Investigation of the selenium species distribution in a human B-cell lymphoma line by HPLC- and GC-ICP-MS in combination with HPLC-ESIMS/MS and GC-TOFMS after incubation with methylseleninic acid

Abstract

A methodology using the on-line combination of micro-HPLC with ICP-MS and electrospray ionisation (ESI) MS/MS, GC-ICP-MS and GC-TOFMS is proposed for investigation of the Se species distribution in lysates and headspace of human lymphoma cell lines after incubation with methylseleninic acid (MSA), which is known as a precursor of methylselenol, a metabolite with anti-cancer properties. The HPLC-ICP-MS results showed significant differences in speciation results of control (untreated) and Se-exposed DHL-4 cells, revealing the presence of three Se compounds in the cell extracts after 10 min incubation with MSA. These compounds were preliminarily identified as Se-methylselenocysteine (SeMC), selenomethionine (SeMet) and gamma-glutamyl-methylselenocysteine (γ-glutamyl-SeMC) by HPLC-ICP-MS on the basis of retention matching with standards. Quantification of Se-compounds in cell lysates was performed by reversed-phase HPLC coupled directly to ICP-MS. Calibration was by the standard additions technique, using peak area measurements of the chromatographic signals by monitoring the ⁸²Se signal. Verification of the presence of SeMC, the major Se compound in the cell extracts, was achieved by on-line HPLC-ESIMS/MS in selected reaction monitoring (SRM) mode. Total selenium concentrations in cell lysates were determined by flow injection analysis coupled with ICP-MS. The capabilities of solid phase micro-extraction (SPME)-GC in combination with ICP-MS and GC-TOFMS were investigated for studying the distribution of volatile Se species in the headspace of the lymphoma DHL-4 cell cultures. A cryogenic oven cooling (COC)-GC system, which led to a significant improvement in the chromatographic performance (retention time, peak shape and consequently, chromatographic selectivity) of compounds with a low boiling point such as dimethylselenide (CH₃SeCH₃) in comparison with conventional GC was employed for the first time. A number of volatile Se species could be detected by GC-ICP-MS, from which dimethylselenide and dimethyldiselenide (CH₃SeSeCH₃) could be identified by COC-GC-TOFMS in the headspace of DHL-4 cell lines exposed to methylseleninic acid.