ORIGINAL PAPER Capabilities of HPLC with APEX-Q nebulisation ICP-MS and ESI MS/MS to compare selenium uptake and speciation of non-malignant with different B cell lymphoma lines Heidi Goenaga-Infante & Shireen Kassam & Emma Stokes & Christopher Hopley & Simon P. Joel Received: 27 September 2010 / Revised: 19 November 2010 / Accepted: 24 November 2010 / Published online: 8 December 2010 # Springer-Verlag 2010 Abstract The formation of intracellular dimethylselenide (DMSe) as a product of exposure of non-malignant (PBMCs) and lymphoma (RL and DHL-4) cell lines to methylseleninic acid (MSA) at clinical levels is suggested here for the first time. This was achieved by analysis of cell lysates by HPLC coupled to ICP-MS via APEX-Q nebulisation, enabling limits of detection for target methyl-Se species which are up to 12-fold lower than those obtained with conventional nebulisation. Methyl-Se- glutathione (CH 3 Se-SG), although detected in lysates of cells exposed to MSA, was found to be a reaction product of MSA with glutathione. This was confirmed by HPLC- ESI MS (MS) analysis of lysates of control cells (unexposed to Se) spiked with MSA. The MS/MS data obtained by collision-induced dissociation fragmentation of the ion m/z 402 (for [M+H] + 80 Se) were consistent with the presence of CH 3 Se-SG. Formation of DMSe was not detected by HPLC- ICP-MS in these spiked lysates, and it was found to require live cells in cell media containing MSA. Interestingly, the ratio of DMSe to CH 3 Se-SG was significantly higher in lymphoma cells exposed to MSA in comparison to non- malignant cells. Moreover, maximum Se uptake levels in lymphoma cell lines seemed to be reached much earlier (after 10 min of MSA exposure) than in non-malignant cells. Finally, the GC-TOF-MS speciation data obtained for cell headspace suggested that the major Se species (dimethyldiselenide) appeared to be present in lymphoma cell headspace at significantly higher concentrations than in non- malignant cell headspace after only 10 min of exposure to MSA. Evidence for the presence of dimethylselenidesulfide in lymphoma cell headspace is also provided for the first time. Keywords Selenium speciation . Non-malignant cell lines . Cancer cell lines . Intracellular dimethylselenide . APEX-Q nebulisation . Methylseleninic acid . Clinical levels . HPLC-ICP-MS . HPLC-ESI MS/MS Introduction In vitro studies in lymphoma cell lines have shown that low (non-toxic) concentrations of methylseleninic acid (MSA) can increase the efficacy of chemotherapeutic agents [1–3]. The identification of mechanisms by which Se sensitises lymphoma cells to chemotherapy whilst protecting normal cells from toxicity requires the combination of data provided by mass spectrometry techniques with that from biomeasurements (e.g. ELISA, real-time PCR). Such data are essential to understand the association between cancer signal pathways and inhibition of tumour cell invasion by Se drugs and, therefore, to identify the optimum treatment. The identification of differences in intracellular and headspace element speciation between non-malignant and cancer cells exposed to Se drugs at parts per billion levels poses analytical challenges, and to the author ’ s knowledge, it has not been reported yet. The main challenge is the relatively low concentration of intracellular Se species Published in the special issue Speciation Analysis in Healthcare with Guest Editor Heidi Goenaga Infante. H. Goenaga-Infante (*) : E. Stokes : C. Hopley LGC Limited, Queens Road, Teddington, Middlesex TW11 OLY, UK e-mail: Heidi.Goenaga-Infante@lgc.co.uk S. Kassam : S. P. Joel Centre for Experimental Cancer Medicine, Institute of Cancer & Cancer Research UK Clinical Centre, Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK Anal Bioanal Chem (2011) 399:1789–1797 DOI 10.1007/s00216-010-4474-1