 3.

LC/MS/MS fragmentation of vitamin E metabolites.