Development of an Ultraperformance Liquid Chromatography/Mass Spectrometry Method To Quantify Cisplatin 1,2 Intrastrand Guanine-Guanine Adducts
Development of an Ultraperformance Liquid Chromatography/Mass Spectrometry Method To Quantify Cisplatin 1,2 Intrastrand Guanine-Guanine Adducts
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DOI：
10.1021/tx800481j
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发表时间：
2009-05-01
影响因子：
4.1
通讯作者：
Swenberg, James A.
中科院分区：
文献类型：
--
作者：
Baskerville-Abraham, Irene M.;Boysen, Gunnar;Swenberg, James A.
铂类化疗药物已广泛应用于癌症的治疗。顺铂是第一种基于铂的化疗剂，因此自20世纪60年代后期以来一直作为抗肿瘤剂被广泛研究。由于该试剂形成几种DNA加合物，因此需要高度灵敏和特异性的定量测定来将单个加合物的分子剂量与治疗效果相关联。建立了以N-15（10）CP-d（GpG）为内标物的超高效液相色谱-串联质谱（UPLC-MS/MS）法定量测定1，2-鸟嘌呤链内顺铂加合物[CP-d（GpG）]。内标物通过MS/MS表征，其浓度通过电感耦合等离子体质谱法验证。在选择性反应监测模式下通过UPLC-MS/MS定量之前，通过酶水解、离心过滤和HPLC（收集馏分）纯化DNA中含有CP-d（GpG）的样品[CP-d（GpG）的m/z为412.5 -> 248.1;[N-15（10）] CP-d（GpG）的m/z为417.5-253.1]。标准品的回收率> 90%，并且定量不受小牛胸腺DNA浓度增加的影响。该方法每次注射使用25 μ g DNA。定量限为3 fmol或每10（8）个核苷酸3.7个加合物，接近（32）p后标记方法对该加合物的灵敏度。这些数据表明，该方法适用于体外和体内评估由顺铂和卡铂形成的CP-d（GpG）加合物。随后，将该方法应用于使用卵巢癌细胞系和C57/BL 6小鼠的研究，以说明该方法能够使用生物学相关系统和剂量定量CP-d（GpG）加合物。生物标志物的发展，以确定治疗期间的组织特异性分子剂量学将导致更全面的了解顺铂和卡铂的治疗和不良反应。这将有助于完善治疗方案和适当的个性化治疗方案。
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 N-15(10) 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 [N-15(10)] CP-d(GpG)]. The recovery of standards was >90%, and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 mu g of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 10(8) nucleotides, which approaches the sensitivity of the (32)p 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.