Selenosugar determination in porcine liver using multidimensional HPLC with atomic and molecular mass spectrometry

Abstract

A methodology based on liquid chromatography coupled online with atomic and molecular mass spectrometry was developed for identifying trace amounts of the selenosugar methyl 2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (SeGalNAc) in porcine liver, obtained from an animal that had not received selenium supplementation. Sample preparation was especially critical for the identification of SeGalNAc by molecular mass spectrometry. This involved liver extraction using a Tris buffer, followed by sequential centrifugations. The resulting cytosolic fraction was pre-concentrated and the low molecular weight selenium (LMWSe) fraction obtained from a size exclusion column was collected, concentrated, and subsequently analyzed using a tandem dual-column HPLC–ICP-MS system which consisted of strong cation exchange (SCX) and reversed phase (RP) columns coupled in tandem. Hepatocytosolic SeGalNAc was tentatively identified by retention time matching and spiking. Its identity was further confirmed by using the same type of chromatography on-line with atmospheric pressure chemical ionization tandem mass spectrometry operated in the selected reaction monitoring (SRM) mode. Four SRM transitions, characteristic of SeGalNAc, were monitored and their intensity ratios determined in order to confirm SeGalNAc identification. Instrument limits of detection for SeGalNAc by SCX-RP HPLC–ICP-MS and SCX-RP HPLC–APCI-MS/MS were 3.4 and 2.9 μg Se L⁻¹, respectively. Selenium mass balance analysis revealed that trace amounts of SeGalNAc, 2.16 ± 0.94 μg Se kg⁻¹ liver (wet weight) were present in the liver cytosol, corresponding to 0.4% of the total Se content in the porcine liver.