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A species-specific double isotope dilution strategy for the accurate quantification of platinum-GG adducts in lung cells exposed to carboplatin


Platinum-DNA adducts, and in particular Pt-GG, has been identified as the major cytotoxic species during chemotherapy treatment with platinum containing drugs. This paper reports for the first time a strategy based on the use of double species-specific isotope dilution analysis (IDA) for the quantification of carboplatin-GG adducts formed by exposing lung cells to carboplatin. The main challenge posed by the use of carboplatin in this pre-clinical application, in comparison with most previously reported work using cisplatin, include the relatively low reactivity of this drug, thus demanding for improved limits of detection to be achieved in order to perform accurate quantification of the adducts at low ng Pt/mg DNA levels with relatively small uncertainty in micro-volumes of the biological sample. This was alleviated by developing micro-flow HPLC reversed phased methodology coupled to sector field ICP-MS (R=300), showing a limit of detection of 0.2 ng Pt/ mg DNA. To perform IDA, carboplatin-GG calibrants and spikes (194Pt-enriched GG adducts) were synthesized in house and characterised for Pt mass fraction, Pt distribution and structural composition. In order to assess the accuracy of the developed procedure, in the absence of certified reference materials, a reference sample prepared by incubation of calf thymus DNA with carboplatin and characterised in house (e.g. for its total P and Pt contents and Pt-GG concentration) was analysed in parallel. The use of this sample as a quality control of the cleavage of DNA and recovery of Pt adducts from real samples makes the described strategy particularly novel. Moreover, spike recovery experiments on the cell samples with the reference carboplatin-DNA sample were undertaken. The validated methodology was applied to cultured human lung cells exposed to carboplatin; Pt-GG adducts were found at a level of 5.54ng Pt/ mg DNA with a relative expanded combined uncertainty (k=2) of approximately 20%. The major contributing factors to the overall measurement uncertainty were the mass fraction of Pt in the natural carboplatin-GG standard, the measured isotope ratio precision of sample and calibration blends and the blend to blend variation. The SI traceable methodology presented here will be invaluable for the provision of reference values to clinical measurements and and cancer clinical trials.

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Publication details

The article was received on 01 Mar 2017, accepted on 08 Jun 2017 and first published on 08 Jun 2017

Article type: Paper
DOI: 10.1039/C7JA00078B
Citation: J. Anal. At. Spectrom., 2017, Accepted Manuscript
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    A species-specific double isotope dilution strategy for the accurate quantification of platinum-GG adducts in lung cells exposed to carboplatin

    S. Cuello, R. LARIOS, C. L. Deitrich, T. Lekishvili, V. Nischwitz, B. L. Sharp and H. Goenaga-Infante, J. Anal. At. Spectrom., 2017, Accepted Manuscript , DOI: 10.1039/C7JA00078B

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