Method development for selenium speciation analysis in biological samples by using HPLC with atomic and molecular MS

Abstract

The purpose of these studies is to develop methods of coupling high performance liquid chromatography (HPLC) coupling with various mass spectrometry techniques (i.e., ICP-MS, ESI-MS/MS, APCI-MS/MS, ESI - IT/MSn) for the identification and characterisation of selenium species present in biological samples that are of biological interest. This thesis presents the results from the following studies: • Method development for qualitative and quantitative analysis of four urinary selenium species (TMSe+, SeMet, SeGalNAc and SeGluNAc) relevant to selenium metabolism in the human body by using HPLC with atomic and molecular mass spectrometry. • Development and application of HPLC - ICP - MS and HPLC - APCI - MS/MS methodologies for selenium speciation in cytosolic samples of porcine liver. • HPLC separation and mass spectrometric characterisations of Se-containing tocopherol and tocotrienol succinate derivative compounds: α - tocopheryl 2 - phenylselenyl succinate, γ - tocopheryl 2 - phenylselenyl succinate and γ - tocotrienyl 2 - phenylselenyl succinate. First, a LC-ES-MS/MS method was developed for the analysis of four organic selenium species relevant to human urinary metabolism: trimethylselenonium ion (TMSe+), selenomethionine (SeMet) and two selenosugars, methyl 2-acetamido-2- deoxy-1-seleno-β-D- galacto- and - gluco-pyranoside (SeGalNAc and SeGluNAc, respectively). The chromatographic separation of TMSe+, SeMet, SeGalNAc and SeGluNAc was achieved by combining a cation exchange precolumn and reversed phase chromatography under isocratic elution coditions with an aqueous mobile phase. Online detection was performed using sensitive and selective ESI-MS/MS in the selective reaction monitoring (SRM) mode. As a result, SeGalNAc was detected as the major human urinary metabolite of selenium in all samples analysed, whereas TMSe+ was detected in the urine from only one volunteer before and after receiving a selenium supplement. Spiking experiments in the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome this interference isotopically labelled 13CD3 82SeGalNAc was used as an internal standard; whereas in the absence of an appropriate internal standard for TMSe+, the standard addition method was used. Quality control for the accurate quantification of TMSe+ and SeGalNAc was carried out by analysing spiked human urine samples with selenium standards over a concentration range of 10 -50 μg Se/L. The method’s limit of detection was comparable to that of HPLC-ICP-MS detection for the four selenium standards: for TMSe+ it was 1.1 μg Se/L, for SeMet 2.1 μg Se/L, for SeGalNAc 0.1 μg Se/L, and for SeGluNAc 0.1 μg Se/L. Further, matrix effects on the performance of the HPLC-ESI-MS/MS system for the analysis of urine samples. These were defined as the absolute and relative matrix effects. The study revealed a 2 - 6 times of suppression on the SRMs that used for the detection of TMSe+ and SeGalNAc in urine matrices in the current LC – MS conditions. In addition, in the presence of urine matrix, the LODs of HPLC-ESI-MS/MS analysis TMSe+ and SeGalNAc were estimated as 2.8 - 5.9 μg Se/L and 3.6 - 5.5 μg Se/L respectively. Secondly, 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-Dgalactopyranoside (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 fraction (LMWSe) obtained from a size exclusion column was collected, concentrated, and subsequently analyzed using a tamdem dual-column HPLC 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, respectively. The HPLC separation in the above analyses could be further improved by using a PerfectSil Target C8-3 reversed phase column. The tentative identification of SeMet was carried out by using five different HPLC systems with ICP – MS as detector. However, the further tandem MS/MS analysis in SRM mode on SeMet was not sensitive enough for its successful identification and characterisation of this molecule in hepatocytosolic matrix. The application of affinity chromatography – ICP – MS method to the analysis of liver cytosol sample resulted in the identification of selenoprotein P. The determination of total Se content in porcine liver and its cellular fractions and the evaluation of the Se mass balance and the concentration of SeGalNAc, SeMet*, selenoprotein P revealed that elenoprotein P is the major selenium species concerning the selenium content in the liver sample (~ 50 %). Trace amounts of SeGalNAc and SeMet* were present in the liver cytosol as 0.42 % and 0.13 %, respectively of the total Se content in the porcine liver. As the third part of this thesis, the characteristic MSn and tandem MS behaviours of two Se-containing tocopherol and tocotrienol succinate derivative compounds were investigated: α - tocopheryl 2 - phenylselenyl succinate (PSSA - α - tocopherol) and γ - tocopheryl 2 - phenylselenyl succinate (PSSA - γ - tocopherol). A methodology of using HPLC – APCI - MS/MS in the SRM mode was developed for PSSA - α - tocopherol and PSSA - γ - tocopherol. The LOD (estimated as about 5 - 6 μg/L for the two seleno- tocopherol succinate derivative compounds) and the SRM ratios (within the 3 - 11 % RSD of SRM ratios for the standards) that were obtained for this method demonstrated the feasibility of using RP HPLC - APCI - MS/MS in the SRM mode for the determination of PSSA - α - tocopherol and PSSA - γ - tocopherol in cell samples. Additionally, data on the MSn and tandem MS characteristics of two aromatic selenium compounds, PSSA and Methyl PSSA, are also provided in this thesis.