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Here, we report the identificationof dimethylarsinothioyl glutathione(DMMTAV(GS)) as a metabolite in cellular extracts of dimethyarsinousglutathione (Darinaparsin, DMAIII(GS)) treated human multiplemyeloma (MM) cell lines. Co-elution of sulfur and arsenic on the inductivelycoupled plasma mass spectrometer (ICP-MS) indicated the presence ofsulfur along with arsenic in the newly observed unidentified moleculeon the speciation chromatograms of cell lines treated with DMAIII(GS). Liquid chromatography–electrospray ionization–massspectrometry of the unknown peak in the MS and tandem MS modes revealedmolecular ion peaks at m/z= 443.9and 466.0, corresponding to [DMMTAV(GS) + H]+and [DMMTAV(GS) + Na]+, as well as peaks at314.8 for the loss of glutamic acid and 231.1 for the loss of glycine.In addition, peaks were observed at 176.9 corresponding to cysteineand glycine adducts and at 137.1 for the [C2H6AsS]+ion. An increase in the peak area of the unidentifiedpeak was observed upon spiking the cell extracts with a standard ofDMMTAV(GS). Heat deactivation of MM cells prevented theformation of DMMTAV(GS) raising the possibility of itsformation via an enzymatic reaction. Formation studies in DMAIII(GS) treated MM cells revealed the dependence of DMMTAV(GS) formation on the depletion of DMAIII(GS).The presence of 5 mM glutathione prevented its formation, indicatingthat DMAIII, a dissociation product of DMAIII(GS), is likely a precursor for the formation of DMMTAV(GS). DMMTAV(GS) was observed to form under acidic andneutral pH conditions (pH 3.0–7.4). In addition, DMMTAV(GS) was found to be stable in cell extracts at both acidicand neutral pH conditions. When assessing the toxicity by exposingmultiple myeloma cells to arsenicals externally, DMMTAV(GS) was found to be much less toxic than DMAIII(GS) andDMMTAV, potentially due to its limited uptake in the cells(10 and 16% of the uptakes of DMAIII(GS) and DMMTAV, respectively). [ABSTRACT FROM AUTHOR]