Development of an Ultraperformance Liquid Chromatography/Mass Spectrometry Method To Quantify Cisplatin 1,2 Intrastrand Guanine−Guanine Adducts

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• 作者：Irene M. Baskerville-Abraham ; Gunnar Boysen ; J. Mitchell Troutman ; Esra Mutlu ; Leonard Collins ; Kathryn E. deKrafft ; Wenbin Lin ; Candice King ; Stephen G. Chaney ; James A. Swenberg
• 刊名：Chemical Research in Toxicology
• 出版年：2009
• 出版时间：May 18, 2009
• 年：2009
• 卷：22
• 期：5
• 页码：905-912
• 全文大小：190K
• 年卷期：v.22,no.5(May 18, 2009)
• ISSN：1520-5010

文摘

Platinum chemotherapeutic agents have been widely used in the treatment of cancer. Cisplatin was the first of the platinum-based chemotherapeutic agents and therefore has been extensively studied as an antitumor agent since the late 1960s. Because this agent forms several DNA adducts, a highly sensitive and specific quantitative assay is needed to correlate the molecular dose of individual adducts with the effects of treatment. An ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantification of 1,2 guanine−guanine intrastrand cisplatin adducts [CP-d(GpG)], using ¹⁵Nb>10b> CP-d(GpG) as an internal standard, was developed. The internal standard was characterized by MS/MS, and its concentration was validated by inductively coupled plasma mass spectrometry. Samples containing CP-d(GpG) in DNA were purified by enzyme hydrolysis, centrifugal filtration, and HPLC with fraction collection prior to quantification by UPLC-MS/MS in the selective reaction monitoring mode [m/z 412.5→248.1 for CP-d(GpG); m/z 417.5→253.1 for [¹⁵Nb>10b>] CP-d(GpG)]. The recovery of standards was >90%, and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 μg of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 10⁸ nucleotides, which approaches the sensitivity of the ³²P postlabeling method for this adduct. These data suggested that this method is suitable for in vitro and in vivo assessment of CP-d(GpG) adducts formed by cisplatin and carboplatin. Subsequently, the method was applied to studies using ovarian carcinoma cell lines and C57/BL6 mice to illustrate that this method is capable of quantifying CP-d(GpG) adducts using biologically relevant systems and doses. The development of biomarkers to determine tissue-specific molecular dosimetry during treatment will lead to a more complete understanding of both therapeutic and adverse effects of cisplatin and carboplatin. This will support the refinement of therapeutic regimes and appropriate individualized treatment protocols.